Experimental Biology and Medicine

The cuproenzymes lysyl oxidase, cytochrome-c oxidase, and superoxide dismutase are key factors in understanding the cardiac hypertrophy and cardiomyopathy associated with dietary copper restriction. The role of copper in cardiac lipid and energy metabolism as a consequence of changes in some of these enzyme activities in comparison with what is known about normal cardiac substrate utilization is discussed here. While the decrease in the nuclear encoded subunits of cytochrome-c oxidase in hearts from copper-deficient rats is known, new evidence suggests that other factors, such as ATP synthase metabolism may be exerting an influence upon this observation. While this review focuses on newer knowledge about energy and fatty acid metabolism in copper deficiency, the extracellular matrix is considered as well. This complex interplay of extracellular and cellular events in copper restriction is outlined as a model for further studies of this unique model of concentric hypertrophy.

Work on the glucose-6-phosphatase system has intensified and diversified extensively in the past 3 years. The gene for the catalytic unit of the liver enzyme has been cloned from three species, and regulation at the level of gene expression is being studied in several laboratories worldwide. More than 20 sites of mutation in the catalytic unit protein have been demonstrated to underlie glycogenesis type 1a. Inhibition of glucose-6-P hydrolysis by several newly identified competitive and time-dependent, irreversible inhibitors has been demonstrated and in several instances the predicted effects on liver glycogen formation and/or breakdown and on blood glucose production have been shown. Refinements in and additions to the presently dominant “substrate transport-catalytic unit” topological model for the glucose-6-phosphatase system have been made. A new model alternative to this, based on the “combined conformational flexibility-substrate transport” concept, has emerged. Experimental evidence for the phosphorylation of glucose in liver by high-K _m, glucose enzyme(s) in addition to glucokinase has continued to emerge, and new in vitro evidence supportive of biosynthetic functions of the glucose-6-phosphatase system in this role has appeared. High levels of multifunctional glucose-6-phosphatase have been shown present in pancreatic islet β cells. Glucose-6-P has been established as the likely insulin secretagog in β cells. Interesting differences in the temporal responses of glucose-6-phosphatase in kidney and liver have been demonstrated. An initial attempt is made here to meld the hepatic and pancreatic islet β-cell glucose-6-phosphatase systems, and to a lesser extent the kidney tubular and small intestinal mucosal glucose-6-phosphatase systems into an integrated, coordinated mechanism involved in whole-body glucose homeostasis in health and disease.

In the present review, the structure of β-fetoprotein (AFP) is discussed in consideration of AFP membership and position in the albuminoid supergene family in relation to other gene family members. Ontogenetic AFP gene expression is then discussed in view of AFP mRNA presence in various tissues at different times during development. The multiple molecular forms of AFP is also presented in relation to published reports of AFP binding proteins and cell surface receptors. The review proceeds on to present AFP as a potential model of a modular/cassette protein based on sequence comparison with cleaved fragments of prohormones and biological response modifiers. Such cleaved fragments could potentially serve as peptide messengers for vascular, neuroendocrine, and digestive biological activities. Following a discussion on fibrin binding and serine proteases, AFP-cytoskeletal, extracellular matrix, and cellular adhesion interactions are considered. AFP as a carrier/transport protein based on structural relationships is further elucidated by examination of the various ligands bound to AFP and its hormone interaction. Since AFP binds heavy metals, the question is posed of whether AFP could function as an antioxidant. An analysis of transcription factors, tumor suppressors, and homeodomain proteins follows, which is interfaced with the concept of programmed cell death in light of amino acid sequence matches detected on the AFP molecule. Emphasis was naturally placed upon the homeodomain protein sequence stretches since AFP is a fetal, phase-specific protein found throughout embryogenesis, histogenesis, and organogenesis. In keeping with histogenesis, a discussion of AFP and eye lens protein development is presented. Finally, AFP sequence analysis presented in light of members of the immunoglobulin superfamily, autoimmune disorders, and various disease states culminates the review. A closing discussion then summarizes regions of presumptive matched protein identities on each of AFP's three domains.

For centuries, preservation of the human body has been practiced for ceremonial, religious, emotional, prurient, or medical purposes (1, 2). Medical education has depended in large part on the preservative qualities of formaldehyde for cadavers since the turn of the 20th century (3, 4). Unfortunately, formaldehyde has been designated as, and is, a mitogen. Preservation of cadavers is a requirement for teaching gross anatomy to medical students or students in allied medical sciences and requires disease-free specimens containing little to no mitogenic agents. To achieve this end, cadavers must be tested for the presence of human immunodeficiency virus (HIV) and prepared with the minimal use of formaldehyde. The following discourse relates a method of achieving superior preservation of disease-free cadavers and their central nervous systems.

We have recently reported that prolactin (PRL) stimulates iodide uptake into cultured mouse mammary tissues. This effect occurs in both TCA soluble and insoluble tissue fractions. The effect of PRL apparently involves an RNA-DNA-dependent mechanism, since actinomycin D and cyclohexamide abolish the PRL stimulation of iodide uptake and its incorporation into protein. Perchlorate and thiocyanate, inhibitors of the iodide transporter, also abolish the PRL effects on iodide uptake and incorporation. Similarly, propylthiouracil and aminotriazole, inhibitors of peroxidase, abolish both effects of PRL. Finally, the extent of iodide uptake in mammary cells is suppressed by about 50% in sodium-free medium. These studies thus suggest the existence of a sodium-iodide symporter in the mammary gland which has characteristics similar to the iodide transporter in the thyroid gland–-that is, it is sodium dependent and is inhibited by perchlorate and thiocyanate. The fact that both iodide transporter inhibitors and peroxidase inhibitors abolish PRL-stimulated iodide uptake and incorporation suggests that there may be a coupled mechanism involving the iodide transporter and the peroxidase enzyme.

Effects of dietary protein level with and without L-arginine (Arg) infusion on plasma tumor necrosis factor-β (TNF-β) response to endotoxin (lipopolysaccharide [LPS]) as well as plasma concentration and urine output of nitrite and nitrate (NO_x), the stable end products of nitric oxide radical (NO), were studied in beef heifers (275-310 kg body wt). The animals were fed low- (LP; 7.96%) or high- (HP; 13.94%) protein diets for 10 days before LPS administration (Escherichia coli; 0.2 μg/kg, iv). L-Arginine in saline (0.5 g/kg body wt) or saline was infused for 8 hr with one-third of total Arg infused before LPS administration. Plasma TNF-β concentrations increased in all heifers after LPS injection (peak at 1 hr and return to baseline at 4 hr); however, concentrations were lower in HP- than in LP-fed heifers at 1, 2, and 3 hr. Infusion of Arg did not affect plasma TNF-β response to LPS. Plasma NO_x concentrations increased in all heifers after LPS challenge; compared with saline, Arg infusion increased the total response (integrated area under concentration curve) in LP- but not in HP-fed heifers. Relative to pretreatment period, the rate of NO_x output in urine collected 2-6 hr after LPS administration increased in all heifers regardless of dietary protein level and was further amplified by Arg infusion. The rate of NO_x output in urine collected 6-24 hr after LPS challenge was even higher in LP-fed heifers infused with Arg but returned to the basal values in other groups. Activity of hepatic inducible NO synthase was not affected by LPS, Arg, or dietary protein level at the time points studied. The data suggest that dietary protein levels can modulate both TNF-β and NO responses to LPS in cattle; high dietary protein intake decreases TNF-β response and attenuates the conversion of supplemental Arg to NO.

Male Holtzman rats were offered a semipurified low-copper (Cu) diet (0.36 mg Cu/kg) for 5-6 weeks to further characterize cardiac hypertrophy, which accompanies Cu deficiency. Cu-adequate (controls) were given supplemental Cu (20 μg/ml) in their drinking water, and Cu-deficient rats were given deionized water. Cu-deficient rats had lower plasma ceruloplasmin activity, lower hemoglobin levels, higher heart weights, and similar body weights compared with Cu-adequate rats. The relative degree of hypertrophy in the right ventricle of Cu-deficient rats was significantly higher (2.3-fold) than that in the left ventricle and atria (both were 1.9-fold higher than the values in Cu-adequate rats). Edema was not detected. Ventricles and atria of Cudeficient rats had markedly lower Cu and no significant differences in iron concentrations compared with Cu-adequate rats. Heart protein concentrations were not altered consistently by Cu deficiency. Enzyme activities of the cuproenzymes cytochrome-c oxidase (CCO), copper, zinc-superoxide dismutase (SOD), dopamine β-monooxygenase (DBM), peptidylglycine β-amidating monooxygenase (PAM), and the selenoenzyme glutathione peroxidase (GPX) were measured in the atria and ventricles. Cu deficiency resulted in lower specific activities of all cuproenzymes, with the exception of ventricular PAM. GPX was not altered by chamber region or diet. Specific activity of PAM was 200-fold higher in atria than in ventricles in control rats. Catecholamine analyses by HPLC confirmed that, like ventricular tissue, atria of Cudeficient rats had lower norepinephrine and higher dopamine concentrations, consistent with lower DBM activity. Another experiment detected no differences between the two dietary groups in mean arterial blood pressure, heart rates, or responses after challenge with angiotensin II, phenylepherine, or acetylcholine in cannulated rats. In this Cu-deficient rat model, all chambers of the heart exhibit similar and marked hypertrophy. Biochemical alterations following dietary Cu deficiency were also similar in atria and ventricles. The hypertrophic response appears different from the response to simple pressure or volume overload.

The presence of a melatonin-binding component in the membrane fraction of the ovarian granulosa cells of hens that has characteristics of a receptor such as high affinity, limited capacity, reversible binding, and binding specificity was demonstrated by radioligand binding assays. When the cells were incubated in vitro with various concentrations of luteinizing hormone (LH) in the presence and absence of melatonin (1 and 5 nM), the dose-response curve for the LH-stimulated progesterone production was shifted to a higher concentration of LH: ED₅₀ of LH was approximately two times greater in the presence of melatonin. The results suggest a direct action of melatonin on the ovarian granulosa cells of the hen to lower the responsiveness to LH for progesterone production.

Prolactin treatment of NMuMG mammary epithelial cells inhibits the ability of epidermal growth factor (EGF) to transduce a variety of signals, possibly by interfering with receptor tyrosine phosphorylation. However, the mechanism by which prolactin inhibits EGF receptor signaling is unclear. The objective of this study was to evaluate the effects of prolactin on the dynamics of EGF receptor degradation and resynthesis, and on the association of the receptor with the cytoskeleton. EGF decreased the EGF receptor content of NMuMG cells, and this decrease was unaffected by prolactin treatment. Subsequent to the decrease in EGF receptors, cells re-accumulated EGF receptors, and this re-accumulation was also unaffected by prolactin. In other studies, EGF induced a rapid association of EGF receptor with Triton X-100-insoluble (cytoskeletal) elements. The cytoskeletally associated receptors were more heavily tyrosine phosphorylated than soluble receptors in the absence of prolactin. In the presence of prolactin, similar amounts of EGF receptor associated with the cytoskeleton, but both cytoskeletal and soluble receptors exhibited decreased tyrosine phosphorylation. These studies indicate that the effects of prolactin on EGF receptor signaling are not likely to be due to altered receptor dynamics or cytoskeletal association but are more likely due to an alteration in receptor kinase activity.

Several lines of evidence suggest that 1,25-dihydroxyvitamin D₃ (1,25D₃) may be important in chemoprevention of human cancer. Here, we show that human promyelocytic leukemia cells HL60 cultured in the presence of 30 nM 1,25D₃ (30A cells) for 3 years exhibited a reduced rate of tumor growth when injected into nu/nu mice, while cells grown in 40 nM 1,25D₃ (40AF cells) failed to form detectable tumors in 11 out of the 12 inoculated mice. Interestingly, both 30A and 40AF cells grew approximately twice as fast as the parental HL60-G cells under tissue culture conditions, even in the presence of 1,25D₃, to which they developed resistance. Tests of the susceptibility of these cells to natural killer (NK) cell cytotoxicity showed that 40AF, but not HL60-G or 30A cells, were targets for the murine spleen NK cells. However, lysis of 30A cells was also detected when human NK cells were used in this assay, though the effector-to-target cell ratio necessary to obtain significant lysis above background levels was higher for 30A (80:1) than for 40AF (10:1) cells. These results suggest a mechanism for the reported chemopreventive effects of sunlight-generated 1,25D₃ or dietary vitamin D₃.

We assessed changes in transcriptional activation of the inducible isoform of nitric oxide synthase (iNOS) in a model of macrophage-dependent proliferative glomerulonephritis in the rat resembling human forms of rapidly progressive nephritis. By the use of a cDNA probe derived from rat glomerular RNA and an RNase protection assay, iNOS expression was assessed at early and late stages of the disease and was correlated with the extent of macrophage infiltration. Prominent iNOS expression occurred in isolated glomeruli 24 hr after onset of immune injury when marked glomerular infiltration by macrophages also occurred. Treatment of animals with immune injury with the arachidonic acid cyclooxygenase inhibitor, indomethacin, potentiated iNOS expression. iNOS expression was short-lived; it was markedly reduced on Day 2 of injury and undetectable on Days 4 and 10, despite sustained infiltration of glomeruli by macrophages. These observations suggest that in glomerular immune injury the enhanced expression of iNOS is not sustainable possibly due to downregulatory factors generated in the course of injury.

Estrogens stimulate proliferation and differentiation of uterine epithelial cells in vivo. Mitogenic action of estrogens may be mediated by growth factors such as insulin-like growth factor-1 (IGF-I). This study was designed to determine whether IGF-I and insulin affect proliferation of uterine epithelial cells obtained from 3- to 4-week-old immature female mice in a serum-free culture system. The epithelial cell number on Day 5 in culture was significantly increased by adding IGF-I (10 and 100 ng/ml) or insulin (100 and 1000 ng/ml) to the culture media, indicating that IGF-I is more effective than insulin in inducing the epithelial growth. The epithelial DNA synthesis was significantly stimulated by IGF-I (1 and 10 ng/ml), suggesting that both the epithelial proliferation and their detachment from substratum are stimulated by 1 ng/ml of IGF-I, but that the former is more accelerated than the latter by 10 ng/ml of IGF-I. These results demonstrate that both IGF-I and insulin directly stimulate the growth of uterine epithelial cells, and suggest that insulin may act via IGF-I receptors. IGF-I immunoreactivity was detected in the cytoplasm of the cultured cells, indicating that the cells synthesize IGF-I. Estradiol-17β (E₂) at lower concentrations (0.001-0.1 nM) tended to increase the number of epithelial cells, while E₂ at higher concentrations (1 to 100 nM) did not affect it. It is highly probable that IGF-I produced in endometrial cells induces their proliferation by an autocrine or paracrine mechanism.