Experimental Biology and Medicine

Further experimentation with the steer thyroid cell line indicates that the formation of “follicles” is enhanced by exposure to 8.9 × 10^-7 M thyroxine (T₄) or triiodothyronine (T₃) for 96 hr. The production of mucosubstances by the cultured thyroid cells is also increased by treating with T₄ or T₃, effects demonstrable after staining with PAS-alcian blue at pH 2.5. It is suggested that these in vitro effects bear on morphological organization and synthetic activity of the thyroid gland as well as on autoregulation and intraglandular homeo-stasis that may occur in situ.

Magnesium chloride was infused into the renal artery of anesthetized dogs in order to determine its effect on renal function. Natriuresis and diuresis were observed during MgCl₂ infusion, but there appeared to be no effect on glomerular filtration rate (GFR), or plasma sodium or potassium concentrations. Although mean arterial blood pressure and renal plasma flow (RPF) decreased throughout the experiment, the fall was not significant until after stopping MgCl₂ infusion. A significant stimulation of renin secretion occurred during magnesium administration.

UO₂ ²⁺ 1.3 mM added as UO₂(NO₃)₂ to the mucosal solution consistently inhibited the P.D. and I _sc evoked by 11 mM glucose and 35 mM 3-O-methyl glucose across isolated strips of bullfrog small intestine bathed by symmetrical Ringer solutions in which SO₄ ^2- was the major anion. The average degree of inhibition in the presence of glucose was 42 ± 7 (SEM) percent. P.D. and I _sc in the absence of transported solutes were not significantly altered by mucosal UO₂ ²⁺ at this concentration. Increasing the mucosal UO₂ ²⁺ concentration to 2.6 mM did not significantly increase its inhibitory action on glucose-evoked P.D. and I _sc. Further increasing the UO₂ ²⁺ concentration to 13 mM completely inhibited glucose-induced P.D. and I _sc but also markedly reduced these parameters in the absence of glucose. Serosal UO₂ ²⁺ (1.3 mM) had no effect on the P.D. and I _sc evoked by glucose and 3-O-methyl glucose. It is suggested that the inhibitory action of UO₂ ²⁺ involves binding of this ion to anionic sites located in the apical membrane of the absorptive cells. Mucosal or serosal UO₂ ²⁺ (1.3 mM) had no effect on the P.D. and I _SC elicited by 20 mM valine, indicating that under the conditions of these experiments UO₂ ²⁺ selectively inhibits sugar-induced P.D. and I _SC and, by implication, mucosal sugar uptake.

Glucose was administered intravenously to 50- and 100-day-old GF and CV rats. Fasting blood glucose levels in GF and CV rats were found to be comparable. Glucose tolerance tests showed that GF and CV rats clear glucose from the blood at a similar rate. Although insulin concentrations in 100-day-old GF rats tended to be somewhat lower than in CV rats, the percentage increase during the 30-min period after glucose administration was similar, and matched the increase in blood glucose. Levels of plasma catecholamines were analyzed fluorometrically and were found to be comparable in 100-day-old GF and CV rats. It was concluded that insulin insufficiency plays no role in the syndrome of metabolic anomalies demonstrated by the germfree rat.

Bilirubin, an organic anion and breakdown product of heme, is excreted chiefly by the liver by a process involving uptake by the hepatocyte and subsequent conjugation to form a more polar compound (1). The availability of purified preparations of bilirubin, especially of radiolabeled bilirubin, has greatly facilitated studies of its metabolism (1-5). Study of the transport of bilirubin from blood to bile, however, must also include consideration of the mechanisms for excretion of bilirubin conjugates. Such study has been limited because of difficulty in obtaining conjugated bilirubin free of significant quantities of contaminants (6-19).

Recent studies of albumin-agarose gel affinity chromatography have revealed that the gels have a high affinity for bilirubin and a relatively low affinity and capacity for tri-and dihydroxy bile acids (20, 21), suggesting that this method might effectively separate bilirubin conjugates from other components in bile. We describe here, a rapid and relatively simple method for purification of conjugated bilirubin from bile.

Materials and methods. Albumin-agarose gel was prepared by a modification of the cyanogen bromide method (20, 22), and packed in small columns of 0.9-cm i.d. Bile acids were quantitated in bile and column eluates by gas-liquid chromatography (23), cholesterol by the method of Zak et al. (24), and phospholipids by a modification of the method of Chen et al. (25, 26).

Absorption spectra were obtained with a Beckman Acta III spectrophotometer, and fluorimetric analyses with a Farrand Mark I spectrofluorimeter.

Tritium was quantitated by adding up to 0.5 ml of sample to 15 ml of Aquasol (New England Nuclear) and by counting in a liquid scintillation counter (Nuclear Chicago).

All samples were recounted after addition of [³H]toluene (New England Nuclear) and results were corrected for background and quenching.

Myocardial protein synthesis was elevated 7 days after rats were subjected to experimental aortic outflow obstruction. Although RNA synthesis was not increased at this time, RNA concentration was elevated and may have provided for the observed increase in protein synthesis. A possible basis for the persistence of the high RNA levels was a decrease in the degradation of RNA. The increase in intracellular calcium observed in hypertrophied tissue may be involved in the maintenance of RNA concentration and in the increased rate of protein synthesis.

We have shown that a potent agonist of the luteinizing hormone injected on either Day 11 of pseudopregnancy or Day 15 of pregnancy causes luteinization of ovarian follicles and a rapid regression of functional corpora lutea. Serum levels of progesterone declined markedly within 3 days of peptide injection in pregnant animals. In the presence of regressing corpora lutea, fetal resorption occurs which can be prevented by estradiol (500 ng, twice a day) or progesterone. Daily levels of estradiol below 1 μg did not maintain either corpora lutea weight or fetal survival, whereas 4 μg of the steroid maintained corpora lutea weight, but not pregnancy.

Mice were given a lethal dose of whole-body γ radiation and injected with 10⁵ or 10⁶ marrow cells from 10- to 143-week-old syngeneic donors. Nine days later, colony-forming units (CFU) were counted in the spleens of mice given 10⁵ cells, and 21 days after irradiation, spleen and thymus weights were determined in the recipients of 10⁶ cells. It was found that there were no significant changes with age in marrow CFU or in the ability of marrow cells to repopu-late host spleens. In contrast, thymic regeneration was significantly impaired when the recipients Received marrow cells from donors 117 weeks of age or older. These observations suggest that with aging there is a decrease in marrow T-cell progenitors and/ or a decline in their proliferative capacity.

Granules of human peripheral blood leucocytes contain four well-characterized elastase isozymes and one or two slow-moving elastaselike esterases (SE) which have not been as well characterized. SE are capable of hydrolyzing typical elastase synthetic substrates such as N-acetyl-DL-alanine-α-naphthyl ester (Ac-DL-Ala-1-ONap) and N-t-butyloxycarbonyl-L-alanine-p-nitrophenyl ester (Boc-Ala-ONp), but unlike the highly basic elastase isozymes, SE barely migrate into 13% acrylamide gels during cationic electrophoresis at pH 4.3. Hydrolysis of Ac-DL-Ala-1-ONap by SE requires the presence of Triton in the gel, and hydrolysis of Boc-Ala-ONp by the same enzyme(s) is also enhanced in the presence of the detergent. Triton is not required for these activities, in the case of the elastase isozymes. Diisopropylfluorophosphate (Dip-F) inactivates both SE and the elastase isozymes, whereas Ac-(Ala)₂-Pro-AlaCH₂Cl (a powerful inactivator of the leucocyte elastase isozymes at 10^-4 M concentration) does not inactivate SE at the same concentration. Immunochemical studies revealed antigenic cross-reaction between the rapidly migrating leucocyte elastase isozymes and SE. Two preparations of leucocyte granules from nonleukemic bone marrow cells showed no activity of the rapidly migrating elastase isozymes, but did contain SE activity. SE may be a precursor or zymogen form of the elastase isozymes, present in immature cells and partly retained through later stages of development.

Plasma lysosomal enzyme levels and hepatic phagocytosis were determined following Noble-Collip drum trauma in the rat. Circulating cathepsin and acid phosphatase activity increased after suble-thal trauma (300 rev), reaching maximal levels at 1-3 hr and returning to pretrauma levels at 24 hr after trauma. Hepatic phagocytosis was decreased maximally at 1 hr and recovered to control levels at 24 hr after sublethal trauma. Increasing trauma intensity (100-500 rev) resulted in a progressive failure in hepatic Kupffer cell phagocytosis and a progressive increase in plasma lysosomal enzyme levels when tested at 60-min post-trauma. A significant inverse correlation was found between the plasma lysosomal enzyme levels and Kupffer cell phagocytosis after trauma. The functional significance of the relationship between these two parameters and its importance in shock survival remain to be determined.

Isolated perfused fed rat livers spontaneously liberated glucose and ortho-phosphate to the medium; 24-hr fasted rat livers did not exhibit these phenomena. In perfused fed rat livers, glucagon (2 μg) increased glucose output and promoted ortho-phosphate incorporation. In perfused fed rat livers, insulin (250 or 500 mU) inhibited the spontaneous liberation of glucose and orthophosphate. Comparable doses of insulin significantly reduced the glucagon (2 μg)-induced increase in glucose output from perfused fed rat liver, but did not affect orthophosphate uptake by the organ.

O₂ extraction was measured simultaneously in right and left ventricles of dogs. Extraction was about 2.5 vol% higher in left ventricle. This figure, together with flow measurements of others (1-3), indicates that VO₂/100 g is at least twice as great in left ventricle as in right.

Mitochondria were prepared from bovine corpora lutea by differential centrifugation and were purified by iso-pycnic zonal centrifugation. A marked increase in specific cytochrome oxidase activity and a marked decrease in specific DNA and RNA content indicate that the procedure resulted in a highly purified preparation of mitochondria. These organelles had a higher rate of conversion of [4-¹⁴C]cholesterol to [4-¹⁴C]progesterone than did mitochondria separated only by differential centrifugation, suggesting that luteal mitochondria contain the enzyme systems required for progesterone synthesis.

Since age-dependent diminished macrophage function has been related to the increased susceptibility of newborn mice to herpes simplex virus (HSV) infection, the effect of several agents which might activate macrophages or other cells involved in immunity was investigated. BCG, typhoid vaccine, brucella vaccine, levamisole, or staphage lysate were administered to newborn mice prior to challenge with HSV-2. Of these agents, only BCG, administered ip or id 6 days prior to challenge, was found to increase the survival rate of newborn mice. The possible use of BCG, alone or in combination with other modalities, for the prevention or treatment of neonatal HSV infections is discussed.

Early polyoma (Py) virus-specific RNA synthesis was examined in cells infected with different concentrations of Py-virus. The effect of various multiplicities of infection (m.o.i.) on the rate of Py-RNA synthesis is different at 30 hr as compared to 65 hr. Thirty hours after infection at 27°, in the presence of 5-fluoro-2-deoxyuridine (FdU), an increase in input multiplicity was not associated with a quantitatively commensurate increase in the amount of virus-specific RNA synthesized. At 65 hr, the amount of viral RNA synthesized was roughly proportional to the number of infecting virus particles.

The following points summarize these findings: (i) there are 2 glutamine utilizing enzyme systems in the rat kidney; (ii) the cytoplasmic glutamyltransferase system hydrolyzes either glutamine isomer while the mitochondrial localized glutamin-ase 1 is specific for the ι-isomer; (iii) the cytoplasmic pathway contributes 70% of the total renal ammonia production in the normal kidney; (iv) chronic metabolic acidosis results in a 20-fold activation of the mitochondrial glutaminase 1 pathway.

In mice infected with mouse-adapted influenza A/PR/8/34, hepatic car-bamyl phosphate synthetase (CPS) activity was reduced to 88%, and ornithine trans-carbamylase (OTC) was reduced to 83% of control values. In mice infected with mouse-adapted B/Lee/40, CPS activity was 98% and OTC was 94% of control values. These limited reductions in enzyme activity were attributed to a nonspecific debilitating effect of acute influenzal pneumonia. These findings suggest that the pronounced reduction of CPS and OTC activities reported in Reye's syndrome in man are not a general manifestation of the severity of influenza infection.

The effect of the administration of sheep anti-LHRH gamma globulin (anti-LHRH-G) on implantation of fertilized ova was investigated in rats. Daily injections of 1 ml of anti-LHRH-G from Days 1 through 7 of pregnancy uniformly inhibited implantation of fertilized ova on Day 8, but viable sites, though considerably smaller in size than in control rats, became distinguishable on Day 14 in most rats. In some of these rats resorption of fetuses occurred, and others delivered pups 7-8 days after term. When the rats were given anti-LHRH-G from Days 3 through 5, the implantation was delayed by 5 days, but the gestation was not terminated. A single injection of 1 ml of anti-LHRH-G on Day 4 inhibited implantation on Day 8, but injection on Day 3 or 5 did not. The delayed implantation by anti-LHRH-G injected on Day 4 was nullified by concomitant administration of 2 sc injections of 1 μg of LHRH, or a single dose of 1 μg of estradiol. The data indicate that the hypothalamic LHRH is essential on Day 4 of pregnancy for timely implantation of fertilized ova, probably by maintaining LH and, consequently, estrogen secretion.

Serum-free supernatant fluids from monolayer cultures of B-16 mouse melanoma cells were found to contain a soluble membrane associated tumor-specific antigen. The 100,000 g supernatant of the culture fluid induced an antibody response to the B-16 cells both in rabbits and in the mouse strain of origin (C57B1/6J).

Similar supernatant fluids derived from an unrelated cell line (L-929) or from normal C57B1/6 fibroblasts did not contain the B-16 specific material. Preliminary results indicate that the B-16 specific material is a protein of low molecular weight which is released into the culture fluid chiefly by living cells and, to a lesser extent, by autolysing cells.

Perchloric acid-extractable whole blood spermidine and spermine concentrations were determined over a 4-week period in three men, four women, and one ovariectomized woman. Individual male spermidine/spermine ratios showed little fluctuation and similar values were obtained for each of the three males studied. Male spermidine and spermine concentrations, although stable for each male, varied from one subject to the next. Individual female spermidine/spermine ratios, as well as individual concentrations, fluctuated substantially when compared to those values obtained for males; female ratios appear to rise and fall as a function of the menstrual cycle. The spermidine/spermine ratios obtained from a normal female receiving oral contraceptive, as well as those from an ovariectomized female, were characteristic of values obtained from men. It is suggested that a sex-related hormone(s) influences both the spermidine/spermine ratio and spermidine and spermine concentrations in females.

Ovarian follicles isolated from bovine ovaries at the 16th and 17th days of the estrous cycle were minced, pooled, and incubated in Krebs-Ringer bicarbonate buffer at 37° for 2 hr. Samples were analyzed for testosterone, estrogens, and progesterone by radioimmunoassays. Incubation alone failed to cause an increase in testosterone (12.1 vs 29.3 ng/g of tissue; P > 0.05). Additions of 5 μg/ml of bovine LH or prostaglandin F₂α caused increases in testosterone synthesis to 125.0 (P < 0.01) and 103 ng/g (P < 0.01). Estradiol synthesis was also stimulated by LH (P < 0.01). However, stimulation due to addition of PGF₂α (5.1 to 15 ng/g) was not significant (P > 0.05). PGF₂α also caused a slight but nonsignificant increase in estrone secretion. Incubation failed to increase the progesterone concentration (623 vs 774 ng/g of tissue; P > 0.05). There was no increase in progesterone synthesis due to addition of either LH or PGF₂α (P > 0.05). The results show that bovine follicles produce much more testosterone than estrogen and that both LH and PGF₂α significantly stimulate testosterone synthesis in bovine follicular tissue without increasing net progesterone biosynthesis.

Thyroidal radioiodine release increased shortly after a single injection of small doses of PTU, while moderate doses of MMI produced a similar increase of thyroidal radioiodine release with a latency of 7-9 hr. Large doses of PTU and MMI failed to augment thyroidal radioiodine release for at least 29 to 34 hr after the initial administration of goitrogens, although plasma TSH increased significantly because of goitrogen administration. An increase of thyroid hormone release in response to exogenous TSH was depressed by PTU and MMI in rats and mice treated with T₄. Since this depression of TSH action only continued for a short period in spite of continuous administration of goitrogens, and since final thyroidal radioiodine release rate was similar to that produced by small doses of PTU, the effects mentioned were not simply due to general toxic action of goitrogens. It is suggested that large doses of PTU and MMI not only block thyroid hormone synthesis but also interfere with the action of TSH on thyroid hormone secretion.

To examine if hypothalamic or pituitary hormones are involved in the induction of the natriuresis which follows the injection of hypertonic saline or norepi-nephrine into the third ventricle, lesions were placed in the median eminence and the responses to intraventricular norepineph-rine or hypertonic saline were evaluated. Sham lesions in which the electrode was lowered into the brain but stopped short of the hypothalamic region did not interfere with the natriuresis, kaliuresis, and antidi-uresis induced by the third ventricular injection of either hypertonic sodium chloride or norepinephrine. Lesions in the median eminence which induced diabetes insipidus as evidenced by an increase in water consumption to approximately four times normal completely abolished the natriuresis and kaliuresis in response to intraventricular hypertonic saline or norepinephrine and diminished the antidiuresis. The observations suggest the possibility that a natriuretic hor-mone(s) is involved in the induction of central natriuresis.

Mouse saliva contains a potent inhibitor of complement activity. The secretion of this inhibitor appears to be regulated by action on α-adrenergic receptors for two reasons. First, an α-agonist (norepineph-rine) elicited saliva with a 260-fold higher specific activity of the inhibitor than that obtained with a cholinergic agent (pilocar-pine). Second, the α-agonist elicited saliva having a 43-fold greater specific activity than that obtained following administration of a β-adrenergic agonist (isoproterenol). This anticomplementary factor probably proteolytically degrades one or more of the complement components since it is inhibited by several protease inhibitors. The salivary anticomplementary factor is more potent than trypsin, chymotrypsin, thrombin, or Kallikrein. The anticomplementary factor has a pattern of inhibition like that of Kallikrein but unlike those of trypsin or chymotrypsin.

Partial purification of chicken epiphyseal PDE activity by centrifugation and column chromatography has defined two distinct peaks of PDE activity. The faster eluting peak (I) has a higher apparent K_m for cyclic AMP than the slower eluting major peak (II_s and II). Peak I has greater activity towards cyclic AMP as a substrate than towards cyclic GMP but does use both substrates. Peak I is not inhibited by T-3 or indomethacin at physiological concentrations. Substrates studies demonstrate the presence of at least two overlapping PDE species in the major peak (II_s and II). There is suggestive evidence that indomethacin is a more potent inhibitor of peak II_s which can use either cyclic AMP or cyclic GMP as substrates, whereas T-3 is a more potent inhibitor of fractions eluting where the enzyme only has activity with cyclic AMP.

Ovalbumin-pulsed spleen cells were found to stimulate thymidine uptake of lymph node cells of syngeneic mice immunized with ovalbumin in complete Freund's adjuvant after treatment of spleen cells with Mitomycin C but not after heating the spleen cells at 56° for 30 min. Ovalbumin-pulsed spleen cells of allogeneic mice failed to stimulate the immune lymph node cells more than unpulsed cells, although a net increase in the thymidine uptake above the allogeneic stimulation was observed when free ovalbumin was added to the mixed culture. To eliminate the high background of the mixed lymphocyte reaction, F₁ mice were made chimeric with bone marrow of one of the parental strains. Using lymph node cells of the immunized chimeras, the stimulation by pulsed spleen cells was much greater when antigen was presented on cells of the parental strain used for bone marrow injection than when presented on cells of the other parental strain.

The non-oncogenic rat C-type virus WF-1, isolated from a Wistar-Furth rat embryo cell line, was characterized biochemically. The purified virus has a buoyant density of 1.15 to 1.16 g/cm³ in sucrose, RNA-dependent DNA polymerase (reverse transcriptase) activity, and RNA with a sedimentation coefficient of 62 to 68 S. The viral RNA is single-stranded, and, upon treatment with heat, yields components with sedimentation coefficients of 36 S, 18 to 20 S, and 4 to 12 S.

Femoral vein (FV) pressure-volume relationships were measured in vitro in 14 dogs with chronic (more than 4 weeks), one-kidney perinephritic hypertension and in 13 unilaterally nephrectomized normotensive control dogs. Segments of FV were also examined histologically and analyzed for their water and electrolyte contents. Compared to controls: (i) the FV pressure-volume curves of hypertensive dogs were shifted toward the pressure axis (P < 0.05); (ii) calculated venous compliance in the pressure range of 0-15 mm Hg was decreased (P < 0.05); and (iii) the water and sodium contents of veins from hypertensive dogs were increased (P < 0.05). Histological examination of the FV from hypertensive and control dogs did not reveal significant differences. The findings indicate that the decreases in venous compliance that we have previously observed in the early stages (less than 4 weeks) of perinephritic hypertension in dogs persist into the chronic stage of hypertension. Venous wall “edema” may account for the decreased venous compliance in this form of hypertension.

The ability of various adeno-sine analogs to inhibit cholera toxin activation of the intestinal epithelial cell adenylate cyclase-cyclic AMP system was investigated. After incubation of cells with cholera toxin for 6 hr, large increases in cellular cyclic AMP content were observed. Addition of 2′,5′-dideoxyadenosine during the last 30 min of this 6-hr incubation resulted in 70% reduction in elevated cyclic AMP content. Other analogs were not effective inhibitors. 2′,5′-Dideoxyadenosine was also a potent inhibitor of cholera toxin-activated intestinal cell adenylate cyclase activity with half-maximal inhibition occuring at 16 μM. NaF-stimulated cyclase was less susceptible to inhibition. The data suggest that inhibition by 2′,5′-dideoxyadenosine is due at least in part to direct inhibition of the cholera toxin-activated intestinal adenylate cyclase activity.

A small group of human volunteers given live SV40 virus, and who sero-converted to SV40, did not develop any heterologous antibody response to BK virus, even when BK antibody was demonstrated in preinfection sera. SV40 infection does not appear to contribute significantly to the patterns of BK antibody seen in human populations.

A prospective study was conducted to define the content, significance, and source of lysozyme present in the pleural fluid in human diseases. The pleural fluid lysozyme activity is similar in various malignant and nonmalignant transudates and exudates, and is of limited diagnostic value. The pleural fluid activity correlated well with that of paired serum samples but it had poor correlation with the disease state, the pleural fluid granulocyte counts, and total white blood cell counts. The data suggest that the pleural fluid lysozyme may be derived primarily from the blood and that it is not the product of inflammatory or neo-plastic cells in the pleural fluid itself.

A histo-immunofluorescence technique using anti-actin antibodies has been applied to various subcellular fractions of bovine adrenal medulla and rabbit blood platelets. In the adrenal medulla only the membranes of the chromaffin granules, but not the fractions containing other subcellular particles (microsomes, mitochondria) showed marked immunofluorescence. In platelets, considerable fluorescence was present in the membranes of the 5-hydroxy-tryptamine organelles as well as in other subcellular particles. It is concluded that in the adrenal medulla, actin is specifically associated with the membranes of the amine storage organelles, whereas in platelets the protein shows a rather general subcellular distribution.