Experimental Biology and Medicine

Holding a Ph.D. or other doctoral degree in the biological sciences used to allow for only one main career path. Faculty positions at either research-intensive universities or at predominantly teaching institutions defined the career path very well. The situation is considerably more complex now. Many individuals decide to pursue careers in for-profit organizations, either by personal preference, geographic needs, or simply as an expedient way to continue in biology in a meaningful way. This article arises from the need to define a better understanding of the nature of corporate job seeking for potential job applicants. The author draws upon his experience over a thirty-year scientific career in industry and academics. The distinctions he makes are helpful for job-seekers in this new environment.

Responses to steroids initiated from non-nuclear receptors Impinge on a wide variety of cellular responses and utilize nearly all known signal transduction webs. While the mechanisms by which steroid receptors localize in the membrane are still unclear, it is apparent that this alternative localization allows steroid receptors to participate in a wide range of complex functions influencing cell proliferation, death, and differentiation. The central debate still remains the identity of the protein class or classes that mediate membrane-initiated (nongenomic) responses. The data thus far have supported several possibilities, including: nuclear steroid receptor-like forms in non-nuclear locations; other known (nonsteroid) membrane receptors or channels with additional steroid-binding sites; enzymes; transporters; receptors for serum steroid-binding proteins; unique and previously undescribed proteins; or chimeras of typical steroid receptor domains with other unique or known protein domains. Categorizing membrane steroid receptor proteins based exclusively on the actions of antagonists and agonists, without considering cell context and protein partnering Issues, may mislead us into predicting more receptor subtypes than really exist. However, the plethora of signaling and functional outcomes may indicate the participation of more than one kind of steroid-binding protein. Resolving such unanswered questions will require future investigative focus on this alternative arm of steroid action, which is likely to yield as many therapeutic opportunities as have nuclear steroid mechanisms.

GATA transcription factors are emerging as critical players in mammalian reproductive development and function. GATA-4 contributes to fetal male gonadal development by regulating genes mediating Müllerian duct regression and the onset of testosterone production. GATA-2 expression appears to be sexually dimorphic being transiently expressed in the germ cell lineage of the fetal ovary but not the fetal testis. In the reproductive system, GATA-1 is exclusively expressed in Sertoli cells at specific seminiferous tubule stages. In addition, GATA-4 and GATA-6 are localized primary to ovarian and testicular somatic cells. The majority of cell transfection studies demonstrate that GATA-1 and GATA-4 can stimulate inhibin subunit gene promoter constructs. Other studies provide strong evidence that GATA-4 and GATA-6 can activate genes mediating gonadal cell steroidogenesis. GATA-2 and GATA-3 are found in pituitary and placental cells and can regulate α-glycoprotein subunit gene expression. Gonadal expression and activation of GATAs appear to be regulated in part by gonadotropin signaling via the cyclic AMP-protein kinase A pathway. This review will cover the current knowledge regarding GATA expression and function at all levels of the reproductive axis.

In previous minireviews in this journal, we discussed work on induction of tetrahydrobiopterin biosynthesis by cytokines and its significance for nitric oxide (NO) production of intact cells as well as functions of H₄-blopterin identified at this time for NO synthases (Proc Soc Exp Biol Med 203: 1–12, 1993; Proc Soc Exp Biol Med 219:171–182, 1998). Meanwhile, the recognition of the importance of tetrahydrobiopterin for NO formation has led to new insights into complex biological processes and revealed possible novel pharmacological strategies to Intervene in certain pathological conditions. Recent work could also establish that tetrahydrobiopterin, in addition to its allosteric effects, is redox-active in the NO synthase reaction. In this review, we summarize the current view of how tetrahydrobiopterin functions in the generation of NO and focus on pharmacological aspects of tetrahydrobiopterin availability with emphasis on endothelial function.

Dietary dehydroepiandrosterone (DHEA) inhibits the proliferation of syngeneic bone marrow cells (BMC) infused into lethally irradiated mice. Potential mechanisms for suppression of hematopolesis were evaluated and the findings were as follows: (i) depletion of NK, T, B or macrophage cells failed to reverse suppression by DHEA; (ii) stem cell stimulation by erythropoietin, growth hormone, interieukin-2, Friend leukemia virus, or cyclophosphamide failed to reverse suppression; (iii) supplementation of fatty acids, mevalonate, or deoxyribonucleotides, which are dependent upon glucose-6-phosphate dehydrogenase function, did not enhance BMC growth in mice fed DHEA; (iv) DHEA downstream metabolites 4-androstenedione and 17β-estradiol, as well as the synthetic steroid, 16α-chioroeplandrosterone (but not testosterone or 5-androstene-3β,17β-diol), also inhibited BMC growth. Tamoxifen antagonized the effects of 17β-estradiol but not DHEA; (v) dietary DHEA causes hypothermia, but housing of DHEA-fed mice at 34°C to maintain normal body temperature did not reverse suppression; (vi) DHEA leads to a decrease in food intake in rodents. Pair-feeding control diet to mice fed DHEA mimicked the effects of dietary DHEA; (vii) adrenalectomy and orchiectomy decrease the levels of stress and sex hormones, respectively. Neither procedure affected the ability of food restriction or DHEA feeding to inhibit hematopolesis; (viii) growth of GR-3 NM pre-B leukemia cells in unirradiated mice was also suppressed by DHEA or food restriction. We conclude that DHEA, by reducing food intake in mice, inhibits bone marrow and leukemia cell growth. The precise mechanism(s) by which reduced food intake per se inhibits hematopolesis is not known, but may involve an increased rate of cellular apoptosis.

Metallothionein (MT) is a low-molecular-weight, cysteine-rich protein that contains heavy metals such as cadmium and zinc. The biological function of MT in platelets is not yet understood. Therefore, the aim of this study was to systematically examine the inhibitory mechanisms of metallothionein in platelet aggregation. In this study, metallothionein concentration-dependently (1-8 μM) inhibited platelet aggregation in human platelets stimulated by agonists. Metallothionein (4 and 8 μM) inhibited phosphoinositide breakdown in [³H]-inositol-labeled platelets, intracellular Ca⁺² mobilization in Fura-2 AM-loaded platelets, and thromboxane A₂ formation stimulated by collagen. In addition, metallothionein (4 and 8 μM) significantly Increased the formation of cyclic GMP but not cyclic AMP in human platelets. Rapid phosphorylation of a protein of Mr 47,000 (P47), a marker of protein kinase C activation, was triggered by PDBu (100 nM). This phosphorylation was markedly inhibited by metallothionein (4 and 8 μM) in phosphorus-32-labeled platelets. In an in vivo thrombotic study, platelet thrombus formation was induced by irradiation of mesenteric venules in mice pretreated with fluorescein sodium. Metallothionein (6 μg/g) significantly prolonged the latency period for inducing platelet plug formation in mesenteric venules. These results indicate that the antiplatelet activity of metallothionein may involve the following pathways: (1) metallothionein may inhibit the activation of phospholipase C, followed by inhibition of phosphoinositide breakdown and thromboxane A₂ formation, thereby leading to inhibition of intracellular Ca⁺² mobilization; (ii) Metallothionein also activated the formation of cyclic GMP in human platelets, resulting in inhibition of platelet aggregation. The results strongly indicate that metallothionein provides protection against thromboembolism.

Extracts or supernatants from cultures of Lactobacilli are used for their medicinal effects, including wound healing and immune system stimulating activity. We have studied the in vivo and in vitro effects of supernatants from bacterial cultures of two strains of Lactobacillus (LS) on tissue repair and angiogenesis. Subcutaneous injection of LS into rodent ears led to proliferation of blood vessels that also exhibited strong immunostaining for Flk-1 receptor. Some inflammatory cells were scattered among the blood vessels. The continuous influx of polymorphonuclear leukocytes (PMNs) and macrophages into transcutaneous wounds in mice treated with LS resulted in prolonged inflammatory phase of wound healing and delayed wound closure, including reepithellalization. Subcutaneous injection of Matrigel Impregnated with LS into the abdominal wall led to rapid and transient influx of PMNs in the vicinity of the gel. LS stimulated the proliferation of murine macrophage J774.A1 cell line and porcine lymphocytes but not that of murine fibroblast AKR-2B cells. LS also induced production of TNF-α by J774.A1 cells and by porcine kidney epithelial LLC-PK1 cells. LS did not appear to have an effect on collagen production. In conclusion, our study demonstrates the potential of LS to function as a stimulator of the inflammatory stage of tissue repair, TNF-α production, and of angiogenesis.

We previously reported that treatment of streptozotocin-Induced diabetic rats with zinc plus cyclo (his-pro) (CHP) decreased fed blood glucose levels and water intake. The present study was conducted to examine the dose-dependent, acute, and chronic treatment effects of CHP on oral glucose tolerance (OGT), fed blood glucose levels, water intake, and plasma Insulin levels in young and aged Sprague-Dawley (S-D) rats, nondiabetic Wistar rats, and genetically diabetic Goto-Kakizaki (G-K) rats. Acute gastric gavage of 10 mg zinc plus 1.0 mg CHP/kg body weight significantly improved OGT in 4- and 13-month-old nondiabetic S-D rats and in 2-month-old diabetic G-K rats. Young S-D and G-K rats returned to pretreatment OGT values 1 week after acute gavage of zinc plus CHP (ZC), but improved OGT values persisted for at least 1 week after gavage in aged S-D rats. OGT values and fed blood glucose decreased to the greatest extent among other treatments when G-K rats were given free access to drinking water containing 1.0 to 1.5 mg CHP/L plus 10 mg zinc/L for 2 weeks. Although food and water intake showed a tendency to decrease, no statistically significant differences were observed in young G-K rats. Plasma insulin levels and blood glucose levels in both normal and diabetic G-K rats decreased with 2-week treatment with ZC. To test the direct effects of ZC on muscle tissue, we observed the effect of various doses of ZC on normal and G-K rat muscle slices. The optimal level of CHP alone for maximal muscle glucose uptake in muscle slices from normal rats was 10 μg/mL and 5.0 μg/mL in G-K rats, and ZC stimulated glucose uptake. However, no statistically significant difference was demonstrated between normal and G-K rat tissues in this study. These results indicate that oral intake of an optimal dose of ZC stimulates blood glucose metabolism, probably by stimulating muscle glucose utilization.

Apoptotic death of CD4+ T lymphocytes is a major cause of the immunodeficiency caused by human immunodeficiency virus (HIV), but it is still unclear how this process precisely occurs. To characterize a potentially useful cellular model, we have analyzed the tendency of chronically HIV-infected CD4+ human cell lines of different origin to undergo apoptosis. We studied ACH-2 and U1 lines, derived from the CD4+ T-cell A301 and the promonocytic U937 cell lines, respectively, and induced apoptosis via several stimuli that trigger different pathways. Their capacity to regulate plasma membrane CD95 expression and to produce soluble CD95 was also analyzed. Using staurosporine, TNF-α plus cycloheximide, and 7-radiations, we observed that ACH-2 were more sensitive to programmed cell death than A301, while U1 were less sensitive than U937. Both infected cell types had a lower sensitivity to CD95-induced apoptosis; the analysis of changes in mitochondrial membrane potential corroborated these observations. Plasma membrane CD95 was similarly regulated in all cell types, which, however, presented a different capacity to produce soluble CD95 molecules.

Our in vitro results may offer a new perspective for developing further studies on the pathogenesis of HIV infection. A chronically infected cell line of lymphocytic origin is more susceptible to apoptosis than its parental cell type, while infected monocytic cells are less sensitive than their uninfected counterpart. Thus, it is possible to hypothesize that one of the reasons by which circulating monocytes survive and represent a viral reservoir is the capacity of HIV to decrease the sensitivity to apoptosis of this cell type. However, further studies on ex-vivo collected fresh cells, as well as on other cell lines, are urgently needed to confirm such hypothesis.

Animal models of Human African Trypanosomiasis (HAT) have been developed to understand the pathogenic mechanisms leading to the passage into the neurological phase, most of them referring to histological aspects but not clinical or behavioral data. Our study aimed at defining simple clinical and/or behavioral markers of the passage between the hemolymphatic phase and the meningo-encephalitic stage of the disease. Sprague-Dawley rats (n = 24) were infected with Trypanosoma brucel brucai AnTat 1.1E. Food intake and body weight were measured daily from the day of infection until death. Hematocrit was measured twice a week. Behavioral disturbances were evaluated through an Open-field test. A sudden weight loss occurred on the twelfth day after Infection, due to a significant drop of food intake starting two days before. The rats developed an anemic state shown by the hematocrit measurements. The Open-field test showed them to be less active and reactive as soon as the second week after infestation. A complementary histological study observed trypanosomes and inflammatory cells in the choroid plexus at the same period. These results are in favor of central nervous system functional disturbances. The observed weight loss is discussed as being a parameter of the entry in the meningo-encephalitic phase. The rat model reproduces neurological symptoms observed in the human disease and may prove to be useful for further neurohistological and therapeutic studies.