Expression of transforming growth factor-beta 1 messenger ribonucleic acid and the modulation of deoxyribonucleic acid synthesis by transforming growth factor-beta 1 in human endometrial cells

OBJECTIVE: The purpose of this study was (1) to evaluate the potential sites of transforming growth factor-beta 1 synthesis in human endometrium by analyzing separated endometrial glands and stromal cells for transforming growth factor-beta 1 messenger ribonucleic acid by Northern analysis of total ribonucleic acid and (2) to investigate the effects of transforming growth factor-beta 1 on deoxyribonucleic acid synthesis in endometrial epithelial and stromal cells in culture. STUDY DESIGN: Endometrial glands and stroma from proliferative and secretory endometrium were isolated after collagenase treatment of endometrial tissue minces and were analyzed for transforming growth factor-beta 1 messenger ribonucleic acid by Northern analysis. We studied the effects of estradiol-17 beta and transforming growth factor-beta 1 on deoxyribonucleic acid synthesis in endometrial epithelium and transforming growth factor-beta 1 on stromal cells in culture by evaluating tritiated thymidine incorporation into trichloroacetic acid-precipitable material. RESULTS: Transforming growth factor-beta 1 messenger ribonucleic acid was detected for Northern analysis in separated endometrial stromal cells in levels that were greatest during the secretory phase and in greater levels than in epithelial cells from that same tissue. Transforming growth factor-beta 1 messenger ribonucleic acid in glandular epithelium in culture was not increased to detectable levels by treatment with transforming growth factor-beta 1. Deoxyribonucleic acid synthesis in endometrial glandular epithelium was inhibited by transforming growth factor-beta 1, but transforming growth factor-beta 1 stimulated deoxyribonucleic acid synthesis in endometrial stromal cells in culture. After treatment for 5 days with estradiol-17 beta (10(-8) mol/L), deoxyribonucleic acid synthesis in endometrial glands in culture was decreased by 40%. Transforming growth factor-beta 1 (1 ng/ml) did not alter this effect of estradiol-17 beta on deoxyribonucleic acid synthesis. CONCLUSIONS: Transforming growth factor-beta 1 acts to decrease deoxyribonucleic acid synthesis in epithelial cells and to increase it in stromal cells isolated from human endometrium and maintained in monolayer culture. Transforming growth factor-beta 1, potentially of stromal cell origin, could participate in the regulation of endometrial cell proliferation and differentiation in vivo.     

Treatment of articular cartilage defects of the knee with autologous chondrocyte implantation

alpha 1-Adrenergic receptor-mediated responses are overwhelming in adult rat hepatocytes. Inversely, beta-responses are predominant over alpha 1-responses in the hepatocytes that have been cultured at a low cell density (10(4) cells/cm2) for 24 h. The insulin-EGF-induced DNA synthesis in the beta-response-dominant hepatocytes was doubled by beta-agonists or cAMP-generating agents added far behind (16-20 h) the addition of insulin/EGF; i.e., immediately before the entry into the S-phase of the cell cycle. Agonists of alpha 1-adrenergic or other Ca2+, mobilising receptors added to the alpha 1-response-dominant hepatocytes increased DNA synthesis only if they were added within 1-2 h after the addition of insulin/EGF, at the early stage of G1-phase. Agonists of "non-dominant" receptors were rather antagonistic to agonists of "dominant" receptors. Thus, agonists of alpha 1-adrenergic (and other Ca2+ mobilising) receptors and agonists of beta-adrenergic (and other cAMP-generating) receptors acted as comitogens in their own particular manners in the presence of growth factors in hepatocytes in which the respective receptor functions were dominant.     

A simple electrode for metallic replication

The effect of ritodrine upon uterine artery blood flow (UtBF) and umbilical vein blood flow (UmBF) was investigated in near-term chronic sheep preparations. During intravenous ritodrine infusions to the ewe in sequential dose rates from 100 to 800 mug per minute, UtBF fell progressively to 43 per cent below control levels and mean maternal arterial pressure (MMAP) declined 20 per cent. During constant infusions of 100, 400, or 800 mug per minute of ritodrine to the ewe for 120 minutes,, UtBF decreased 10, 37, and 31 per cent, respectively, and MMAP decreased 6, 20 and 25 per cent respectively. Dose-related maternal tachycardia and hyperglycemia occurred. There were no significant changes in UmBF, mean fetal arterial pressure, or fetal heart rate. During all infusions, maternal and fetal arterial pH, PCO2, and PO2 remained within normal physiologic limits. Simultaneous infusion of ritodrine (400 mug per minute) and propranolol (100 mug per minute) blocked the maternal tachycardia, but decreases in UtBF, MMAP, and UmBF were observed. Ritodrine infusions to the fetus (25 mug per minute) resulted in fetal tachycardia and a variable increase in UmBF.     

Liver regeneration and lymphocyte activation

A 70 per cent partial hepatectomy in the rat results in a massive deoxyribonucleic acid synthesis not only in the remaining liver cells but also in the lymphoid organs. Serum factor present in the rat after hepatectomy stimulated both hepatocytes and lymphocytes in culture. Lethally irradiated rats, followed by a partial hepatectomy, could be restored their regeneration ability only when the lymphocytes taken from rats after hepatectomy were injected. A hypothesis is that the replications of the regenerating liver cells and the antigen stimulated lymphocytes are triggered by a common pathway, probably through adenosine 3':5'-cyclic phosphate activation.     

Effects of insulin-like growth factor I and II on DNA synthesis and proliferation in primary cultures of adult rat hepatocytes

We compared the effects of insulin-like growth factor I (IGF-I) and II (IGF-II) on DNA synthesis and proliferation and investigated various signal transduction mechanisms involved in insulin-like growth factor-induced mitogenesis in primary cultures of adult rat hepatocytes. IGF-I stimulated hepatocyte DNA synthesis and proliferation with an EC50 of 75 ng/ml within 4 h of culture. These effects were sensitive to the IGF-I concentration and cell density. Hepatocyte proliferation induced by IGF-I was potentiated by metaproterenol (10(-6) M) as well as by 8-bromo-cAMP, phorbol 12-myristate 13-acetate (PMA; 10(-8) M) and was inhibited by U-73122 (1-(-[[17beta-3-methoxyestra-1,3,5(10)-triene-17-yl]amino]hexyl]-+ ++1Hpyrrol-2,5-dione)), genistein, wortmannin, PD98059 (2'-amino-3'-methoxyflavone) and rapamycin. The IGF-I effect was independent of pertussis toxin (100 ng/ml). IGF-II also dose dependently stimulated hepatocyte DNA synthesis and proliferation with an EC50 of 0.75 ng/ml within 4 h of culture. However, these effects were not dependent on the initial plating density. The stimulatory effects of IGF-II were potentiated by UK-14304 (5-bromo-6-[2-imidazolin-2-ylamino]-quinoxaline) (10(-5) M) and inhibited by phenylephrine, PMA, metaproterenol, 8-bromo-cAMP, PD98059, rapamycin, and pertussis toxin. The IGF-II effects were not affected by genistein, U-73122, and wortmannin. These results suggest that IGF-I and IGF-II rapidly stimulate the DNA synthesis and proliferation of adult rat hepatocytes by separate mechanisms.     

The effect of donor and recipient age on engraftment of tissue-engineered liver

A novel treatment for end-stage liver disease using heterotopic hepatocyte transplantation on biodegradable polymers has been investigated. Survival and repopulation of adequate cell mass to replace hepatic function has been the principal difficulty of this method. Hence the authors have begun to investigate the role of donor and recipient age on the efficiency of hepatocyte transplantation. Lewis rats were used as donors and recipients. Hepatocytes were isolated with a collagenase digestion, both for the adult and fetal livers (17 days estimated gestational age). After digestion, the hepatocytes were seeded onto 95% porous poly-(L)-lactic acid matrices. The polymer-cell constructs with adult or fetal cells were then implanted between mesenteric leaves of three different recipient groups: adults (approximately 200 g), 2-week, and 4-week neonates (two to five animals per group, depending on litter size). The specimens were harvested at 4 weeks, stained with Hematoxylin and Eosin (H&E), and the cell area of each specimen (24 sections per group) was quantitated using morphometric analysis. Results were statistically analyzed using an unpaired, two-tailed Student's t test. At 4 weeks, all specimens showed survival of groups of hepatocytes, especially along the periphery of the polymers and near blood vessels. The hepatocyte cell area for the six groups was calculated in square micrometers: the adult cells transplanted into adult recipients, 0.16 x 10(5) microns2; fetal cells into adults, 0.47 x 10(5) microns2; adult into 4-week neonates, 1.17 x 10(5) microns2; fetal into 4-week neonates, 4.54 x 10(5) microns2; adult into 2-week neonates, 2.98 x 10(5) microns2, and fetal into 2-week neonates, 5.81 x 10(5) microns2. In all three recipient groups, the area of fetal hepatocytes was approximately two to three times the area of the adult hepatocytes (P < .05 for 2-week and 4-week neonatal recipients, P = .06 for adult recipients). Also, as the recipient age decreased, there was an increase in the hepatocyte cell area (P < .05 for fetal or adult groups). The authors conclude that fetal hepatocytes heterotopically transplanted have a significant survival advantage over adult hepatocytes, independent of recipient age. The authors further conclude that the neonatal environment is more favorable than the adult environment for implantation of hepatocytes.     

Downregulation of polo-like kinase correlates with loss of proliferative ability of cardiac myocytes

Cardiac myocytes rapidly increase the cell number during the fetal and early neonatal period, but they lose their proliferative ability soon after birth. To understand the mechanism of how cardiac myocytes exit from the cell cycle, we examined the role of a newly identified serine/threonine kinase, polo-like kinase (Plk), in the process of proliferation of cardiac myocytes. Northern blot analysis revealed that Plk gene was abundantly expressed in cardiac myocytes and non-myocytes of fetal and neonatal rats but not in cardiocytes of adult rats. Western blot analysis showed that Plk protein was also detected only in fetal and neonatal hearts. During the early stage of cardiac differentiation. Plk expression was well correlated with the proliferative ability of cardiocytes. Plk mRNA was most abundant in undifferentiated embryonic stem (ES) cells and the mRNA levels decreased along with cardiac differentiation in the developing ES cell system. Once serum was deprived from the culture media, expression levels of Plk were markedly decreased and DNA was not synthesized in both cardiac myocytes and non-myocytes of neonatal rats. Re-addition of serum stimulated Plk gene expression and DNA synthesis in non-myocytes but not in cardiomyocytes. All these results taken together with the critical role of Plk in DNA synthesis in many cell types suggest that downregulation of Plk is important for the permanent withdrawal of cardiomyocytes from the cell cycle.     

DNA synthesis is dissociated from the immediate-early gene response in the post-ischemic kidney

OBJECTIVE: Fetal growth and development are closely related to normal placental growth and function. We performed a study to determine the effect of a 10-day period of fetal hypoxemia induced by umbilical-placental hypoperfusion on tissue deoxyribonucleic acid synthesis rates in the 0.84 to 0.91 of gestation ovine fetus and placenta. STUDY DESIGN: Daily fetal placental embolization was performed in four chronically catheterized sheep fetuses until fetal arterial oxygen content decreased by approximately 30% compared with preembolization values. Five control fetuses received vehicle only. On experimental day 10, the deoxyribonucleic acid synthesis rate was determined by injecting tritiated thymidine (1 mCi/kg) intravenously approximately 8 hours before the end of the study. RESULTS: Fetal arterial oxygen decreased from 3.2 +/- 0.1 (SEM) mmol/L preembolization to 2.2 +/- 0.2 mmol/L on day 10 (p < 0.001) and remained unchanged in controls. On day 10 deoxyribonucleic acid synthesis rates were significantly reduced in embolized fetuses compared with controls, by 38% in cotyledons (83.0 +/- 15.1 vs 133.7 +/- 9.9 disintegrations/min/micrograms deoxyribonucleic acid, p < 0.05), 28% in the left ventricular wall (36.8 +/- 3.7 vs 51.0 +/- 4.7 disintegrations/min/micrograms deoxyribonucleic acid, p < 0.05), and 45% in the quadriceps muscle (15.4 +/- 4.0 vs 28.1 +/- 3.0 disintegrations/min/micrograms deoxyribonucleic acid, p < 0.05). Tritiated thymidine autoradiography demonstrated that cotyledonary deoxyribonucleic acid synthesis occurred exclusively in the fetal trophoblasts cells. CONCLUSION: We concluded that a reduction in cotyledonary, quadriceps muscle, and left ventricular myocardium deoxyribonucleic acid synthesis rates are the earliest adaptive mechanisms of fetal growth associated with development of umbilical-placental insufficiency. We speculate that alteration in the myocardial deoxyribonucleic acid synthesis rate could be a major contributing factor in the deterioration of fetal myocardial function associated with increased placental vascular resistance.     

A boy with X-linked hyper-IgM syndrome and natural killer cell deficiency

OBJECTIVE: To compare the efficacy of nifedipine with ritodrine in the management of preterm labor. METHODS: One hundred eighty-five singleton pregnancies with preterm labor were assigned randomly to either ritodrine intravenously (n = 90) or nifedipine orally (n = 95). The principal outcome assessed was delay of delivery. RESULTS: Ritodrine was discontinued in 12 patients because of severe maternal side effects, and their results were excluded from further analysis. More women in the ritodrine group delivered within 24 hours (22 versus 11, P = .006), within 48 hours (29 versus 21, P = .03), within 1 week (45 versus 36, P = .009), and within 2 weeks (52 versus 43, P = .005) compared with those receiving nifedipine. There were significantly fewer maternal side effects in the nifedipine group. Apgar scores and umbilical artery and vein pHs were similar in both groups. The number of admissions to the neonatal intensive care unit (NICU) in the nifedipine group was significantly lower than in the ritodrine group (68.4 versus 82.1%, P = .04). CONCLUSION: Nifedipine in comparison with ritodrine in the management of preterm labor is significantly associated with a longer postponement of deliver, fewer maternal side effects, and fewer admissions to the NICU.     

Ultrastructural characteristics of intercellular contacts and bile canaliculi in neonatal rat hepatocytes in primary culture

The ultrastructure of the cellular contacts and bile canaliculi was examined in cultured neonatal (day 5) rat hepatocytes to elucidate the development of cellular polarity. A new scanning electron microscopic technique for cultured hepatocytes allowed a view of cell-cell attachment and the entire cell surface, including the underside on plastic dishes. At 3 h after plating, neonatal hepatocytes were shown to be round, with loss of the preferential localization of cell organelles. After 6 h of culture, the cells had become oblong; they were aggregated in groups of several cells and the cellular contacts were not as rigid or as straight as those in adult hepatocytes. Transmission electron microscopy showed the biliary functional polarity to be like that in vivo. On the undersurfaces of adjacent neonatal hepatocytes a hemicanalicular structure lined with microvilli was found, which probably corresponds to the ultrastructure of bile canaliculi in vivo. However, no canaliculi or orifices of bile channels were found in adult hepatocytes. These results suggest that in neonatal rat hepatocytes the formation of tight rigid cellular contacts was suppressed. Modulation of cell membranes appeared on the undersurfaces of neonatal hepatocytes in early culture stages. The differences in the development of cellular polarity could be caused by the proliferating activity of neonatal hepatocytes.     

In vivo electrochemical studies of dopamine clearance in subregions of rat nucleus accumbens: differential properties of the core and shell

The effects of the rodent hepatocarcinogens clofibric acid and diprofibrate on the activity of the peroxisomal fatty acyl-CoA oxidase, DNA synthesis, and apoptosis were compared in cultured rat and human hepatocytes. Rat hepatocytes expressed a 10-fold greater level of the peroxisomal fatty acyl-CoA oxidase compared to human hepatocytes. At the highest concentration (1.0 mM), both drugs induced a two- to threefold increase in this enzyme activity in both rat and human hepatocytes. Ciprofibrate (0.1 and 0.2 mM) caused a twofold increase in DNA synthesis in rat hepatocytes, whereas clofibric acid had no effect on DNA synthesis in these cells. In contrast, increasing concentrations of both clofibric acid and ciprofibrate produced inhibition of DNA synthesis in human hepatocytes. By using the terminal transferase dUTP-biotin nick end labeling technique, it was observed that 0.1 and 0.2 mM clofibric acid and ciprofibrate suppressed transforming growth factor-beta (TGF beta)-induced apoptosis by 50% in rat hepatocytes, but they had no effect on TGF beta-induced apoptosis in human hepatocytes. Although clofibric acid and ciprofibrate diminished TGF beta-induced apoptosis, they had no effect on the basal apoptotic levels in the rat hepatocyte cultures. However, both drugs significantly increased the percent of apoptotic cells in the human hepatocyte cultures. It is concluded that primary rat and human hepatocyte cultures respond differently to peroxisome proliferators. The differences in effects on DNA synthesis and apoptosis support the hypothesis that human liver cells are refractory to peroxisome proliferator-induced hepatocarcinogenesis.     

Cholera toxin stimulates type II pneumocyte proliferation by a cyclic AMP-independent mechanism

Cholera toxin (CT) stimulated DNA synthesis by low-density primary cultures of adult rat type II pneumocytes (T2P) in a dose-dependent manner, either in the presence or the absence of serum. In the presence of 1% rat serum, 1 microgram/ml CT also stimulated a 50% increase in cell number over 8 days of incubation (P<0.01); this was in addition to a 2-fold increase in cell number induced by the serum alone (P<0.05). The same dose of CT also elevated intracellular cAMP and the total activity of protein kinase A (both P<0.01), suggesting toxin stimulation of T2P proliferation by a cAMP-dependent mechanism. However, the effect of CT on DNA synthesis could not be mimicked by 8-bromoadenosine 3':5'-cyclic monophosphate (8-bromo-cAMP), nor by N6,2'-O-dibutyryladenosine 3':5'-cyclic monophosphate (dibutyryl-cAMP), each tested over a wide range of concentrations. l-Isoproterenol stimulated surfactant secretion by over 5-fold (P<0. 01), but neither the beta-agonist, forskolin nor 3-isobutyl-1-methylxanthine had any significant effect on DNA synthesis. The purified B-subunit of CT stimulated DNA synthesis to the same degree as did the holotoxin, either in the presence or the absence of rat serum. In contrast, the purified A-subunit had no significant effect. These data suggest that cholera toxin stimulates type II pneumocyte proliferation through a mechanism that is independent of cAMP, protein kinase A and toxin-catalyzed ADP-ribosylation.     

Effects of activin-A, inhibin-A, and transforming growth factor-beta 1 on stage-specific deoxyribonucleic acid synthesis during rat seminiferous epithelial cycle

Activin and inhibin are members of the transforming growth factor-beta (TGF beta) gene family. They are expressed in various organ systems, where they possess regulatory functions. Inhibin, activin, and TGF beta have been reported to also be expressed in the adult rat testis. We studied in vitro the action of these growth factors on premitotic and premeiotic DNA synthesis during the rat seminiferous epithelial cycle. Two-millimeter rat seminiferous tubule segments were isolated by transillumination-assisted microdissection from stages V, VIIa, VIII-IX, and I of the cycle and incubated in vitro in the presence of activin-A, inhibin-A, or TGF beta 1. During 24-, 48-, and 72-h incubation spontaneous progression of spermatogenesis was noted. The staged samples allowed us to selectively quantitate DNA synthetic activity of specific germ cell types. At the end of the culture, the tubules were pulse labeled with [3H]thymidine, and DNA synthesis was quantified by liquid scintillation counting, and the activated cells were detected by autoradiography. Activin-A stimulated preleptotene spermatocyte DNA synthesis in a dose-dependent manner. DNA synthesis of intermediate spermatogonia was also stimulated by activin-A, whereas inhibin-A inhibited DNA synthesis of these cells. TGF beta 1 had a small, but significant, stimulatory effect on DNA synthetic activity at stage VII. These results support the view that activin-A, inhibin-A, and TGF beta 1 take part in the regulation of DNA synthesis during rat spermatogenesis.     

Atypical beta-adrenergic receptors in rat liver: evidence for transient expression during aging

This study was designed to determine whether functional beta 3-adrenergic receptors exist in the rat liver and whether there are alterations in the beta 3-adrenergic response with age in a manner similar to that which occurs for beta 2-adrenergic receptors. The beta 3 stimulation of adenylyl cyclase activity was assessed using the novel beta 3-specific agonist, CGP-12177A. Adenylyl cyclase activation by CGP-12177A was seen only in the adults. Isoproterenol-stimulated adenylyl cyclase activity was high in the neonates, declined by 45% in the adults, and was high again in the senescent rats. ICI 118551, a beta 2-selective antagonist, attenuated the isoproterenol-stimulated activity by two-thirds but had no effect on the CGP-12177A-stimulated activity. These data demonstrated the presence of the beta 3-adrenergic receptor in the rat liver, although only at the adult stage of development. In addition, these data confirm earlier findings that beta 2-adrenergic activation of adenylyl cyclase is biphasic with age and indicate that the emergence of the beta 3-stimulated activity coincides with the attenuation of beta 2-stimulated activity.     

Preventive effect of ritodrine hydrochloride and/or urinary trypsin inhibitor against lipopolysaccharide-induced preterm delivery in mice

BACKGROUND: The purpose of this study was to confirm the preventive effect of ritodrine hydrochloride (ritodrine) alone or ritodrine plus urinary trypsin inhibitor (UTI) in a mouse model of preterm delivery. METHODS: On day 17 of pregnancy, female C3H/HeN mice impregnated by male B6D2F1 mice were given two intraperitoneal injections of lipopolysaccharide (LPS) (50 micrograms/kg) at a 3-hour interval, which induced a 100% incidence of preterm delivery within 25 hours of the second dose. Ritodrine (1, 3, or 10 mg/kg, p.o.), UTI (25 X 10(4) units/kg, i.p.), ritodrine (3 mg/kg, p.o.) plus UTI (25 x 10(4) units/kg, i.p.), distilled water (10 ml/kg, p.o.), or distilled water (10 mg/kg, p.o.) plus saline solution (10 ml/kg, i.p.) were administered to the pregnant animals 10 times at 1-hour intervals from 8:00 AM to 5:00 PM on day 18 of pregnancy. In addition, the preventive effect of ritodrine, UTI, or ritodrine plus UTI was examined on LPS-induced contraction of uterine muscle strips isolated from pregnant mice on day 17 of gestation. RESULTS: The incidence of preterm delivery decreased significantly in a dose-dependent fashion with ritodrine treatment, and there was a significant and synergistic decrease after combined treatment with ritodrine plus UTI. The in vitro uterine contraction induced by LPS was significantly suppressed by both ritodrine and UTI. CONCLUSIONS: Combination therapy with ritodrine plus UTI may be helpful for preventing preterm delivery in humans without the cardiovascular side effects that often accompany treatment with ritodrine alone.     

Growth response of adult germ cells to rat androgen-binding protein and human sex hormone-binding globulin

Androgen-binding protein (ABP) is produced by Sertoli cells depending on the development and the stage of the spermatogenic cycle. Germ cell proliferation is at its peak when ABP is at its peak and secreted towards the testicular basal compartment containing spermatogonia and premeiotic spermatocytes. Rat isolated adult germ cell DNA synthesis was studied in vitro in the presence of ABP with and without steroids and in the presence of pure or recombinant sex steroid hormone-binding globulin (SHBG) using thymidine incorporation. Results are: SHBG is able to promote DNA synthesis in the absence of cofactors. Testosterone reacted negatively to the stimulatory effect of SHBG. We conclude that ABP, the physiological steroid-binding protein, should be considered as a paracrine regulator of spermatogenic DNA synthesis in the adult rat.     

Effects of progesterone on proliferation and differentiation of fetal rat calvarial osteoblasts in vitro

OBJECTIVES: To investigate the influence of progesterone on proliferation and differentiation of osteoblast at the levels of gene expression and cell functions. METHODS: Fetal rat calvarial osteoblasts were cultured in vitro in the presence of (10(-9) mol/L-10(-6) mol/L) progesterone. Cell proliferation, alkaline phosphalase (ALP) activity, osteocalcin mRNA expression and osteocalcin secretion in the medium and bone nodule formation were analyzed. RESULTS: Progesterone did not influence cell proliferation; Progesterone enhanced the ALP activity in rat osteoblasts; Progesterone stimulated osteocalcin mRNA expression in a dose-dependent manner and increased the amount of osteocalcin in the culture medium; Progesterone increased both number and area of bone nodule formation. CONCLUSION: Progesterone has a multi-stimulating effect on the differentiation of fetal rat calvarial osteoblast, hut no effect on cell proliferation.     

Tumour radiosensitization by high-oxygen-content gases: influence of the carbon dioxide content of the inspired gas on PO2, microcirculatory function and radiosensitivity

The rat testis is considered to be an immunologically privileged site because of its reduced capacity to support antigen-specific immune responses. To understand this phenomenon, it is essential to characterize both the lymphocyte subpopulations normally present in the testis and their regulation by testicular cytokines. Peripheral blood was obtained from adult male Dark Agouti or Sprague-Dawley rats, and testicular interstitial tissue was collected after perfusion of the testes to remove blood. Blood and testis lymphocytes were isolated using discontinuous Percoll density gradients, and the testicular lymphocytes were further purified by selective adherence to remove mononuclear phagocytes. The isolated lymphocytes were analyzed by flow cytometry using specific monoclonal antibodies and fluorescein labeling and were enumerated as total T cells, CD4+ T cells, CD8+ T cells, B cells, and natural killer (NK) cells. In contrast to peripheral blood, in which the CD4+ T-cell subset was the major lymphocyte subset, rat testis T cells were predominantly of the CD8+ subset, and a large population of NK cells also were present. Subsequently, peripheral blood lymphocytes were stimulated with the polyclonal T-cell activator, phytohemagglutinin, and cultured in the presence of activin, inhibin, or transforming growth factor beta (TGFbeta) prior to flow cytometric analysis. Activin and TGFbeta suppressed T-cell proliferation without any selective effect on either T-cell subset, and inhibin had no effect. The predominance of CD8+ T cells and NK cells, and the relatively minor proportion of CD4+ T cells, are consistent with both increased cellular immune surveillance and a reduced capacity for initiating antigen-specific immune responses in the adult rat testis.     

Mapping of the binding of platelet-derived growth factor to distinct domains of the basement membrane proteins BM-40 and perlecan and distinction from the BM-40 collagen-binding epitope

Iron is required for cell proliferation of all living species. Moreover, iron excess may be involved in the development of hepatocellular carcinoma. In this study we analyzed the effects of deferoxamine, an iron chelator, on normal porcine hepatocyte proliferation. We confirmed that hepatocytes isolated from young pigs proliferate in the presence of insulin and fetal calf serum as shown by [3H] methyl-thymidine incorporation, presence of mitotic figures and increase in cell number. This was paralleled by nuclear expression of p34cdc2 and its associated histone H1 kinase activity. In the presence of deferoxamine, [3H] methyl-thymidine incorporation, expression of nuclear proteins (p34cdc2 and PCNA) and H1 kinase activity were drastically reduced. In addition, in contrast with control cultures, cells in S-phase were not detected by flow cytometry. These data suggest that iron chelation by deferoxamine can arrest the progression of porcine hepatocytes in the G1 phase of the cell cycle.