The effects of neurotrophins on several forms of neuronal degeneration in murine cortical cell cultures were examined. Consistent with other studies, brain-derived neurotrophic factor, neurotrophin-3, and neurotrophin-4/5 all attenuated the apoptotic death induced by serum deprivation or exposure to the calcium channel antagonist nimodipine. Unexpectedly, however, 24-hour pretreatment with these same neurotrophins markedly potentiated the necrotic death induced by exposure to oxygen-glucose deprivation or N-methyl-D-aspartate. Thus, certain neurotrophins may have opposing effects on different types of death in the same neurons.
LONG-TERM potentiation (LTP) in hippocampus is a type of synaptic plasticity that is thought to be involved in learning and memory(1). Several lines of evidence suggest that LTP involves 3',5'-cyclic GMP (cGMP), perhaps as an activity-dependent presynaptic effector of one or more retrograde messengers (refs 2-12, but see ref, 13). However, previous results are also consistent with postsynaptic effects of cGMP. This is difficult to test in hippocampal slices, but more rigorous tests are possible in dissociated cell culture(14-17). We have therefore developed a reliable method for producing N-methyl-D-aspartate (NMDA) receptor-dependent LTP at synapses between individual hippocampal pyramidal neurons in culture, We report that inhibitors of guanylyl cyclase or of cGMP-dependent protein kinase block potentiation by either tetanic stimulation or low-frequency stimulation paired with postsynaptic depolarization. Conversely, application of 8-Br-cGMP to the bath or injection of cGMP into the presynaptic neuron produces activity-dependent long-lasting potentiation, The potentiation by cGMP involves an increase in transmitter release that is in part independent of changes in the presynaptic action potential. These results support a presynaptic role for cGMP in LTP.
We have reported that members of the bcl-2 gene family are expressed and gonadotropin regulated in ovarian granulosa cells during follicular maturation and atresia. Because Bcl-2, a protein that prevents apoptosis in several cell types, is reported to function as an antioxidant or free radical scavenger, the present studies were designed to investigate if oxidative stress plays a role in granulosa cell apoptosis during follicular atresia in the immature rat ovary. In the first series of experiments, the role of oxidative stress in the induction of granulosa cell apoptosis was directly tested using a defined in vitro follicle culture system. Healthy antral follicles obtained from equine CG (eCG)-primed immature (27 day old) rats were incubated in serum-free medium for 24 h in the absence or presence of FSH (100 ng/ml; a control for inhibiting apoptosis), superoxide dismutase (SOD; 10-1000 U/ml), ascorbic acid (0.01-1 mM; a free radical scavenger), N-acetyl-L-cysteine (25-100 mM; a free radical scavenger and stimulator of endogenous glutathione peroxidase activity), or catalase (10-1000 U/ml). Granulosa cells within follicles incubated in medium alone exhibited extensive apoptosis after 24 h of incubation, and this onset of apoptosis was blocked by treatment with FSH (29 +/- 4% of controls; P < 0.001, n = 3). Moreover, apoptosis in follicles was also inhibited by treatment with SOD (44 +/- 4% of controls at 1000 U/ml; P < 0.01, n = 3), ascorbic acid (55 +/- 9% of controls at 1 mM; P < 0.05, n = 3), N-acetyl-L-cysteine (24 +/- 7% of controls at 100 mM; P < 0.001, n = 3), or catalase (35 +/- 6% of controls at 1000 U/ml; P < 0.001, n = 3). In the second series of experiments, complementary DNAs corresponding to secreted (SEC-SOD), copper/zinc-containing (Cu/Zn-SOD), and manganese-containing (Mn-SOD) forms of rat SOD, rat seleno-cysteine glutathione peroxidase (GSHPx), and rat catalase were isolated and used to synthesize antisense RNA probes for Northern and slot blot analysis of changes in SOD, GSHPx, and catalase gene expression during follicular maturation. In vivo priming of 25-day-old female rats for 2 days with 10 IU eCG, which promoted antral follicular growth and survival, increased levels of messenger RNA encoding SEC-SOD (216 +/- 9% of saline-treated controls, P < 0.05, n = 3) and Mn-SOD (222 +/- 14% of saline-treated controls, P < 0.05, n = 3) vs. saline-treated controls. However, gonadotropin priming did not alter expression of Cu/Zn-SOD, GSHPx, or catalase messenger RNA in the ovary. Nevertheless, the induction of SEC-SOD and Mn-SOD expression by eCG provided further evidence that gonadotropins may promote granulosa cell survival in developing antral follicles via activation of an oxidative stress response. Collectively, these data suggest that the gonadotropin-mediated inhibition of follicular atresia involves enhanced expression of oxidative stress response genes whose products may then function to protect granulosa cells from the damaging effects of reactive oxygen species.
A system for tetracycline-regulated inducible gene expression was described recently which relies on constitutive expression of a tetracycline-controlled transactivator (tTA) fusion protein combining the tetracycline repressor and the transcriptional activation domain of VP16 [Gossen, M. \& Bujard, H. (1992) Proc. Natl. Acad. Sci. USA 89, 5547-5551]. This system yielded only low levels of transactivator protein, probably because tTA is toxic. To avoid this difficulty, we placed the tTA gene under the control of the inducible promoter to which tTA binds, making expression of tTA itself inducible and autoregulatory. When used to drive expression of the recombination activating genes 1 and 2 (RAG-1 and RAG-2), the autoregulatory system yielded both substantially higher levels of variable (diversity) joining [V(D)J] recombination activity (70-fold on average) and inducible expression in a much larger fraction of transfected cells (autoregulatory, 90%, vs. constitutive, 18%). In addition, this system allowed the creation of transgenic mice in which expression of a luciferase transgene was inducible tens to hundreds of times the basal levels in most tissues examined. Induced levels of expression were highest in thymus and lung and appear to be substantially higher than in previously reported inducible luciferase transgenic mice created with the constitutive system. With the modified system, inducible transactivator mRNA and protein were easily detected in cell lines by RNA and Western blotting, and transactivator mRNA was detected by RNA blotting in some tissues of transgenic mice. This autoregulatory system represents an improved strategy for tetracycline-regulated gene expression both in cultured cells and in transgenic animals.
Cells from transgenic mice expressing a human mini-gene for collagen I were used as markers to follow the fate of mesenchymal precursor cells from marrow that were partially enriched by adherence to plastic, expanded in culture, and then injected into irradiated mice. Sensitive PCR assays for the marker collagen I gene indicated that few of the donor cells were present in the recipient mice after 1 week, but 1-5 months later, the donor cells accounted for 1.5-12% of the cells in bone, cartilage, and lung in addition to marrow and spleen. A PCR in situ assay on lung indicated that the donor cells diffusely populated the parenchyma, and reverse transcription-PCR assays indicated that the marker collagen I gene was expressed in a tissue-specific manner. The results, therefore, demonstrated that mesenchymal precursor cells from marrow that are expanded in culture can serve as long-lasting precursors for mesenchymal cells in bone, cartilage, and lung. They suggest that cells may be particularly attractive targets for gene therapy ex vivo.
Release of 92‐kd type IV collagenase/gelatinase, also known as gelatinase B, by inflammatory and tumor cells is increasingly recognized and is believed to facilitate cellular migration across basement membranes. It has been implicated in the pathogenesis of many diseases, but little is known of its cellular origin(s) and function in liver. In this study we have demonstrated synthesis and release of gelatinase B by human and rat Kupffer cells in primary culture. Northern analysis of RNA extracted from Kupffer cells stimulated with phorbol ester demonstrated a 2.8 kb transcript for gelatinase B. Immunoblotting and zymography of serum‐free Kupffer cell–conditioned media demonstrated extracellular release of immunoreactive enzyme and gelatinase activity, Mr 92,000 (95,000 from rat cells). The organomercurial 4‐aminophenyl mercuric acetate (APMA) activated the enzyme in vitro, indicating secretion primarily as a proenzyme. Stimulation of Kupffer cells by phorbol ester markedly induced gelatinase B release, which was inhibited by cycloheximide. In contrast, cycloheximide had no effect on constitutive secretion in culture, suggesting that there is some intracellular storage. Kupffer cell–derived gelatinase B was also partially purified and characterized. After separation by gelatin sepharose and gel filtration chromatography, gelatin‐degrading activities of 95, 88, 75, and 65 kd were detected, the three lower‐molecular‐weight species probably representing activated forms. Enzyme activity was inhibited by ethyl‐enediaminetetra‐acetic acid (EDTA), but not by serine‐and thiol‐protease inhibitors, and was restored by zinc. Activity was also inhibited by tissue inhibitor of metallo‐proteinase‐1 (TIMP‐1) and α‐2 macroglobulin. The partially purified enzyme rapidly degraded denatured collagens (gelatin) as well as native types III, IV, and V collagens, but had no activity against casein, types I and VI collagens. (HEPATOLOGY 1995; 22:304–315.)
Astrocytes, a sub-type of glial cell in the central nervous system, can release the excitatory transmitters glutamate and aspartate in response to elevated levels of internal calcium. To investigate potential release mechanisms that may be present in these cells we have determined whether protein components of the neuronal secretory apparatus are expressed in astrocytes. Western blots, immunocytochemistry and RT PCR demonstrate that astrocytes express cellubrevin, synaptobrevin II and syntaxin, proteins known to form a macromolecular fusion complex. However, SNAP-25 which is another neuronal protein of the fusion complex, was not detected. Astrocyte cellubrevin and synaptobrevin II were also shown to be sensitive to the proteolytic activity of tetanus toxin. Together these data indicate that astrocytes express some proteins that are known to form a fusion complex indicating that regulated exocytosis might mediate calcium-regulated transmitter release from these cells.
We have used suspension-cultured parsley cells (Petroselinum crispum) and an oligopeptide elicitor derived from a surface glycoprotein of the phytopathogenic fungus Phytophthora megasperma f.sp. glycinea to study the signaling pathway from elicitor recognition to defense gene activation. Immediately after specific binding of the elicitor by a receptor in the plasma membrane, large and transient increases in several inorganic ion fluxes (Ca , H , K , Cl ) and H O formation are the first detectable plant cell responses. These are rapidly followed by transient changes in the phosphorylation status of various proteins and by the activation of numerous defense-related genes, concomitant with the inactivation of several other, non-defense-related genes. A great diversity of cis-acting elements and trans-acting factors appears to be involved in elicitor-mediated gene regulation, similar to the apparently complex nature of the signal transduced intracellularly. With few exceptions, all individual defense responses analyzed in fungus-infected parsley leaves have been found to be closely mimicked in elicitor-treated, cultured parsley cells, thus validating the use of the elicitor/cell culture system as a valuable model system for these types of study.