Signalling pathways and the regulation of SUMO modification.

The modification of proteins by SUMO (small ubiquitin-related modifier) conjugation is becoming increasingly recognized as an important regulatory event. Protein SUMOylation can control a whole range of activities, including subcellular localization, protein-protein interactions and enzymatic activity. However, the SUMOylation process can itself be controlled. In the present review, the mechanisms through which protein SUMOylation is regulated are discussed, with particular emphasis on the impact of signalling pathways. A major point of regulation of the SUMO pathway is through targeting the E3 ligases, and a number of different ways to achieve this have been identified. More generally, the MAPK (mitogen-activated protein kinase) pathways represent one way through which SUMOylation of specific proteins is controlled, by using molecular mechanisms that at least in part also function by modifying the activity of SUMO E3 ligases. Further intricacies in signalling pathway interactions are hinted at through the growing number of examples of cross-talk between different post-translational modifications and SUMO modification.

Characterization of the chicken interleukin-1beta converting enzyme (caspase-1) cDNA and expression of caspase-1 mRNA in the hen.

We have cloned and sequenced a chicken homolog to the mammalian interleukin-1beta (IL-1beta)-converting enzyme (caspase-1) cDNA and have evaluated caspase-1 mRNA expression in various tissues from the domestic hen, including ovarian follicle granulosa and theca layers. The deduced amino acid (aa) sequence of chicken caspase-1 is 44.9% identical to human caspase-1, and contains an active site pentapeptide that is characteristic of the caspase family of cysteine proteases. Of interest, however, is that the putative chicken caspase-1 cDNA is predicted to encode a comparatively short (19aa) N-terminal prodomain, as well as two Cys residues within the active pentapeptide (QC162C163RG) as compared to the QACRG pentapeptide found in the mammalian caspase-1 sequence. While the chicken caspase-1 mRNA transcript is widely expressed among different tissues, levels are particularly high in the bursa of Fabricius and comparatively low in ovarian follicles at all stages of development. Finally, treatment of granulosa cells with IL-1beta, the primary if not sole product of caspase-1 activity, fails to either promote apoptotic cell death or enhance viability in granulosa cells. Considering the relatively low levels of caspase-1 mRNA expression in ovarian follicle tissues plus the inability of IL-1beta to alter granulosa cell viability, in vitro, it is concluded that caspase-1 is not an integral part of the apoptotic pathway in granulosa cells. However, the pattern of mRNA expression is consistent with a requirement for caspase-1 mediated IL-1beta production in chicken immune tissues.

Expression and localization of Bcl-2 related proteins in human ovarian cancers.

BACKGROUND: Interactions between Bcl-2 and its related proteins regulate cell death in a number of tissues including the ovary. Some Bcl-2-related proteins (Bcl-2, Bcl-xLong, MCl-1, and Dad-1) function as cell death repressors, whereas others (Bax, Bcl-xShort, Bak, and Bad) facilitate apoptosis. MATERIALS AND METHODS: Immunoblotting and immunohistochemistry using specific antisera were performed with a panel of human ovarian carcinomas and normal human ovaries. RESULTS: All Bcl-2 related proteins were expressed in normal and malignant ovarian tissues. Higher levels of Bcl-2 versus Bcl-xLong were observed in normal ovary by immunoblotting, compared to a majority of cancer samples. Increased levels of death-inducing Bax were related to malignant transformation across all types of ovarian cancer included in this study. CONCLUSIONS: The results provide further evidence that a balance of expression among Bcl-2 family members may regulate ovarian cancer cell survival.

Expression of bcl-2 and nr-13 in hen ovarian follicles during development.

Follicle atresia is initiated within the granulosa cell layer of ovarian follicles and is mediated via the process of apoptosis. In the hen, at least two populations of granulosa cells can be distinguished during follicle development on the basis of their inherent susceptibility or resistance to apoptosis, in vitro. Given the previously established correlation between expression of bcl-xLong and hen granulosa cell resistance to apoptosis, the present studies were conducted to characterize expression of bcl-2 and an avian bcl-2 homologue, nr-13, in follicles at various stages of development. Levels of nr-13 mRNA were significantly higher only in granulosa cells from the largest (F1) preovulatory follicle compared to 3- to 5-mm prehierarchal follicles. By comparison, bcl-2 mRNA levels were 5- to 9-fold higher in granulosa cells from the three largest preovulatory follicles compared to those from follicles 9 to 12 mm in diameter and prehierarchal follicles. The increase in neither nr-13 nor bcl-2 was correlated with the stage of follicle development associated with the acquisition of resistance to apoptosis in granulosa cells (e.g., at the 9- to 12-mm stage). Results from the present studies do not support a close correlation between constitutive expression of nr-13 or bcl-2 mRNA and the transition from a state of apoptosis susceptibility to apoptosis resistance in hen granulosa cells. Thus, it is proposed that nr-13 and bcl-2 play more of a supportive role in regulating additional aspects of ovarian cell function such as cell proliferation and/or differentiation.

N2 Fixation, Carbon Metabolism, and Oxidative Damage in Nodules of Dark-Stressed Common Bean Plants.

Common beans (Phaseolus vulgaris L.) were exposed to continuous darkness to induce nodule senescence, and several nodule parameters were investigated to identify factors that may be involved in the initial loss of N2 fixation. After only 1 d of darkness, total root respiration decreased by 76% and in vivo nitrogenase (N2ase) activity decreased by 95%. This decline coincided with the almost complete depletion (97%) of sucrose and fructose in nodules. At this stage, the O2 concentration in the infected zone increased to 1%, which may be sufficient to inactivate N2ase; however, key enzymes of carbon and nitrogen metabolism were still active. After 2 d of dark stress there was a significant decrease in the level of N2ase proteins and in the activities of enzymes involved in carbon and nitrogen assimilation. However, the general collapse of nodule metabolism occurred only after 4 d of stress, with a large decline in leghemoglobin and antioxidants. At this final senescent stage, there was an accumulation of oxidatively modified proteins. This oxidative stress may have originated from the decrease in antioxidant defenses and from the Fe-catalyzed generation of activated oxygen due to the increased availability of catalytic Fe and O2 in the infected region.

Induction of apoptotic cell death in hen granulosa cells by ceramide.

Recent studies have demonstrated that ovarian follicle atresia occurs extensively before follicle selection into the avian preovulatory hierarchy, and that this process is mediated via granulosa cell apoptosis. Subsequent to follicle selection, granulosa cells are inherently resistant to apoptosis, and such resistance is correlated with increased expression of death suppressor genes such as bcl-xlong. In the present studies we used this avian ovary model system to 1) identify cellular characteristics and mechanisms related to apoptotic cell death of granulosa cells in vitro, and 2) further characterize functional differences between apoptosis-susceptible (4- to 8-mm follicle) and apoptosis-resistant (preovulatory follicle) granulosa cells. Treatment of granulosa cells from the largest preovulatory follicle with N-octanoylsphingosine (C8-ceramide) results in pronounced oligonucleosome formation, a hallmark of apoptosis. That this is indicative of programmed cell death is supported by an increased incidence of pyknotic nuclei and apoptotic bodies in C8-ceramide-treated samples compared to that in control cultured cells. Tumor necrosis factor-alpha, a stimulator of ceramide production, actively promotes oligonucleosome formation in apoptosis-susceptible, but not in apoptosis-resistant, granulosa cells. Induction of apoptosis is also observed after exposure of apoptosis-resistant granulosa cells to sphingomyelinase treatment and UV irradiation, which are known to stimulate endogenous ceramide production, and to the anticancer drug, daunorubicin, which initiates de novo ceramide biosynthesis via activation of ceramide synthase. Although treatment of granulosa cells with fumonisin B1, a specific ceramide synthase inhibitor, blocks daunorubicin-stimulated oligonucleosome formation, UV-induced cell death is unaffected. Taken together, these results demonstrate that pharmacological factors known to mimic the actions of ceramide or stimulate ceramide production can induce oligonucleosome formation and programmed cell death in granulosa cells. More importantly, however, the ability of a physiologically relevant initiator of ceramide biosynthesis, tumor necrosis factor-alpha, to promote cell death is evident only in apoptosis-susceptible granulosa cells collected from atresia-prone prehierarchal follicles. These data provide support for ceramide as an important intracellular signaling mechanism, mediating granulosa cell apoptosis and follicle atresia.

Susceptibility of avian ovarian granulosa cells to apoptosis is dependent upon stage of follicle development and is related to endogenous levels of bcl-xlong gene expression.

Studies were conducted to evaluate the susceptibility of avian ovarian granulosa cells to apoptosis when incubated in vitro and to relate this relative susceptibility to both the stage of follicle development from which granulosa cells were collected (atresia-prone vs. -resistant) and to the expression of a gene previously linked to the regulation of cell viability, bcl-xlong. Granulosa cells from slow growing, prehierarchal (4- to 8-mm diameter; atresia-prone) follicles were found to undergo rapid and progressively extensive apoptosis after incubation in defined medium for 6-24 h (P < 0.05 vs. unincubated controls). By contrast, cells from the largest preovulatory (F1) follicle, as well as from follicles most recently recruited into the follicle hierarchy (9- to 12-mm diameter), showed significantly less low molecular wt labeling at 6 h of incubation (P < 0.05 vs. 4- to 8-mm follicles). Furthermore, the amount of low molecular wt labeling did not significantly increase in cells from either stage of follicle development at 12 or 24 h of incubation (P < 0.05 vs. 6 h incubation). This biochemical indication of ongoing apoptosis in prehierarchal follicle granulosa cells was confirmed by an increased incidence of pyknotic nuclei detected by morphological analysis. Thus, increased susceptibility to apoptosis in incubated prehierarchal follicle granulosa cells is correlated with the high rate of follicle atresia that is known to occur at this stage of development in vivo. Recombinant human FSH (100 mIU) and transforming growth factor-alpha (3.3 nM) partially suppressed apoptosis in prehierarchal follicle granulosa cells after 6 h of incubation (by 46-57%; P < 0.05 vs. control), as did the cAMP analog, 8-Br-cAMP (1 mM; by 59%; P < 0.05). A single form of the bcl-2-related gene, bcl-x, was detected in hen ovarian tissues; this transcript corresponded to bcl-xlong, the death-suppressing form of bcl-x. The highest levels of bcl-xlong messenger RNA were found in granulosa tissue from preovulatory follicles, with significantly lower levels detected in prehierarchal follicle granulosa tissue (P < 0.05). Elevated expression of bcl-xlong in preovulatory follicles was correlated to increased resistance to the process of apoptosis, in vitro, and the virtual absence of follicle atresia at this stage of development, in vivo. We conclude that there is a direct relationship between the inherent susceptibility of avian granulosa cells to apoptosis and the high rate of follicle atresia in follicles not yet selected into the preovulatory hierarchy. Moreover, our results are consistent with the proposal that the expression of death-suppressing genes, including bcl-xlong, is capable of rendering cells resistant to the process of apoptosis. The findings reported herein provide the foundation for a novel model with which to further elucidate molecular mechanisms related not only to the initiation of follicle atresia, but also events associated with the process of follicle selection.

Parathyroid hormone-induced resorption in fetal rat limb bones is associated with production of the metalloproteinases collagenase and gelatinase B.

The role of matrix metalloproteinases in parathyroid hormone (PTH)-induced bone resorption was assayed using a fetal rat limb bone culture system. Cotreatment of bones with PTH and recombinant inhibitor of metalloproteinases, TIMP-1, in vitro, inhibited the PTH-stimulated 45Ca release from the limb bones without affecting beta-glucuronidase release. TIMP-1 was fully effective when added during only the final 24 h of a 72 h culture with PTH but was ineffective when added for only the first 24 h of the 72 h culture. In contrast, calcitonin (CT) was effective when added for either the first 24 or the final 24 h of the culture. Using in situ hybridization, the mRNA for collagenase was detected in mononuclear cells of cultured bone. Treatment of the bones with PTH resulted in an increase in the number of cells producing collagenase mRNA, some of which had osteoclastic morphology, PTH also caused a dramatic induction of the mRNA for the 92-kD gelatinase B metalloproteinase in both mononuclear and osteoclastic cells. There was no detectable mRNA for the metalloproteinases stromelysin-1, stromelysin-2, or matrilysin in PTH-treated or control cultures. These results suggest that PTH-induced bone resorption is mediated, at least in part, by the induction of collagenase and gelatinase B mRNA in bone cells.

Matrix metalloproteinases are expressed during ductal and alveolar mammary morphogenesis, and misregulation of stromelysin-1 in transgenic mice induces unscheduled alveolar development.

The matrix-degrading metalloproteinases stromelysin-1, stromelysin-3, and gelatinase A are expressed during ductal branching morphogenesis of the murine mammary gland. Stromelysin-1 expression in particular correlates with ductal elongation, and in situ hybridization and three-dimensional reconstruction studies revealed that stromelysin-1 mRNA was concentrated in stromal fibroblasts along the length of advancing ducts. Transgenic mice expressing an activated form of stromelysin-1 under the control of the MMTV promoter/enhancer exhibited inappropriate alveolar development in virgin females. Ultrastructural analysis demonstrated that the basement membrane underlying epithelial and myoepithelial cells was amorphous and discontinuous compared with the highly ordered basal lamina in control mammary glands. Transgenic mammary glands had at least a twofold increase in the number of cells/unit area and a 1.4-fold increase in the percent of cycling cells by 13 wk of age compared with nontransgenic littermates. In addition, transgenic glands expressed beta-casein mRNA, but not protein, and resembled the proliferative and differentiated state of an animal between 8 and 10 days pregnant. An analysis of metalloproteinase expression in the glands of normal pregnant females demonstrated that the same matrix metalloproteinase family members, including stromelysin-1, were expressed in connective tissue cells surrounding epithelial clusters during the time of lobuloalveolar development. These results suggest that metalloproteinases may assist in remodeling ECM during normal ductal and alveolar branching morphogenesis, and that disruption of the basement membrane by an activated metalloproteinase can affect basic cellular processes of proliferation and differentiation.

Decreased tumor formation in 7,12-dimethylbenzanthracene-treated stromelysin-1 transgenic mice is associated with alterations in mammary epithelial cell apoptosis.

To determine the role of a specific member of the metalloproteinase family, stromelysin-1, in mammary carcinogenesis and tumor progression, transgenic mice expressing activated rat stromelysin-1 under the control of the mouse mammary tumor virus promoter/enhancer were treated with the carcinogen 7,12-dimethylbenzanthracene (DMBA) to induce mammary tumors. Surprisingly, the expression of stromelysin-1 during the time of DMBA treatment reduced the number of mice developing mammary tumors, in particular adenoacanthomas, from 65 to 32% (P = 0.02). In contrast, when transgenic mice expressing both transforming growth factor alpha and stromelysin-1 under the control of the mouse mammary tumor virus long terminal repeat were treated with DMBA, there was no significant difference in the number of mice that developed tumors compared to transforming growth factor alpha controls. A 4-fold increase in the number of apoptotic cells was detected in stromelysin-1 transgenic mice compared to littermate controls at the time of DMBA administration, suggesting that the reduction in DMBA-induced tumorigenicity is likely to be due, at least in part, to an increased rate of cell turnover in stromelysin-1 transgenic mice. When malignant adenocarcinomas developed in the stromelysin-expressing mice, there was no detectable alteration in the extent of invasion or in the metastatic potential of these tumors compared to tumors from control mice. These results suggest that the expression of a single metalloproteinase, stromelysin-1, is insufficient for the progression of mammary adenocarcinomas to an invasive and metastatic phenotype, but that matrix degradation by metalloproteinases can alter basic processes of cell proliferation and apoptosis.

Modulation of matrilysin levels in colon carcinoma cell lines affects tumorigenicity in vivo.

The expression of the metalloproteinase matrilysin in the human colon carcinoma cell lines SW480 and SW620 correlates with the ability of the SW620 cells to invade an artificial basement membrane in vitro and metastasize to the liver following injection into the cecum of nude mice in vivo. Transfection of either wild-type or activated forms of matrilysin into the SW480 cells, which do not express endogenous matrilysin, did not reproducibly increase in vitro invasion but increased the tumorigenicity of the cells when injected into the cecum of nude mice. Antisense reduction of matrilysin levels decreased the tumorigenicity of the SW620 cells and subsequent metastasis to the liver. These results suggest that matrilysin gene expression by colon adenocarcinoma cells is not sufficient for tumor invasion and metastasis but contributes to the tumorigenicity and progression of colorectal tumors.

Expression and localization of matrix-degrading metalloproteinases during colorectal tumorigenesis.

The metalloproteinase matrilysin is widely expressed in the epithelial tumor cells of malignant colorectal adenocarcinomas. Approximately 50% of benign adenomas also express low levels of matrilysin that is focally localized. The expression of stromelysin-1, stromelysin-3, and gelatinase A was observed in the stromal component of several carcinomas and was not present in adenomatous tissue. The expression of interstitial collagenase and gelatinase B was observed in occasional adenomas and carcinomas. Stromelysin-2 transcripts were not detectable in any of the samples examined. Tissue inhibitor of metalloproteinase-1 gene expression was widespread and was observed in both epithelial and stromal cells of adenomas and carcinomas. These results indicate that matrilysin gene expression is an early event in colorectal tumorigenesis and that the expression of stromelysin-1, stromelysin-3, and gelatinase A is primarily a late event. The observed gene expression patterns suggest that matrilysin may participate in early events in tumor progression and that multiple members of the metalloproteinase family may work in concert to facilitate late-stage tumor invasion and metastasis.

Matrix-degrading metalloproteinases in tumor progression.

The matrix-degrading metalloproteinases (MMPs) have been implicated in tumor invasion and metastasis. Recently it has become clear that the expression of MMPs in tumors is frequently localized to stromal cells surrounding malignant tumor cells. In the mouse skin model of multi-stage carcinogenesis, the MMP stromelysin is expressed in stromal fibroblast-like cells surrounding benign and malignant squamous cell carcinomas. Conversion of these tumors to highly invasive and metastatic spindle-cell tumors is however, associated with the expression of stromelysin-1 mRNA in the tumor cells themselves. The analysis of MMPs in human colon adenocarcinomas at different stages of tumor progression revealed that matrilysin was the only MMP expressed in the tumor cells, while stromelysin-1 and stromelysin-3 mRNA was detected in stromal cells surrounding malignant tumor cells. Matrilysin mRNA is detected in benign tumors as well as malignant tumor cells, and the relative level and percent of tumors expressing matrilysin correlates with the stage of tumor progression. These results suggest that both stromal and tumor cell metalloproteinases may contribute to tumor invasion and metastasis, and also suggests that MMPs may play a role in earlier events in the tumor progression pathway. A potential role for MMPs in tumor growth is illustrated by results which suggest that the expression of matrilysin in human colon cancer-derived cells increases tumorigenicity following injection into the cecum, and that transgenic mice expressing matrilysin mRNA show a marked proliferative response. MMPs may therefore play multiple roles in tumor progression.

An approach to consumer feedback in an out-patient specialty service.

Details an approach to consumer feedback in an out-patient specialty service. Outlines the approach to undertaking a consumer survey and how feedback from people using the Genito-Urinary Medicine services can be obtained. The aim of the survey was to assess the level of individual/personal care. Gives the objectives and methodology, main findings and recommendations, together with a cost benefit analysis of the work.

Reversible o(2) inhibition of nitrogenase activity in attached soybean nodules.

Various forms of stress result in decreased O(2) permeability or decreased capacity to consume O(2) in legume root nodules. These changes alter the nodule interior O(2) concentration (O(i)). To determine the relationship between O(i) and nitrogenase activity in attached soybean (Glycine max) nodules, we controlled O(i) by varying external pO(2) while monitoring internal H(2) concentration (H(i)) with microelectrodes. O(i) was monitored by noninvasive leghemoglobin spectrophotometry (nodule oximetry). After each step-change in O(i), H(i) approached a new steady state, with a time constant averaging 23 s. The rate of H(2) production by nitrogenase was calculated as the product of H(i), nodule surface area, and nodule H(2) permeability. H(2) permeability was estimated from O(2) permeability (measured by nodule oximetry) by assuming diffusion through air-filled pores; support for this assumption is presented. O(i) was nearly optimal for nitrogenase activity (H(2) production) between 15 and 150 nm. A 1- to 2-min exposure to elevated external pO(2) (40-100 kPa) reduced H(i) to zero, but nitrogenase activity recovered quickly under air, often in <20 min. This rapid recovery contrasts with previous reports of much slower recovery with longer exposures to elevated pO(2). The mechanism of nitrogenase inhibition may differ between brief and prolonged O(2) exposures.

Pulmonary edema induced by betamimetic drugs.

We present six cases of pulmonary edema related to betamimetic (beta sympathomimetic amine) agents (three to ritodrine and three to terbutaline). The hemodynamic data from three patients suggest that this may be a form of noncardiogenic pulmonary edema. A seventh case, resulting in a maternal death, is included because of the association with ritodrine therapy and the instructive complications of invasive monitoring. We also propose an outline for the management of betamimetic-drug-induced pulmonary edema.

Irradiation of liver metastases due to gestational choriocarcinoma.

One of the major unresolved problems in high-risk choriocarcinoma is the prevention of hemorrhage from tumor necrosis during chemotherapy. The authors report a successfully treated patient who developed intrahepatic bleeding while receiving systemic chemotherapy and concomitant whole liver irradiation. Patients with trophoblastic liver metastases are at risk for bleeding from tumor necrosis during chemotherapy but, because there are few reported cases, the degree of risk cannot be predicted. Radiation therapy for the prevention of this complication may not be as successful for liver metastases as it is for brain metastases; reports are too scarce to evaluate its efficacy. As demonstrated in the present patient, radiation therapy does not completely eliminate the risk of hemorrhage from chemotherapy. The hypothesis that patients with liver metastases from gestational choriocarcinoma are more susceptible to treatment failure and life-threatening complications than other members of the high-risk category needs support. Other investigators are encouraged to report their results with trophoblastic liver metastases to clarify the type and degree of risk, to determine the role of adjuvant radiation therapy, and to establish optimum treatment protocols.