Experimental Biology and Medicine

Parainfluenza Type 3 (Para 3) viremia has been demonstrated in hamsters by recovering virus from erythrocytes during pulmonary infection produced by the intranasal inoculation of virus. In attempts to isolate virus from blood, the plasma, erythrocytes and leukocytes were separated and cultured prior to freezing. Fetal bovine serum rather than calf serum was used in the tissue culture medium because the latter frequently contained Para 3 antibody. In addition, erythrocytes and leukocytes were allowed to remain in prolonged contact with tissue culture cells. The nonspecific CPE produced by plasma was avoided by washing tissue culture sheets 4 hr after the addition of plasma.

The authors gratefully acknowledge the assistance of Dr. C. K. Y. Fong who examined tissue culture preparations in the electron microscope and Dr. G. D. Hsiung who provided valuable criticism during the preparation of the manuscript. The excellent technical assistance of Mrs. Barbara Meek and Mrs. Jeana Richo is appreciated.

Exposure of mice to water of either 28 or 20° stimulated in about 2 min a maximal oxygen consumption value (12–130 ml/kg/min) similar to swimming in thermoneutral (36°) water. 20° water was considered an excessive stress because of a relatively rapid decline in rectal temperature of 1.2°/min which resulted in metabolic depression. Respiratory exchange ratios during stress in 21% O₂ averaged 0.94 suggesting submaximal loads on the aerobic system. During the additional stress of hypoxia (12% O₂) R rose above 1.00 possibly indicating a true maximal aerobic stress. The ability of these tests to detect aerobic defects was demonstrated by the 26% lower VR _{O2 max} in mice breathing 12% O₂ during stress as compared to those in 21% O₂. It seems both the swimming and cold stress tests may be used interchangeably. Each is of short duration and neither require training.

The authors thank Mr. Joseph F. Pitt for his assistance.

A significant increase was induced by lathyrism in the small fraction of collagen-like material solubilized from rat glomeruli. An increased affinity and staining by PTA of fibrils within the GBM of lathyritic rats was found by electron microscopy, and also probably reflects an alteration in the collagen-like moiety of the GBM by APN, a material known to affect the cross-linking of collagens. No change in total proteinuria was found in lathyritic rats, nor other major changes in renal structure by conventional microscopy, electron microscopy, or specialized stains of the sialoprotein layer of the glomerulus. Further study of physicochemical alterations induced in the GBM combined with methods to evaluate glomerular permeability, may reveal the location and nature of the filtration barrier.

Erythromycin estolate (EE) in concentration as low as 5 × 10^–6 M led to the leakage of enzymes from Chang liver cells exposed to the drug. Two other erythromycin derivatives (propionate and base), which differ from EE in that they do not produce hepatic injury in patients, did not lead to leakage of enzyme from the Chang cells until concentrations of these two derivatives reached 5 × 10^–4 M.

After in vitro incubation of rat liver Golgi membranes with ¹⁴C-CMP-sialic acid, the membranes were extracted by treatment with 2 × 10^–4 M Tris, pH 8.6, and lipoproteins were isolated by ultracentrifugation. The distribution of labeled lipoprotein was found to be 42% in VLDL, 14% in LDL, and 44% in HDL after sequential isolation of each density fraction. The addition of unlabeled whole rat serum to the extracted Golgi resulted in a decrease in label recovered in VLDL which was attributed to exchange of sialopeptides between VLDL and HDL. However, the addition of serum doubled the amount of recovered labeled lipoprotein and the extra label was associated with HDL. The labeled peptides were similar to those normally found in HDL as judged by DEAE-cellulose chromatography of the apo-HDL peptides. These results suggest the presence of a precursor pool of apolipoproteins within Golgi vesicles.

The survival of young BALB/c mice after abrupt restriction to a single 4-hr span of daily food accessibility can depend on the temporal placement of this feeding span in relation to the lighting regimen. Housing conditions are an important code-terminant of this response.

Sulfapyridine potentiates the analgesic effect of morphine. It also produces analgesia when administered to morphinepretreated mice long after the analgesic effect of morphine has worn off. Morphine possibly binds to blood and/or tissue components, most likely in the brain, forming pharmacologically inactive complexes, and sulfapyridine prevents and/or reverses the binding, thus freeing active morphine.

Antagonism of the antinociceptive action of morphine in mice by prior parenteral administration of cAMP has been confirmed using two additional analgesic testing procedures, the hot-plate test and the stretching test. The analgesia-antagonistic property, however, is not specific for cAMP because it also occurred after pretreatment with compounds which probably increased the concentration of adenosine or adenine in the body. Preliminary indications are that an unsubstituted purine ring is a requirement for the adenine derivative that is primarily responsible for the antagonism of the analgesic action of morphine.

The CV1 strain of vaccinia virus replicates poorly in human embryo kidney cells whereas most vaccinia strains such as the U.S. Reference smallpox vaccine replicate well in this host system. Absorption by the cell and production of viral antigen proceed at the same rate for both strains. Electron micrographs reveal that the CV1 strain produces immature forms of particles which do not possess a dense nucleoid of DNA material. Very few normal mature particles are formed and bizarre particles are found in the cytoplasm. CV1 DNA synthesis seems unable to proceed in the normal fashion in these cells.

The interest and counsel of Dr. L. F. Barker is deeply appreciated by the author. I am also grateful for the technical assistance of Mrs. Glennelle C. Washington.

C₅₇BL mouse urine CSF was found to be of lower molecular weight than normal or postendotoxin mouse serum CSF. In addition the fall in activity of postendotoxin serum CSF was found to be unaltered by bilateral nephrectomy. Because of these findings, a reevaluation of previous data was made. It was concluded that the kidney may play little or no part in the clearance of serum CSF. The origin of urinary CSF was not established.

Bacteriophages were isolated from 11 of 60 samples of live virus vaccines for human use. One of the phages isolated gave similar plaques and had similar morphologic characteristics to the phage found most frequently in fetal bovine serum.

We appreciate the support and encouragement given to us by R. L. Kirschstein, B. L. Elisberg, H. E. Hopps, and H. M. Meyer, Jr.

A low-molecular-weight ⁶⁵Zn complex was identified in the intestinal lumen and mucosa following oral administration of ⁶⁵Zn to rats. Analysis of the ⁶⁵Zn complex by thin layer chromatography indicates that the complex possesses properties markedly different from those of zinc salts and simple zinc-amino acid complexes. These experiments demonstrate that zinc is bound to a low-molecular-weight ligand as well as a variety of macromolecules in the intestine of the rat.

Female Holtzman rats were treated for 3–14 days with daily doses of phenobarbital (37 mg/kg, ip) or chlordane (25 mg/kg, ip) which caused a 10-fold increase in the hydroxylation of acetanilide by hepatic microsomes. Mature females treated with phenobarbital continued to cycle, exhibited preovulatory increases in serum luteinizing hormone (LH), and ovulated normally during a 2-week treatment period. In similar rats, ovariectomized 1 month earlier, the rate of disappearance of LH and follicle stimulating hormone (FSH) from the serum following hypophysectomy was unaltered by 2 weeks of phenobarbital or chlordane treatment. In immature females in which ovulation was induced with pregnant mare's serum, the preovulatory increase in serum LH concentration and subsequent ovulation were not altered by activation of hepatic microsomal enzymes. These results suggest that treatment of rats with phenobarbital does not block ovulation by mechanisms involving hepatic enzymes and/or estrogen metabolism.

Simian virus 40 uptake by the nuclei of permissive monkey kidney cells was enhanced by infecting in the presence of MgSO₄, DEAE-dextran, or arginine-rich histones, but not in the presence of lysine-rich histones. Nuclear uptake of virus was found to be more efficient in aged monolayer cultures than in freshly formed cell monolayers. Inhibition of cellular protein, RNA, or DNA synthesis did not prevent virus uptake. “Empty” capsids, which are essentially devoid of viral DNA, were taken up by the nuclei of permissive cells even more efficiently than infectious virus. Undegraded polypeptides of parental virus could be detected in the nuclei up to 24 hr after infection.

We thank M. McFalls for technical assistance. This study was supported by U.S. Public Health Service Research Grants CA-10594 and CA-10815 from the National Cancer Institute.

A very low cyclic GMP concentration (5 × 10^–11 M) causes the actively “cycling” lymphoblasts in isolated rat thymic lymphocyte populations to start synthesizing DNA by a calcium-independent process. These stimulated cells are able to progress subsequently through the DNA-synthetic phase and enter mitosis. The possible significance of the cyclic GMP-induced stimulatory action in the regulation of lymphoblast proliferation is discussed. An attempt is also made to interrelate the similar actions of cyclic GMP and cyclic AMP.

When myelomonocytic leukemia cells were admixed to spleen cells immune to a fibrosarcoma and the fibrosarcoma target cells, the growth of the leukemic cells was not inhibited. This was demonstrated in in vivo adoptive transfer experiments involving syngeneic animals. It was also shown that T cells are essential for the transfer of immunity to the fibrosarcoma. The results demonstrate that tumor cell rejection was a completely immunologically specific process.

The authors thank Drs. Harris, Metcalf, and Szenberg for their interest in the work, and Misses P. Bell and G. Cousins for their excellent technical assistance.

The lifespan of the quokka, a small marsupial, was shown to be markedly reduced after neonatal thymectomy, but this was not associated with the development of a wasting syndrome. The normal lifespan of intact animals is more than 10 years; however, most neonatally thymectomized quokkas died before 3 years.

We thank Messrs. W. Evans, L. Smallacambe, G. Benwell, W. Bartbolomaeus, and M. Price for animal husbandry, and Mr. N. A. Campbell for statistical advice.

Female rat pups injected sc with 1.0 mg testosterone propionate in 0.1 ml sesame oil on day 5 of life were given a single dose of FSH (200 μg) or LH (25 μg) iv or LHRH (3.0 μg) sc at 1:30 pm on day 30, 45, 60, 75, 90, 120, or 150. Normal cyclic rats were given FSH or LH in the same manner on the second day of diestrus at the same days of age. The data indicate that follicles in androgenized rats are ready to respond to exogenous and induced-endogenous gonadotropin, and that the ovarian threshold to gonadotropin decreases with age. However, the androgenized rats require more gonadotropin than their cyclic rat contemporaries receiving exogenous gonadotropin in order to produce the same ovulatory response.

The author thanks Dr. Roy O. Greep for his interest and support during the course of this study.

Candida extract caused significant tritiated-thymidine uptake (> 5,000 cpm, S/C > 4) in lymphocyte cultures from all normal adults tested. In contrast, in 22% of hospitalized patients, the antigen produced transformation less than any normal subject, and S/C < 4. The difference in candidainduced transformation between normal and hospitalized subjects was statistically significant, while no significant difference in conventional PHA stimulation could be demonstrated.

The author thanks Mr. B. B. Cartwright and Miss S. E. Hieny for technical assistance, and Dr. R. Vander Zwaag for performing statistical analyses.

A method for specific radioactivity analysis of alanine in blood is described. This analysis involves the separation of alanine by an ion exchange column technique and its subsequent isolation as a gravimetrically assay able sample (acetaldehyde-dimedon). Mass contamination of the acetaldehyde-dimedon from valine, which elutes from the ion exchange column close to alanine, is demonstrated to be negligible. Activity contamination of the acetaldehydedimedon from lactate and glucose, two significant products of alanine metabolism, is likewise demonstrated to be negligible. Sample counting rates have been demonstrated to be significantly above background (X 3) 2 hr following a single injection of 25 μCi l-[¹⁴C] alanine to a human subject.

The major detectable pyrrole metabolite of the pyrrolizidine alkaloid, monocrotaline, present in the liver, blood, and urine of rats, has been identified as dehydroretronecine. The subcutaneous injection of this metabolite into rats produces ulceration and hemorrhage of the stomach wall. Inhibition of hepatic parenchymal cell mitosis in regenerating liver was recorded within 52 hr following its administration and persisted for at least 6 weeks.

Pentobarbital-anesthetized rats of both sexes received intravenous infusions of isotonic saline, with or without gelatin and/or salmon calcitonin. Each animal served as its own control. Diuresis, natriuresis and increased GFR followed the addition of gelatin to saline infusions. Similar effects were observed when gelatin plus calcitonin were added to the saline infusion. Calcitonin may have increased sodium excretion, but did not affect urine flow rate or GFR when added to infusions of gelatin. No effect on potassium excretion was seen. Therefore gelatin is diuretic and natriuretic and calcitonin may be natriuretic but not diuretic when infused into the anesthetized rat. The changes in urine flow rate and sodium excretion were not caused by changes in GFR, suggesting altered tubular function as the cause.

This research was supported by National Science Foundation Grant GB 35263, and grants from the Michigan Heart Association and Kidney Foundation of Michigan.

Australia antigen, now termed hepatitis B antigen or HB antigen, has been demonstrated to be closely associated with the serum hepatitis virus and to be an indicator for the presence of the infective agent. The development of HB antigen in cultured human fetal hepatic parenchymal cells (hepatocytes) after exposure to acute-phase sera containing the antigen is described. Hepatitis B antigen was demonstrated in the cultured cells by immunofluorescence methods and specificity determined by agar gel diffusion, counterelectrophoresis, blocking and absorption experiments, and examination of frozen sections of liver containing the antigen. Development of HB antigen in the cultured fetal hepatocytes was slow and occurred in a limited number of cells. Hepatitis B antigen was detected initially as irregularly shaped granular deposits in the nuclei of the hepatocytes and was first observed 2 weeks after exposure. These observations support the concept of the hepatocyte nucleus as the primary site of viral replication. Hepatitis B antigen was observed in the cytoplasm of cultured hepatocytes beginning 4 weeks following exposure. Five to six weeks after exposure, cytoplasmic-antigen-containing hepatocytes were detected in increased numbers, occurring as foci or plaques of 2-4 cells. The foci of antigen-containing cells continued to increase slowly in size. These findings may imply a secondary cytoplasmic phase in the development of hepatitis B virus and also suggest cell to cell transfer. The small number of cultured hepatocytes in which HB antigen developed may be due to a limited capacity of hepatocytes from fetuses in early stages of gestation to support viral synthesis, or to a blocking effect exerted by the large ratio of noninfective to infective virus particles present in the inoculum.

Grateful acknowledgment is given to the Blood Grouping Laboratory of the Buffalo General Hospital for serum specimens and to the Children's Hospital of Buffalo for fetal tissue and to Dorothy C. Noyes for invaluable assistance.

Investigation was made of what role histamine plays in the gastric secretion induced by a new avian pancreatic polypeptide (APP). Use of an anticholinergic agent (glycopyrrolate bromide) was made to block the gastric effects of histamine in adult chickens. However, this agent was without effect on the stimulatory action of APP as measured by secretory volume, H⁺, pepsin, and protein levels. The pancreatic polypeptide action, therefore, is not mediated by histamine release in chickens, and, in fact, APP may be avian gastrin.

A possible relationship between certain inherited behavior patterns and susceptibility to hypertension was studied in rats. An “exploration score” was devised to quantify explorative behavior.

Susceptibility to DOCA-salt hypertension was similar in two strains that had been selectively bred for their disparate explorative behavior. Conversely, explorative behavior was similar in two other strains, selected for their respective susceptibility and resistance to DOCA-salt hypertension. Inheritance of a hypertensive trait appears therefore to be unrelated to inheritance of a specific explorative behavior in these rats.

The author is indebted to Dr. J. J. Groen for his encouragement and support, and to Dr. L. K. Dahl for his reviewing of the manuscript of this paper.

The plasma cholesterol levels of 6-month-old 500-g male retired breeder rats were significantly higher than the plasma cholesterol levels of 71-day-old 200-g adult male rats of the same strain. Oxandrolone administration daily for 21 days reduced the plasma cholesterol of the retired breeder rats 41% to a level below that of the young adult males. There was no difference in weight between the oxandrolone-treated retired breeder rats and their controls. The hypocholesterolemic properties of oxandrolone and the virtues of retired breeder rats as an experimental model are discussed.

This research was supported in part by the Veterans Administration Hospital and G. D. Searle and Company.

The postmenopausal state appears to be associated with an increased incidence of atherosclerotic vascular disease in women. Connective tissue and cellular changes in the female rat thoracic aorta were studied in oophorectomized mature rats with and without estrogen replacement. In one study, no attempt was made to control the increased growth of the oophorectomized female. However, because of the possible contribution of different growth rates to results, in another study, weights of the treated and nontreated groups were balanced by pair-feeding. Both studies showed the same qualitative results. Total DNA contents of anatomically-defined thoracic aortic segments were not affected by oophorectomy or by estrogen replacement. Significant and similar increments in total aortic elastin and collagen were seen in untreated oophorectomized groups compared to estrogen-treated ones; a significant increase in percentage of collagen was found. Accelerated accumulation of connective tissue in the rat aortic wall is associated with reduction of endogenous estrogen levels and is unrelated to changes in body weight; estrogen replacement prevents these changes.

The author is grateful to Mrs. Lisa Kasak and Mr. Richard D'Eletto for their assistance with the biochemical determinations and balance experiments. Mrs. Fay Ricksy helped in preparation of the manuscript.

Eight strains of Streptococcus faecalis var. liquefaciens were classified as bacteriocin producing or nonproducing on the basis of their effect on microbial L-forms. Four strains produced bacteriocin, and four did not. There was a significant association between bacteriocin production and ability to produce hematogenous pyelonephritis in the rat.

Isolation of cytomegalovirus (CMV) has been simplified using monolayers of fibroblasts in plastic cell-culture trays. The technique is at least as efficient as the conventional tube technique and may be more efficient with specimens containing low concentration of virus such as blood. Cytomegalovirus in blood has been detected only in association with the buffy-coat layer, but not with plasma or red cells. Pretreatment of fibroblasts with 5-iodo-2′-deoxyuridine (100 μg/ml) has not increased the CMV isolation rate. Using the new technique, CMV viremia was detected in 30% and CMV viruria in additional 20% of renal allograft recipients. Ninety strains of CMV, ten strains of herpes simplex, and two strains of varicella-zoster were isolated from 1,720 specimens.

We thank Dr. A. Nahmias, Emory University, and Dr. E. H. Lennette, California State Health Department, for identification by immunofluorescence of herpes simplex and varicella-zoster viruses. Supported by contract NHLI-72-3 from the National Heart and Lung Institute.

The results of the experiments described herein suggest that the role of a high fat diet in the rerepression of the genes which code for G6PD and ME is to provide either the corepressor(s) or a precursor of the corepressor(s). It appears that this substance is not normally present in rat liver, despite the large accumulation of fat which occurs after starvation and refeeding an inducer diet. Synthesis of the aporepressor(s) or an enzyme system necessary for the formation of the corepressor(s), or both, requires dietary protein but not carbohydrate.

After stimulation in vivo with heat-killed Staphylococcus aureus, rat peritoneal granulocytes and macrophages produced leukocytic endogenous mediator (LEM). When this LEM was injected into normal rats, it lowered plasma iron and zinc concentrations and markedly increased blood neutrophils. Incubation of macrophages. blood leukocytes, or Kupffer cells with S. aureus in tissue culture media and rat plasma in vitro gave low yields of LEM. Further evidence was presented to show that serum taken from rats 4 hr after an ip injection of Diplococcus pneumoniae contained LEM.

Lysine-rich and arginine-rich histories were extracted from erythroid precursors of bone marrow obtained from three patients with chronic erythremic myelosis. On polyacrylamide gel electrophoresis, the histone patterns resembled the patterns of his-tones extracted from erythroid precursors in patients with treated pernicious anemia. Amino acid analyses of histone fractions demonstrated that the erythremic myelosis histones were arginine rich. Relationships between these findings and the pathogenesis of megaloblastoid erythropoiesis are discussed.

Streptococcal peptidoglycan causes toxic effects in experimental animals, including changes in the skin of rabbits (1-3), rheumatic-like changes in the hearts of mice (4, 5), and persistent extensive carditis in rabbits (3). No one has yet determined whether or not streptococcal peptidoglycan plays a role in rheumatic carditis in man. In order to investigate this possibility, we attempted to detect the presence of antipeptidoglycan in the sera of the rheumatic fever patients with and without carditis.

For the detection of antipeptidoglycan, we used a heterologous system involving a test antigen derived from Micrococcus lysodeikticus and inhibitors obtained from Staphylococcus aureus. The basis of reactivity of peptidoglycan with the antibody in this system is the presence of two determinants: glycan with an immunodominant β-N-acetylglucosamine (6, 7) and peptide with an immunodominant C-terminal d-alanyl-d-alanine (8, 9), presented in Table I. In the peptide determinant, l-alanyl-γ-(α-d-glutamyl)-l-lysyl-d-alanyl-d-alanine, the α-carboxyl group of d-glutamic acid is substituted by glycine in M. lysodeikticus, and by an amide in Streptococcus pyogenes and S. aureus. The glycan determinant in all three bacterial species of the test system is composed of alternating units of N-acetylglucosamine and N-acetylmuramic acid linked β-1,4.

Materials and Methods. The human sera studied for the presence of antipeptidoglycan were obtained from patients with acute rheumatic fever, patients with streptococcal impetigo, and healthy individuals. Fifteen rheumatic fever patients, seven with carditis and eight without, were under treatment at The House of The Good Samaritan. All 15 patients met the revised Jones criteria for rheumatic fever (10). Rheumatic fever patients with definitely significant murmurs were considered to have carditis; those without significant murmurs were considered not to have carditis (11). In order to provide an adequate amount of serum for the tests, it was necessary to pool several serum specimens from each patient.

The antitumor effect of interferon was assayed in mice pretreated with ALS to depress lymphocytes or with silica to depress macrophages. Pretreated DBA/2 mice were inoculated intraperitoneally with interferon-sensitive or interferon-resistant L 1210 cells, and Balb/c mice were inoculated intraperitoneally with Ehrlich ascites cells. The efficacy of interferon was determined by its effect either on survival time in L 1210 tumor-inoculated mice, or on the multiplication of EA cells. Interferon proved as effective in ALS-pretreated tumor-inoculated mice as in mice not receiving ALS. Pretreatment of mice with silica diminished somewhat, but did not abolish the antitumor effect. It is concluded that interferon can exert an antitumor effect in lymphocyte-and macrophagedepressed mice.

One of us (I.G.) is indebted to Dr. Sidney Farber, Director of the Childrens Cancer Research Foundation, Boston, Mass., for his continued interest and support.

We are grateful to Dr. A. C. Allison for his advice in the experiments on silica, to Drs. A. C. Allison and K. Roböck for their generous gifts of silica, and to Dr. Pernilla Lindahl for many helpful discussions.

The comparative evaluation of the endotoxin-detoxifying capacity of diverse populations of rat macrophages was undertaken in order to define the relative role of macrophages in endotoxemia. Actinomycin D-treated mice were employed in the bioassay of submicrogram amounts of endotoxin. Sonicate preparations of rat pulmonary alveolar, peritoneal, hepatic, and splenic macrophages were found to possess significant endotoxin-detoxifying activity. Kupffer cells manifested the greatest activity. In contrast, liver parenchymal cell sonicates did not detoxify endotoxin. The demonstrated endotoxin-detoxifying capacity of macrophages further accept the protective role of macrophages in host defense against endotoxins.

Intact male Holstein calves were fed a purified diet (8.5 ppm Zn) with and without 350 ppm Cd in amounts to provide approximately equal nitrogen balances. The added Cd reduced ⁶⁵Zn absorption, showing that the earlier established effect on Zn absorption was not due to the indirect influence on feed intake and nitrogen balance. Whether the Cd effect was caused by changes at the tissue or intestine level was not established. However, Cd had a major influence on ⁶⁵Zn metabolism after absorption, as indicated by a differential influence on ⁶⁵Zn in several tissues.

The authors thank Mr. M. S. Ansari, Mr. L. O. Branch, Mr. J. D. Brown, Mr. J. C. Carter, Jr., and Miss Beverly Purswell for technical assistance, and Mrs. Shirley Gentry for editorial assistance.

In spinal cats vagotomized and administered sufficient propranolol to block cardiac beta-receptors, the effect of vagal stimulation of the myocardium was studied. With this stimulation, a bradycardia was sustained for 1- to 4-hr intervals. Examination of the myocardial tissue from atria and ventricles revealed, however, no myofibrillary or vascular changes. Since these results indicate that this vagal stimulation did not produce myocardial necrosis, alterations in cardiac tissue attributed by other investigators to parasympathetic activation would appear to involve secondary sympathetic effects.

The amino acid composition of the calculus matrix of various types from different species and the two fractions of urine polyelectrolytes were analyzed by gas chromatography. All matrices and the urine polyelectrolytes had more acidic amino acids than basic amino acids, and, thus, are negatively charged. Nevertheless, there were variations of amino acid compositions between individual calculi and among calculi from different types and species. Apparently, calculus matrix is not composed of preformed compounds.

Immunoelectrophoretic tests revealed that part of the calculus matrix was common to the high-molecular-weight urine polyelectrolytes. Because of the readily available carboxyl groups of aspartic acid and glutamic acid in the small-molecular-weight urine poly-electrolytes for cation binding, they could initiate the aggregation of the charged molecules. Therefore, the participation of the aggregated urine polyelectrolytes, including the high-and the low-molecular-weight fractions, in the formation of calculus matrix is feasible.

The rate of disappearance of ³H-labeled cholesterol and its conversion into bile acids were measured in rats subjected to hypothalamic damage to their ventral medial nuclei, fornices, and the medial portions of their lateral hypothalamic areas. A significant reduction was found in both processes in the experimental rats. Also, the rate of incorporation of acetate-³H into newly synthesized bile acids was found to be reduced in hypothalamus-injured rats. These findings suggest that the hypercholesterolemia regularly appearing after this type of hypothalamic injury stems from a relative lag in the catabolic conversion of cholesterol into bile acids.

Adult cycling rats hypophysectomized on the morning of metestrus (day 1) were pituitary-autotransplanted or treated with 2.0 IU of prolactin twice daily by subcutaneous injection for 15 days. These two types of animals received either LH antiserum (LH A/S) or normal rabbit serum (NRS) on days 5 and 6 after hypophysectomy. Multiple blood samples were drawn by heart puncture from individual animals throughout the study, and the sera were analyzed for progesterone by radioimmunoassay. The results show that LH A/S was not effective in reducing serum progesterone levels maintained by exogenous or endogenous prolactin.

The authors express their appreciation to Miss Johanna McCann for her skilled assistance.

Polyethylene intrauterine devices sutured into the uterine cavity of CF-1C mice for 21 days produced an alteration in peritoneal fluid cell distribution as shown by the percent increase in polymorphonuclear leukocytes and histiocytes, but with no change in animal movement.

Histamine and norepinephrine have a concentration-dependent growth-enhancing effect on fibroblast growth in vitro that is different from the response to serotonin. These distinct dose-response curves suggest different mechanisms of action. Furthermore, histamine depresses growth of human embryo lung fibroblasts while enhancing growth of an established cell line (3T6) and serially cultured strains of mouse skin fibroblasts. The acquired nature of the histamine response is in contrast to the consistent growth-enhancing effect of serotonin and norepinephrine on all fibroblast lines or strains.

At micromolar concentrations, histamine, norepinephrine, and bradykinin antagonize the action of serotonin, while at a nanomolar level norepinephrine potentiates the effect of serotonin. The spatial configuration of bradykinin is critical for the fibroblast growth response. Prostaglandin E₂ inhibits fibroblast growth.

The authors gratefully acknowledge the assistance of Mr. T. Ralph Alvarez and the gift of prostaglandin E₂ from Dr. Vincent A. Ziboh.

Chronic administration of hydrochlorothiazide (10 mg/kg, po for 12 months) to beagle dogs failed to produce any alterations in blood pressure and heart rate, despite a significant reduction in cardiac output, stroke volume, and stroke work. Analysis of ventricular function curves indicates a diminution in contractility of the myocardium in the treated animals. It is concluded from this investigation that the adverse effects of HCT on the myocardium in the face of increased total peripheral resistance noted in this study may warrant careful reevaluation of this agent in cardiovascular therapy.

This study was supported by Public Health Service Grant GM-15587. The authors express their appreciation to Dr. John E. Baer of Merck Institute of Therapeutic Research, West Point, PA, for generously supplying hydrochlorothiazide, and to Messrs. Charles Parham and David Snyder for their valuable technical assistance.

The importance of the monocyte and its derivative, the macrophage, is becoming increasingly apparent in many areas of biologic research. In addition to its well-known role in phagocytosis, it likely plays a part in processing antigen in cellular immunity (1) and facilitating the response of lymphocytes to antigen (2), pokeweed mitogen (3), and phytohemagglutinin (4). It is a rich source of colony-stimulating factor for the in vitro growth of colonies of granulocytes and mononuclear cells from human and murine marrow (5). This cell may of use in the immunotherapy of human neoplastic processes. The monocyte is produced in the bone marrow (6), but its precursor cell is yet to be identified. Measurement of the physiological, immunological, and biochemical activities of the normal human monocyte has been hampered by the difficulty of obtaining pure samples in sufficient quantity. Most methods have resulted in monocyte monolayers adherent to a surface where they cannot be accurately counted or easily handled for study. The present study reports the separation of monocytes in high purity and large quantities from normal human blood. It results in cells that are in suspension and, therefore, can be easily quantitated. They can be easily transferred for radioisotopic, morphologic, and immunologic investigations.

Materials and Methods. Two hundred and fifty milliliters of venous blood from normal volunteers was anticoagulated with heparin (12 U/ml of blood) and the erythrocytes sedimented with 5% dextran (Sigma Chem. Co., St. Louis, MO) for 40 min at 37°. The cells in the supernatant were harvested by centrifugation at 190g for 8 min and washed in sterile phosphate-buffered saline (PBS, pH 7.25). The mononuclear cells were initially separated by the method of Bennett and Cohn (7). The cells were suspended in 27% bovine albumin (Cohn fraction V) (Sigma Chem. Co.) in PBS at a cell concentration of 75 × 10⁶ leukocytes/ml.

The immune responses elicited during a primary immunization in DBA/2J, C3Hf/HeHa, and AKR mice by allografts of the C57BL/6 lymphoma EL4 were studied in vitro. The ⁵¹Cr release assays were used to measure the complement-independent cellular cytotoxicity (C'ICC), the complement-dependent cellular cytotoxicity (C'DCC), and the serum cytotoxicity (SA). The following observations were made: (a) C'ICC was detected against the major histocompatibility antigens (H-2) in all three strains; (b) no C'ICC was detected against the θC3H alloantigen in AKR mice or against the D-region “public” antigens of H-2 (called here “C”) in C3H or AKR mice; (c) C'DCC and/or SA were detected against each of the three antigens in the appropriate responder strains; (d) no major interference was observed between the concomitant complement-dependent humoral response to a nontransplantation alloantigen (θC3H) and H-2, and the cell-mediated response (C'ICC) to H-2; (e) the order of appearance of the C'ICC and C'DCC were different in the three responder strains.

The authors thank Mr. Gabor Papp for his proficient technical assistance.

Serial circulating levels of FSH were higher in five female than in four male infant (0.4-2.0 years) chimpanzees. A rhythm in FSH levels was observed in the females with a periodicity of 8–25 days. There was no sex difference in mean LH levels. However, greater day-to-day variability was observed in the females.

The animals used in the study were handled in accordance with the “Guide for Laboratory-Animal Facilities and Care” established by The National Academy of Science-National Research Council. This work was supported in part by M. R. C. Canada Grant MA-2997 and by N. I. H. Contract DRR-73-2100. The LER-907 human pituitary gonadotropin standard was a gift of the National Pituitary Agency. We thank Dr. F. S. Chebib for help with the statistical analyses and Mrs. R. Poturnak, Mrs. E. Dudeck, and Mr. D. Bookout for technical assistance.

A radioimmunassay was developed for quantifying rat prolactin in blood serum, but the assay detected small amounts of immunoreactive prolactin in serum from hypophysectomized (Hx) rats. Prolonged anesthesia of these Hx rats with urethane significantly decreased their serum prolactin values, while prolonged anesthesia with pentobarbital (PB) had no significant effect.

Transplantation of pituitary tissue (AP) to the renal capsule of Hx rats elevated serum prolactin, and this effect was produced with as little as I/8 AP. Prolonged PB or urethane anesthesia combined with multiple blood sampling of Hx rats bearing ectopic AP significantly increased serum prolactin. The absence of stable prolactin concentrations an these rats suggests that prolactin release by ectopic AP may still be influenced by hypothalamic hypophysiotrophic hormones in the systemic circulation.

The inhibitory effects of various ergot alkaloids on pseudopregnancy (1), decidualization (2), early pregnancy (3), and lactation (4) in the rat have been well documented by several investigators. In general, these effects were attributed to a specific inhibition of prolactin secretion, as prolactin was shown to partially reverse the effects of ergocornine (1), and plasma prolactin levels decreased after treatment with the drug (5). Recently, Wuttke et al. (9) reported a significant decrease in the plasma LH levels in rats also treated with 50 or 200 μg of ergocornine (5). The present study was undertaken to examine the effects of one of the ergot alkaloids, ergotamine tartrate, on ovulation, estrus, and LH secretion in adult cycling rats.

Materials and Methods. Adult female Charles River rats weighing 180-210 g were used in the present experiments. The animals were housed in rooms with constant temperature and a 14-hr light (5 AM to 7 PM EST) and 10-hr darkness schedule. The estrous cycle was checked by daily vaginal smears, and only animals exhibiting two consecutive cycles of 4 days were used in the study.

Ergotamine tartrate (Sigma Chemical Co.) was administered intraperitoneally as a suspension in 0.9% saline at 12:30 PM on the day of proestrus. Control rats received saline at this time. Blood samples were obtained under light ether anesthesia between 5:30 and 6 PM on the day of proestrus, as well as at 11 AM on the day of estrus. Luteinizing hormone, when given, was injected intraperitoneally at 4 PM during proestrus. One group of treated animals was subjected to mechanical stimulation of cervix at 6:30 PM on the day of proestrus.

All the animals were autopsied between 11 AM and 12 noon on the day of estrus.

During the luteal phase of the menstrual cycle, rhesus monkeys (M. mulatto) were treated with 50 IU HCG in saline or saline alone, administered intramuscularly. The ten daily treatments were initiated on the day when implantation would have occurred had the cycle been fertile. Plasma estrogen was determined by radioimmunoassay, and progestin was measured by the competitive protein-binding method. Within 24 hr of the first HCG treatment, progestin levels in the blood reached a peak and then declined gradually despite continued treatment. Estrogen levels also increased but did not reach a peak until the fifth day of HCG administration. Like progestin, the estrogen levels then declined even though treatment was continued. Onset of menstruation in these animals was delayed by several days. The saline-treated animals had normal steroid patterns and cycle length. It is proposed that the injection of HCG brings about biochemical changes in the corpus luteum similar to those occurring during early pregnancy.

The skillful laboratory help of Messers. Robert J. Mandetta and James H. Brinkerhoff is gratefully acknowledged. The metyrapone used in this study was kindly supplied by Ciba-Geigy Pharmaceuticals, Inc., Summit, NJ.

Plasma from 48-hr nephrectomized or sham-nephrectomized rats was incubated with homologous ³²P-labeled PE (renin “preinhibitor”). After extraction of total lipids from the incubate and thin layer silica gel chromatography of the extract, radioactivity was recovered as PE or its deacylated derivative LPE (renin “inhibitor”). Nephrectomy did not cause a reduction in the proportion of counts recovered as LPE, suggesting that its rate of formation and degradation is normal. This suggests that plasma from nephrectomized rats should have no lack of renin “inhibitor” if it is formed from what are known to be undiminished circulating levels of “preinhibitor”. If the kidney is not the major source of circulating “preinhibitor” and does not affect availability of the “inhibitor”, its proposed renin-inhibitory role may be questioned. Alternatively, the phospholipid theory of renin inhibition is untenable in its present form.

Hamster embryo cells infected with GPHLV showed morphological cellular transformation. Virus strains isolated from leukemic guinea pigs showed somewhat higher transforming capacity than the isolates from “normal” Hartley guinea pigs. Virus specific antigen was detected in the cytoplasm of 7-20% of the transformed hamster cells. Infectious virus could be recovered from the transformed cells by co-cultivation with guinea pig kidney cells. Quantitative analysis showed that in these cell lines 12-47/100,000 transformed hamster cells yielded infectious virus.

The authors are grateful to Dr. R. H. Green for his valuable suggestions during the preparation of this manuscript. The excellent technical assistance of JoEllyn Bradley and Judy Wnek is appreciated.

A histopathologic study of serumtransmitted immune polioencephalomyelitis in C58 mice showed that the inflammatory nature of the CNS lesions in the serumtransmitted disease did not differ significantly from the disease actively induced by inactivated syngeneic malignant lymphocytes. The histopathology was characterized by perivascular lymphocytic infiltrates in the gray matter of the spinal cord and brain stem with accompanying microglial proliferation and neuronal destruction. Demyelination, inclusion body formation, astrocyte proliferation, and cerebral and cerebellar involvement were absent. Evidence was summarized indicating that the active serum component was not an immunoglobulin but probably a processed antigen or a lymphokine. The disease appears to result from an age-dependent autoimmune response to theta or theta-like antigens common to CNS tissues and lymphoid cells.

Alcohol, in large amounts, increases the blood flow to brains of man and other animals. To what extent blood flow to specific regions of the brain varies with smaller, nonanesthetizing doses has been the subject of the present study. A new radioisotopic method was employed which permitted the simultaneous measurement of blood flow to each of ten regions of the brain in unrestrained, unanesthetized animals as small as rats. Three doses of alcohol were used: a low dose causing no behavioral change, an intermediate dose causing lack of coordination but no loss of consciousness, and a high toxic dose producing anesthesia. The blood levels of alcohol causing various signs of intoxication were the same in rats and man. At the low dose, no changes were seen in brain blood flow. At the intoxicating dose slight, but insignificant, increases were seen in the basal ganglia, frontal cortex, and olfactory bulb, while hippocampal blood flow decreased. At the anesthetic dose, blood flow increased to the olfactory bulb and the brain stem and actually decreased slightly to the parietal cortex. Support of the depressed ventilation rate observed at this dose of ethanol restored the pCO₂ and pO₂ values to control levels but did not reverse the observed acidosis; cardiac output fell, signaling increased peripheral resistance. Under these circumstances, the flow of blood to all brain regions fell, most notably to the hippocampus, cerebellum, and parietal cortex. The effects of ethanol and pentobarbital are compared.

We are indebted to Dr. D. Knowlton for the analyses of blood levels of ethanol. This work was supported in part by a grant from The Licensed Beverage Industries and by Public Health Service Grant NB05526.

Samples of whole blood were collected at 20–min intervals for a period of several hours from individual cannulated proestrous rat. Plasma luteinizing hormone and prolactin concentrations were determined by radioimmunoassay. Either pentobarbital or pentothal anesthesia maintained during the critical period in proestrus rats blocked the preovulatory increase in serum LH and prevented subsequent ovulation. Pentobarbital anesthesia induced a rapid increase in serum prolactin concentrations whereas pentothal anesthesia did not, but both anesthetics prevented the spontaneous preovulatory increase in serum prolactin. In addition, pentothal anesthesia prevented the response to subsequent injections of pentobarbital.

In steroid–blocked ovariecto–mized rats, increases in serum LH concentrations were induced, both before and after pentobarbital anesthesia, by systemic injections of crude extracts of rat hypothalamic tissue (0.1–2.0 hypothalamic equivalents/rat) or by similar injections of purified porcine luteinizing hormone–releasing hormone (LH–RH) at doses of 0.032–0.080 μg/rat. Increases in serum LH concentrations were greater during pentobarbital anesthesia than they had been in the same animals prior to anesthesia. Under comparable conditions, acid extracts of rat cerebral tissue (2.5 mg equivalents/rat) did not cause an increase in serum LH concentrations. These data reveal an increased responsiveness to LH–releasing activities during pentobarbital anesthesia and suggest that barbiturates probably do not block LH release in the proestrous rat by limiting the pituitary response to such stimuli.

Estradiol (E₂) and progesterone (P) were measured by radioimmunoassay in peripheral blood serum on the second day of diestrus (D–2) and estrus (E) in normal cycling rats, in ovariectomized (OVX) animals, and in PMS–hCG– and OVX–E₂–P–treated rats. PMS–hCG and OVX–E₂–P animals had significantly higher estradiol and progesterone levels than cycling rats on D–2 or E. Progesterone was significantly higher in the PMS–hCG group while estradiol was higher in OVX–E₂–P–treated rats. These results may provide an explanation of previous findings relative to LH dynamics in OVX–E₂–P– and PMS–hCG–treated animals.

We thank Mrs. Ruth Penardi, Miss Katherine Bangs, and Mrs. Frances Smith for technical and secretarial help and Mr. Bobby McAlister for drawing the figure.

Long–term ovariectomized rats primed with estrogen and progesterone responded to exogenous LHRH at dosages of 40 and 100 ng with a sharp rise in plasma LH, peaking at 10–20 min after injection and gradually returning to control levels. In contrast, intact rats treated a week earlier with PMS followed by hCG completely failed to respond to even the higher dosage of LHRH: plasma LH levels remained almost undetecable in these animals. Assays of pituitary extracts revealed a similar concentration and content of prolactin but very depressed values for pituitary LH in these PMS–hCG–treated rats compared with earlier studies in OVX–E₂–P animals.

We thank Dr. A. V. Schally for supplying the synthetic LHRH and Dr. A. F. Parlow and the NIAMDD Hormone Distribution Program for making the RIA kits available. Ms. Frances Smith, Virginia Hoover, and Katherine Bangs and Mr. David Whitmoyer supplied valuable technical assistance.

A bioassay for the detection of the thrombopoietic stimulating factor (TSF or thrombopoietin) that utilizes thrombo–cythemic mice has been presented. A single injection of anti–mouse platelet serum (AMPS) to mice produced a characteristic thrombocytopenia that was followed by rebound thrombocytosis. Two to three days later (7–8 days after the AMPS injection), a significantly (p < 0.05) depressed thrombo–poiesis existed in response to the thrombocytosis. Normal and thrombocythemic mice were injected with normal sheep sera or sera from platelet–depleted sheep and Na₂ ³⁵SO₄ incorporation into platelets was measured. A greater difference in ³⁵S uptake existed between AMPS–treated control mice (mice injected with normal sheep serum) and AMPS–treated stimulated mice (mice injected with serum from platelet–depleted sheep) than in normal mice treated with the same sera. Also, TSF in sheep sera was shown to lose activity after storage at −20°.

The author is grateful to Mrs. Marilyn Cottrell and Miss Rose Clift for technical assistance and to Mrs. Ann Beardsley for her art and stenographic services. Appreciation is also extended to Mr. George Creswell for help in bleeding and maintaining the sheep, to Mr. James Dyer for help in performing the mouse assays, and to Dr. T. T. Odell, Jr. and Dr. Shirley Ebbe for their many helpful suggestions.

The purpose of this study was to assess the action of caffeine as a gastric secretagogue in rats, and to compare it with histamine– or pentagastrin-induced secretion. Rats bearing permanent cannulas of the stomach and the vena cava were infused for 24 hr with normal saline alone (control animals) or with caffeine sodium benzoate, histamine, and pentagastrin, in saline, separately or combined. In doses of 30, 60, and 120 μg/kg body weight for 24 hr, caffeine stimulated output of gastric juice, HC1, and pepsin without significantly affecting the concentration of these components. In this respect, caffeine differed from histamine and pentagastrin, known stimulants of parietal and chief cells in rats. Caffeine did not potentiate the secretory action of histamine or pentagastrin on HC1 and pepsin. Following bilateral vagotomy, caffeine–induced gastric secretion was markedly decreased.

The technical assistance of Mrs. T. Hoogen, Mrs. M. McCubbin, and Mr. T. Pachkowski is gratefully acknowledged.

The fixation of human erythrocytes with glutaraldehyde causes a striking reduction in the agglutinability by ABO–specific antisera. However, glutaraldehyde does not appear to destroy the specific antigenic receptors, since the fixed cells retain the capacity to adsorb specific antibodies from solution.

Streptococcal cell wall mucopep–tide administered intravenously to experimental animals provoked marked circulatory changes. A fall of arterial blood pressure and a decrease of cardiac output were observed. These changes are similar to those reported after the administration of bacterial lipopoly–saccharide. Some evidence suggests that both a direct effect of mucopeptide on sensitive structures and a release of pharmacologically active substances may play a role in the hemodynamic disturbances. On the other hand, the central nervous system probably plays no important role in the circulatory effect of the mucopeptide.

A two–step mechanism for iron absorption has been described in man as well as in experimental animals (1–6). The first, relatively rapid, step is uptake of luminal iron by the mucosal cell. Subsequent transfer out of the cell into the circulation or underlying tissues is slower and only partially complete, thereby providing an intestinal mechanism for the regulation of iron absorption. Unab–sorbed iron is postulated to remain sequestered in the mucosal cell until it is lost in the feces when the cell is desquamated (4, 5).

An earlier study (7) with rat duodenal gut sacs demonstrated that the iron in the mucosa reacted as two chemically distinct forms, Fe²+ and Fe³+. In addition, the Fe²+ fraction was the precursor of the Fe³+ fraction, and the properties of the latter were consistent with the aforementioned sequestered portion of mucosal cell iron. Variable, small portions of the trivalent iron have been identified as ferritin (5, 7, 8), but the major fraction of this pool remains unidentified.

These findings suggested the presence of an intracellular mechanism for the oxidation of Fe²+ to Fe³+, and the present report describes the discovery of such a ferrous–oxidizing activity in the intestine. The physical and chemical properties of this activity are consistent with those of an enzyme and suggest a possible role in mucosal cell iron metabolism for this activity.

Methods. Analytical methods. Total iron was determined by the α,α'–dipyridyl method of Ramsay (9), modified as previously described (3). Aliquots of unknown were mixed with 0.5 ml of 0.5% α,α'–dipyridyl in 1.0 M sodium acetate buffer (pH 4.0) and 0.25 ml of 0.2 M sodium sulfite, diluted to a total volume of 3.25 ml, heated 10 min at 100°, then centrifuged in a clinical centrifuge till clear.

The biological activity of interferon was determined after exposure to human secretions and excretions in vitro. Crude interferon lost some activity when incubated for 4 hr with control medium, but was usually inactivated by at least 50% more when incubated in the presence of serum, saliva, or cerebrospinal fluid, and by close to 100% when incubated with stool extract or bile. Interferon incubated for 24 hr with either serum or cerebrospinal fluid lost almost all activity.