Experimental Biology and Medicine

Telomere shortening is correlated with cell senescence in vitro and cell aging in vivo. The telomere hypothesis suggests that telomere length serves as a mitotic clock for timing cellular replicative life span. Expression of telomerase stabilizes telomere length and allows for continual replication, or cell immortality. This article reviews recent evidences for the role of telomere length and telomerase in the regulation of cellular replicative life span. The therapeutic potential of manipulating telomerase expression and telomere length is also discussed.

The production of superoxide (O₂ ⁻) under hyperoxic conditions is markedly accentuated leading to the generation of potent oxidants such as hydrogen peroxide (H₂O₂), hydroxyl radical (HO⁺), and peroxynitrite (ONOO⁻). Superoxide dismutase (SOD), by rapidly removing O₂ ⁻, reduces the tissue concentration of O₂ ⁻ and prevents the production of HO⁺ and ONOO⁻. Three forms of SOD exist in the lung: CuZnSOD, MnSOD, and extracellular SOD. Considerable supportive, though not all conclusive, evidence suggests that all three forms of SOD are essential for the pulmonary defense against oxygen toxicity, and that enhancement of pulmonary SOD has the potential of protecting against oxygen toxicity.

Activin and inhibin, members of transforming growth factor-β (TGFβ) superfamily, have diverse and widespread effects within living organisms at many stages during growth and development. From the initial isolation of these growth factors based on their effects of FSH secretion, the study of these factors, as well as of the activin-binding protein follistatin, has progressed from the localization of the expression of the inhibin α subunit, activin βA and βB subunits, and activin receptors in the tissues of various organisms to the examination of activin and inhibin as autocrine and paracrine agents in cell proliferation and differentiation. The inhibitory effects on cell growth and differentiation that have been observed upon treatment of cells with activin suggest that further understanding of the bioactivity of this molecule and its characterization on a molecular level may aid in a more complete understanding of cell growth and differentiation. This minireview discusses the roles of activin, inhibin, and follistatin in the arenas of cell proliferation, differentiation, and embryogenesis, as well as the roles of these molecules in cancerous cells.

The integrins are a family of cell surface receptors that mediate cell-extracellular matrix and cell-cell interactions. The quantities and activities of these receptors are modulated during a wide variety of biological processes. A variety of agents have been found to affect expression of integrins and their function. These include cytokines, hormones, and pharmacologic agents. Mechanisms regulating integrin expression and function include regulation of protein levels by transcriptional or posttranscriptional mechanisms, alteration of protein structure by alternative splicing of mRNA, mobilization to the cell surface of preexisting intracellular stores of integrins, and modulation of receptor activity (inside-out signaling). We review studies that assess the effects of external agents on integrin levels using the cytokine TGFβ as an example. We also review studies that analyze integrin regulation with an emphasis on the control of integrin gene transcription. This review shows that the strategies for integrin modulation are quite complex. This regulatory sophistication is likely necessary, given the critical role that integrins play in the myriad social interactions of cells.

The objective of the present study was to examine the zonal changes in endometrial proliferation that occur during the late secretory phase, menses, and postmenstrual endometrial regeneration. We used as our model ovariectomized rhesus monkey in which artificial menstrual cycles were simulated. Our marker of proliferation was the immunohistochemical detection of the Ki-67 antigen. On Day 26, as progesterone (P) levels are falling in the late secretory phase, proliferation in zone IV of the basalis decreased compared with Day 23 (peak P level). Proliferation in the upper regions of the endometrium remained suppressed. Three days after a single bolus injection of the potent antiprogestin RU-486 on Day 20, proliferation in zone IV was virtually absent compared with Day 23 of an artificial cycle. No distinct changes in the pattern of proliferation were observed in the upper regions of the endometrium. On Day 1 of menses (P levels undetectable, estradiol [E] levels of 70–100 pg/ml), there was little proliferation throughout the endometrium. On Day 3 or menses, proliferation returned to zones II–III of the basalis and the functionalis. This proliferation was primarily observed in the glandular epithelia whereas little or no proliferation was observed in zone IV of the basalis. By Day 5 proliferation continued in the glandular epithelia of zones I, II, and III, and was now clearly observable in the stromal cells. Only minimal proliferation was observed in glandular epithelia of zone IV. In the absence of basal E stimulation the return of proliferation to the glandular epithelia in zones I, II, and III was dramatically reduced. These data demonstrate a reciprocal pattern of proliferation in glandular epithelia that is dependent on the prevailing hormonal stimulation. Under P dominance, proliferation is inhibited in zones I, II, and III, and maintained in zone IV, whereas under E dominance (Day 3 or 5) proliferation is driven by E stimulation in zones I, II, and III with little or no proliferation present in zone IV. In addition, the inhibition of proliferation in zone IV by the antiprogestin RU-486 and the decline of zone IV proliferation associated with falling P levels provide further evidence that proliferation of glandular epithelia in zone IV is mediated in part by P.

The possibility that pregnancy-specific β1 glycoprotein (PSG) is released by Sertoli cells was investigated by using reverse hemolytic plaque assays which enable the visualization of release from individual cells in culture. We found that the proportions of cells releasing PSG increased gradually in 3-day old cultures prepared from animals of increasing age (10-, 20-, and 40-day old animals). The gradual appearance of PSG-releasing cells during this period differed markedly from that of transferrin (TF)-releasing cells, suggesting that the age-related development of PSG-releasing cells is regulated in a specific manner. PSG cells were also found in Sertoli cell cultures prepared from stage-associated seminiferous tubule segments of adult rat testes. The percentages of PSG plaque-forming cells differed from one stage-associated culture to another with maximal proportions associated with stages III–V and XIII, and minimal proportions found in stages VII, and IX–XI. The abundance of Sertoli cells that released PSG from stage to stage differed markedly from those that released TF indicating that modulatory processes specific for PSG are also present in the adult testis. Finally, PSG cells were also identified immunocytochemically in cultures prepared from staged tubule segments. The proportion of PSG staining cells from stage to stage were found to be virtually indistinguishable from those identified with plaque assays. When taken together, these results show clearly that Sertoli cells in culture release a PSG-like molecule. Moreover, this release appears to be controlled in an age-related and stage-dependent manner, suggesting that Sertoli cells may be central to PSG function in the testis.

An inexpensive and reliable colorimetric microplate version of the Nb2 lymphoma cell proliferation bioassay for prolactin (PRL) was developed and optimized. The useful range of the assay is between 0.1 and 12.8 ng/ml in terms of rat pituitary PRL. The assay can accommodate up to 20 μl sample/well. The physiological relevance of the assay was verified by measuring thyrotropin-releasing hormone (TRH)-induced secretion of PRL in pituitary cultures and in serum samples of neonatal rats. Through the use of the colorimetric Nb2 assay, PRL-like bioactivities were demonstrated in pituitary extracts of the marsupial, Monodelphis domestica (1.47 ng PRL/μg protein) and of the amphibian, Rana pipiens (1.86 ng PRL/μg protein). Marsupial and amphibian PRLs are predicted to have low specific activities in the Nb2 assay. Since the PRL values were calculated in terms of a rat PRL standard, they probably underestimate the amounts of PRL present. Parallel dose-response curves were obtained with these pituitary extracts and standard rat PRL over a wide range of dilutions. The Nb2 bioassay may serve as a tool for the purification of PRL from these species. The colorimetric version of the Nb2 bioassay may be a useful alternative to traditional Nb2 assays that rely on direct cell count or [³H]thymidine uptake.

It has recently been found that prevention of the acidosis of anaerobic exercise blocks β-endorphin release. Because heavy exercise affects secretion of other anterior pituitary hormones, we studied the results of alkali infusion and ingestion upon blood levels of four hormones: luteinizing hormone (LH), follicle-stimulating hormone (FSH), growth hormone (GH), and prolactin (PRL). Eight male subjects were studied after either 2 mEq/kg placebo (NaCl) or alkali (NaHCO₃) administered before and during exercise to exhaustion. Blood samples were obtained before exercise and then 15, 30, 60, 90, 120, and 180 min postexercise. GH and PRL but not FSH or LH increased significantly postexercise, with a peak at 60 min, and subsequently declined back to baseline by 180 min. Base treatment reduced GH at baseline and postexercise (except at 60 min) and increased PRL significantly, particularly at 60 min. While the precise mechanisms on how acid/base changes affect hormone release remain to be defined, there are possible consequences on gonadal function and substrate availability during exercise.

The in vitro effects of prolactin (PRL) on lipoprotein lipase (LPL) activity and on LPL mRNA levels were studied in cultured mammary tissues derived from mid-pregnant mice. Mouse mammary gland tissues were initially incubated for 24 hr in M199 media containing 1 μg/ml insulin and 10⁻⁷ M cortisol. A subsequent treatment of the tissues with 1 μg/ml PRL caused a 76% increase in heparin-releasable LPL (hrLPL) activity after 24 hr. A significant increase in LPL activity was detected 16 hr after PRL addition, but not at earlier times. PRL at 100 ng/ml elicited a maximum stimulation of LPL activity. When Northern hybridization techniques were employed, PRL was also found to increase the tissue content of LPL mRNA; this effect was initially detected after a 6-hr PRL treatment employing PRL concentrations of 50 ng/ml and above. Specificity studies revealed that only lactogenic hormones stimulated LPL activity and LPL mRNA accumulation in cultured mammary tissues. PRL also expressed a small (25% increase), but significant, effect on ATP citrate-lyase activity in mammary tissues cultured for more than 6 hr with the hormone.

The cause of proteinuria in passive Heymann nephritis has been attributed to the activation of the C_5b-9 membrane attack complex following the antibody binding on the glomerular epithelial cell. Previous studies have shown an association between release of prostaglandin thromboxane B₂ (TxB₂) and proteinuria. Whether this release is dependent on antibody binding per se, or on secondary actions subsequent to antibody binding has not been clarified. The present study was designed to address this issue. Antibody binding event was experimentally separated from the proteinuria by employing a rabbit antibody which produces equivalent glomerular binding equal to that produced by a sheep antibody but without causing proteinuria. Comparisons were made with animals injected with the sheep antibody which produces all the hallmarks of the disease, including proteinuria. Animals injected with the rabbit antibody showed glomerular immunofluorescent deposits which were identical to the deposits produced by the control sheep antibody. However, rabbit antibody failed to produce the typical electron-dense subepithelial deposits, complement binding and proteinuria. Comparison of prostaglandin profile in isolated glomeruli revealed that TxB₂ was unchanged in rabbit antibody–injected glomeruli (compared with its nonimmune antibody control). On the other hand, glomeruli from sheep antibody–injected animals released 45% higher TxB₂ compared with their respective nonimmune antibody control. These data suggest that the binding of antibody per se may not be a sufficient stimulus for TxB₂ release. Subsequent events of subepithelial electron dense deposit formation, complement activation, and proteinuria are associated with TxB₂ release.

C57 BL mice were injected daily with either saline or varied doses of cocaine (5–50 mg/kg), and thymocyte subpopulations were analyzed 4 hr after the fifth injection. Mice injected with either 25 or 50 mg/kg of cocaine showed a decrease in the percentage of CD4⁺8⁺ cells and increase of CD4⁻8⁻, CD4⁺, and CD8⁺ cells. The absolute numbers of each subpopulation, calculated by multiplying the percentage of each subpopulation with the total cell number, revealed an extensive decline in CD4⁺8⁺, a decrease in CD8⁺, an increase in CD4⁻8⁻, and no change in the CD4⁺ subpopulation. Flow cytometric analysis of thymocytes and electrophoresis of the thymocyte DNA revealed a dosage-dependent increase in cells undergoing programed cell death with apoptosis. Culturing of thymocytes from control or drug-treated mice demonstrated an inverse relationship between cell viability and cocaine concentrations, suggesting that in vivo cocaine, or its biological products, may damage thymocytes. Incubation of normal cells with cocaine showed a dose-dependent decrease of viability with identical patterns of the alteration of cell subpopulations observed in vivo. A dose-dependent increase of apoptosis was also observed. In summary, we demonstrate a selective in vivo cocaine-induced alteration of the thymocyte subpopulations and identified programed cell death with apoptosis as the likely mechanism mediating this thymic atrophy. The comparable findings observed in vivo and in vitro support the concept that cocaine may directly affect some features of thymocyte biology, and suggest the usefulness of the in vitro system in studying cocaine effects on thymocyte biology.

Transforming growth factor-β1 (TGFβ1) has recently been identified as a promoter of glomerular scarring in glomerulonephritis. The present studies employed polymerase chain reaction (PCR)-based methods to quantify changes in transcriptional activation of TGFβ1 in experimental crescentic glomerulonephritis. The disease was induced in the rat, and total RNA was isolated from glomeruli at stages in which proliferation, crescent formation, and scarring were prominent features. Total glomerular RNA was reverse transcribed (RT) to cDNA, and RT products were subsequently analyzed by comparative and competitive PCR. Enhanced TGFβ1 expression was apparent at the onset of proliferative glomerulonephritis and prominent during the phase of crescent formation. The observations indicate that PCR-based methods allow quantitation of changes in glomerular TGFβ1 expression in the course of glomerulonephritis.