Premature Entry Research Articles

Characterization of B-type cyclins in the smut fungus Ustilago maydis: roles in morphogenesis and pathogenicity.

Pathogenesis, morphogenesis and cell cycle are connected in the fungal pathogen Ustilago maydis. Here we report the characterization of the catalytic subunit of the cyclin-dependent kinase, encoded by the gene cdk1, and the two B-type cyclins present in this organism, encoded by the genes clb1 and clb2. These cyclins are not redundant and appears to be essential for cell cycle. The analysis of conditional mutants in cyclin genes indicates that Clb1 is required for G1 to S and G2 to M transitions, while Clb2 is specifically required for the onset of mitosis. Both Clb1 and Clb2 carry functional destruction boxes, and expression of derivatives lacking D-boxes arrested cell cycle at a post-replicative stage. High levels of Clb1 generated cells with anomalous DNA content that were hypersensitive to microtubule-destabilizing drugs. In contrast, high levels of Clb2 induce premature entry into mitosis, suggesting that Clb2 is a mitotic inducer in U. maydis. In addition, Clb2 affects morphogenesis, and overexpression of clb2 induces filamentous growth. Furthermore, we have found that appropriate levels of Clb2 cyclin are critical for a successful infection. Mutant strains with half a dose of clb2 or high level of clb2 expression are impaired at distinct stages in the infection process. These data reinforces the connections between cell cycle, morphogenesis and virulence in this smut fungus.

Chk1 kinase negatively regulates mitotic function of Cdc25A phosphatase through 14-3-3 binding.

The order and fidelity of cell cycle events in mammals is intimately linked to the integrity of the Chk1 kinase-Cdc25A phosphatase pathway. Chk1 phosphorylation targets Cdc25A for destruction and, as shown here, inhibits interactions between Cdc25A and its mitotic substrate cyclin B1-Cdk1. Phosphorylation of Cdc25A on serine 178 and threonine 507 facilitates 14-3-3 binding, and Chk1 phosphorylates both residues in vitro. Mutation of T507 to alanine (T507A) enhanced the biological activity of Cdc25A. Cdc25A(T507A) was more efficient in binding to cyclin B1, activating cyclin B1-Cdk1, and promoting premature entry into mitosis. We propose that the Chk1/Cdc25A/14-3-3 pathway functions to prevent cells from entering into mitosis prior to replicating their genomes to ensure the fidelity of the cell division process.

572 Kahalalide F (KF), a new marine compound, in vitro radiosensitizes human tumoral cell lines

Chk1 inhibition increases cell sensitivity to both chemotherapy and radiotherapy in several tumour types and is, therefore, a promising anti-cancer approach. Although several Chk1 inhibitors have been developed, their clinical progress has been hampered by low bioavailability and off-target toxicities.We characterized the radiosensitizing activity of CCT244747, the first orally bioavailable Chk1 inhibitor. We used a panel of bladder and head and neck cancer cell lines and monitored the effect of combining CCT244747 with radiation both in in vitro and in vivo models.CCT244747 sensitized cancer cell lines to radiation in vitro and resulted in a growth delay in cancer xenograft models associated with a survival benefit. Radiosensitization was elicited by abrogation of the radiation-induced G2 arrest and premature entry into mitosis.CCT244747 is a potent and specific Chk1 inhibitor that can be administered orally. It radiosensitizes tumour cell lines and represents a new therapy for clinical application in combination with radiotherapy.

Hair-Cycle-Dependent Expression of Parathyroid Hormone-Related Protein and its Type I Receptor: Evidence for Regulation at the Anagen to Catagen Transition

The humoral hypercalcemia factor parathyroid hormone-related protein is a paracrine-signaling molecule that regulates the development of several organ systems, including the skin. In pathologic circumstances such as hypercalcemia and in development, parathyroid hormone-related protein signaling appears to be mediated by the type I parathyroid hormone/parathyroid hormone-related protein receptor. In order to clarify the role of the ligand and receptor pair in cutaneous biology, gene expression was monitored in a series of murine skin samples ranging from embryonic day 14 to 2 y with in situ hybridization and RNase protection. In all samples, high levels of parathyroid hormone-related protein transcripts were exclusively expressed in the developing and adult hair follicle but were not observed in the interfollicular epidermis. In the adult, parathyroid hormone-related protein mRNA expression was dynamically regulated as a function of the murine hair cycle in a way similar to other signaling molecules that regulate the anagen to catagen transition. PTH receptor transcripts were abundantly expressed in the developing dermis. In the adult skin, PTH receptor mRNA was markedly reduced, but again demonstrated hair-cycle-dependent expression. The dorsal skin of the keratin 14-parathyroid hormone-related protein mouse was used to evaluate the impact of overexpression of the peptide on the murine hair cycle. All types of hair were 30-40% shorter in adult keratin 14-parathyroid hormone-related protein mice as compared with wild-type littermates. This appeared to result from a premature entry into the catagen phase of the hair cycle. Finally, the relationship between parathyroid hormone-related protein signaling and other growth factors that regulate the hair cycle was examined by cross-breeding experiments employing keratin 14-parathyroid hormone-related protein mice and fibroblast growth factor-5-knockout mice. It appears that parathyroid hormone-related protein and fibroblast growth factor-5 regulate the anagen to catagen transition by independent pathways.

The attractive Achilles heel of germ cell tumours: an inherent sensitivity to apoptosis-inducing stimuli.

Testicular germ cell tumours (TGCTs) are extremely sensitive to cisplatin-containing chemotherapy. The rapid time course of apoptosis induction after exposure to cisplatin suggests that TGCT cells are primed to undergo programmed cell death as an inherent property of the cell of origin. In fact, apoptosis induction of germ cells in the testis is an important physiological mechanism to control the quality and quantity of the gametes produced. Although p53 protein is highly expressed in the majority of TGCTs, almost no p53 mutations have been detected. Interestingly, p53 overexpression is associated with loss of p21 and gain of mdm2 expression, which might indicate a partial loss in functionality of the p53 regulatory pathway in TGCTs. Besides p21, TGCTs often show low expression of other proteins involved in the regulation of cell cycle progression, such as the retinoblastoma protein and members of the INK4 family. It can be postulated that the deregulated G(1)-S phase checkpoint results in premature entry into the S phase upon DNA damage. In addition to Bcl-2 family members that are involved in the regulation of germ cell apoptosis in the normal testis via the mitochondrial death pathway, the Fas death pathway is also known to regulate apoptosis of germ cells in the testis. Since chemotherapy has been shown to activate the Fas death pathway and TGCTs co-express both Fas and its ligand FasL, TGCT cells might undergo apoptosis upon cisplatin treatment via autocrine or paracrine activation of the Fas system by FasL. The hypothesis suggested here is that the lack of cell cycle arrest following a cisplatin-containing treatment, together with the activation of the Fas death pathway and the mitochondrial death pathway, explains the rapid and efficient apoptosis of TGCT cells. Defining the mechanisms involved in the cisplatin sensitivity of TGCTs will provide tools to increase cisplatin sensitivity in other human tumours with acquired or intrinsic resistance.

Conservation of Mechanisms Controlling Entry into Mitosis: Budding Yeast Wee1 Delays Entry into Mitosis and Is Required for Cell Size Control

Background: In fission yeast, the Wee1 kinase delays entry into mitosis until a critical cell size has been reached; however, a similar role for Wee1-related kinases has not been reported in other organisms. SWE1, the budding yeast homolog of wee1, is thought to function in a morphogenesis checkpoint that delays entry into mitosis in response to defects in bud morphogenesis.Results: In contrast to previous studies, we found that budding yeast swe1Δ cells undergo premature entry into mitosis, leading to birth of abnormally small cells. Additional experiments suggest that conditions that activate the morphogenesis checkpoint may actually be activating a G2/M cell size checkpoint. For example, actin depolymerization is thought to activate the morphogenesis checkpoint by inhibiting bud morphogenesis. However, actin depolymerization also inhibits bud growth, suggesting that it could activate a cell size checkpoint. Consistent with this possibility, we found that actin depolymerization fails to induce a G2/M delay once daughter buds pass a critical size. Other conditions that activate the morphogenesis checkpoint block bud formation, which could also activate a size checkpoint if cell size at G2/M is monitored in the daughter bud. Previous work reported that Swe1 is degraded during G2, which was proposed to account for failure of large-budded cells to arrest in response to actin depolymerization. However, we found that Swe1 is present throughout G2 and undergoes hyperphosphorylation as cells enter mitosis, as found in other organisms.Conclusions: Our results suggest that the mechanisms known to coordinate entry into mitosis in other organisms have been conserved in budding yeast.

Staphylococcus aureus aconitase inactivation unexpectedly inhibits post-exponential-phase growth and enhances stationary-phase survival.

Staphylococcus aureus preferentially catabolizes glucose, generating pyruvate, which is subsequently oxidized to acetate under aerobic growth conditions. Catabolite repression of the tricarboxylic acid (TCA) cycle results in the accumulation of acetate. TCA cycle derepression coincides with exit from the exponential growth phase, the onset of acetate catabolism, and the maximal expression of secreted virulence factors. These data suggest that carbon and energy for post-exponential-phase growth and virulence factor production are derived from the catabolism of acetate mediated by the TCA cycle. To test this hypothesis, the aconitase gene was genetically inactivated in a human isolate of S. aureus, and the effects on physiology, morphology, virulence factor production, virulence for mice, and stationary-phase survival were examined. TCA cycle inactivation prevented the post-exponential growth phase catabolism of acetate, resulting in premature entry into the stationary phase. This phenotype was accompanied by a significant reduction in the production of several virulence factors and alteration in host-pathogen interaction. Unexpectedly, aconitase inactivation enhanced stationary-phase survival relative to the wild-type strain. Aconitase is an iron-sulfur cluster-containing enzyme that is highly susceptible to oxidative inactivation. We speculate that reversible loss of the iron-sulfur cluster in wild-type organisms is a survival strategy used to circumvent oxidative stress induced during host-pathogen interactions. Taken together, these data demonstrate the importance of the TCA cycle in the life cycle of this medically important pathogen.

The Lysosomal Protease Cathepsin L Is an Important Regulator of Keratinocyte and Melanocyte Differentiation During Hair Follicle Morphogenesis and Cycling

We have previously shown that the ubiquitously expressed lysosomal cysteine protease, cathepsin L (CTSL), is essential for skin and hair follicle homeostasis. Here we examine the effect of CTSL deficiency on hair follicle development and cycling in ctsl(-/-) mice by light and electron microscopy, Ki67/terminal dUTP nick-end labeling, and trichohyalin immunofluorescence. Hair follicle morphogenesis in ctsl(-/-) mice was associated with several abnormalities. Defective terminal differentiation of keratinocytes occurred during the formation of the hair canal, resulting in disruption of hair shaft outgrowth. Both proliferation and apoptosis levels in keratinocytes and melanocytes were higher in ctsl(-/-) than in ctsl(+/+) hair follicles. The development of the hair follicle pigmentary unit was disrupted by vacuolation of differentiating melanocytes. Hair cycling was also abnormal in ctsl(-/-) mice. Final stages of hair follicle morphogenesis and the induction of hair follicle cycling were retarded. Thereafter, these follicles exhibited a truncated resting phase (telogen) and a premature entry into the first growth phase. Further abnormalities of telogen development included the defective anchoring of club hairs in the skin, which resulted in their abnormal shedding. Melanocyte vacuolation was again apparent during the hair cycle-associated reconstruction of the hair pigmentary unit. A hallmark of these ctsl(-/-) mice was the severe disruption in the exiting of hair shafts to the skin surface. This was mostly because of a failure of the inner root sheath (keratinocyte layer next to the hair shaft) to fully desquamate. These changes resulted in a massive dilation of the hair canal and the abnormal routing of sebaceous gland products to the skin surface. In summary, this study suggests novel roles for cathepsin proteases in skin, hair, and pigment biology. Principal target tissues that may contain protein substrate(s) for this cysteine protease include the developing hair cone, inner root sheath, anchoring apparatus of the telogen club, and organelles of lysosomal origin (eg, melanosomes).

Ephrin-as cooperate with EphA4 to promote trunk neural crest migration.

Trunk neural crest cells delaminate from the dorsal neural tube and migrate on two distinct pathways: a dorsolateral route, between the ectoderm and somites,and a ventromedial route, through the somitic mesoderm. Neural crest cells that migrate ventromedially travel in a segmental manner through rostral half-somites, avoiding caudal halves. Recent studies demonstrate that various molecular cues guide the migration of neural crest cells, primarily by serving as inhibitors to premature pathway entry orby preventing neural crest from entering inappropriate territories. Trajectories of migrating trunk neural crest are well organized and generally linear in nature, suggesting that positive, migration-promoting factors may be responsible for this organized cell behavior. However, the identity of these factors and their function are not well understood. Here we examine the expression of members of the EphA subclass of receptor tyrosine kinases and ephrins using RT-PCR and immunocytochemistry. Neural crest cells express ephrins and EphA4 at distinct stages during their migration. In functional analyses, addition of ephrin-A2-, ephrin-A5-, and EphA4-Fc disrupted the segmental organization of trunk neural crest migration in explants: neural crest cells entered rostral and caudal halves of somites. Finally, to test the specific effects of these factors on cell behavior, neural crest cells were exposed in vitro to substrate-bound EphA and ephrin-As. Surprisingly, neural crest cells avoided ephrin-A2 or ephrin-A5 substrates; this avoidance was abolished by the addition of EphA4. Together, these data suggest that ephrin-As and EphA4 cooperate to positively promote the migration of neural crest cells through rostral half somites in vivo.

Premature Chromosome Condensation in Humans Associated with Microcephaly and Mental Retardation: A Novel Autosomal Recessive Condition

We report a novel autosomal recessive disorder characterized by premature chromosome condensation in the early G2 phase. It was observed in two siblings, from consanguineous parents, affected with microcephaly, growth retardation, and severe mental retardation. Chromosome analysis showed a high frequency of prophase-like cells (>10%) in lymphocytes, fibroblasts, and lymphoblast cell lines with an otherwise normal karyotype. (3)H-thymidine-pulse labeling and autoradiography showed that, 2 h after the pulse, 28%-35% of the prophases were labeled, compared with 9%-11% in healthy control subjects, indicating that the phenomenon is due to premature chromosome condensation. Flow cytometry studies demonstrate that the entire cell cycle is not prolonged, compared with that in healthy control subjects, and compartment sizes did not differ from those in healthy control subjects. No increased reaction of the cells to X-irradiation or treatments with the clastogens bleomycin and mitomycin C was observed, in contrast to results in the cell-cycle mutants ataxia telangiectasia and Fanconi anemia. The rates of sister chromatid exchanges and the mitotic nondisjunction rates were inconspicuous. Premature entry of cells into mitosis suggests that a gene involved in cell-cycle regulation is mutated in these siblings.

Rapid cold hardening in Antarctic microarthropods

Summary Rapid cold hardening was examined in three common Antarctic microarthropods using differential scanning calorimetry over timescales between 3 and 30 h, under field and controlled laboratory conditions. In fresh field samples and cultures of the springtail, Cryptopygus antarcticus (Willem), and cultures of the mites, Alaskozetes antarcticus (Michael) and Halozetes belgicae (Michael), maintained under summer field‐simulating conditions, supercooling point (SCP) distributions tracked microhabitat temperature variation over the observation period. Controlled acclimation of samples of summer‐acclimatized C. antarcticus caused significant cold hardening after 12 h at temperatures around 0 °C (+3 to −2 °C). No response was obtained at higher or lower temperatures, or in field‐fresh winter‐acclimatized animals. The latter did not lose cold hardiness when held at positive temperatures for 12 h. Gradual cooling of C. antarcticus over 20 h from +5 to −5 °C caused a considerable increase in cold tolerance. Rewarming partially but non‐significantly reversed this effect. The greatest response occurred between +3 and +1 °C. Maximum faecal pellet production also occurred in this interval, but gut clearance alone was not sufficient to explain observed cold hardening. It is hypothesized that these species possess a hitherto unrecognized capacity to alter cold hardiness in summer in response to environmental temperature cues over a shorter timescale than previously thought, by a mechanism that relies on neither gut clearance nor concentration of body fluids via water loss. This ability may reduce the developmental costs of premature entry into an inactive, cold‐hardy state.

P53 Involvement in the Control of Murine Hair Follicle Regression

p53 is a transcription factor mediating a variety of biological responses including apoptotic cell death. p53 was recently shown to control apoptosis in the hair follicle induced by ionizing radiation and chemotherapy, but its role in the apoptosis-driven physiological hair follicle regression (catagen) remains to be elucidated. Here, we show that p53 protein is strongly expressed and co-localized with apoptotic markers in the regressing hair follicle compartments during catagen. In contrast to wild-type mice, p53 knockout mice show significant retardation of catagen accompanied by significant decrease in the number of apoptotic cells in the hair matrix. Furthermore, p53 null hair follicles are characterized by alterations in the expression of markers that are encoded by p53 target genes and are implicated in the control of catagen (Bax, Bcl-2, insulin-like growth factor binding protein-3). These data suggest that p53 is involved in the control of apoptosis in the hair follicle during physiological regression and imply that p53 antagonists may be useful for the management of hair growth disorders characterized by premature entry into catagen, such as androgenetic alopecia, alopecia areata, and telogen effluvium.

Genetic ablation of orexin neurons in mice results in narcolepsy, hypophagia, and obesity.

Orexins (hypocretins) are a pair of neuropeptides implicated in energy homeostasis and arousal. Recent reports suggest that loss of orexin-containing neurons occurs in human patients with narcolepsy. We generated transgenic mice in which orexin-containing neurons are ablated by orexinergic-specific expression of a truncated Machado-Joseph disease gene product (ataxin-3) with an expanded polyglutamine stretch. These mice showed a phenotype strikingly similar to human narcolepsy, including behavioral arrests, premature entry into rapid eye movement (REM) sleep, poorly consolidated sleep patterns, and a late-onset obesity, despite eating less than nontransgenic littermates. These results provide evidence that orexin-containing neurons play important roles in regulating vigilance states and energy homeostasis. Orexin/ataxin-3 mice provide a valuable model for studying the pathophysiology and treatment of narcolepsy.

Functional Conservation of Phosphorylation-specific Prolyl Isomerases in Plants

The phosphorylation-specific peptidyl prolyl cis/trans isomerase (PPIase) Pin1 in humans and its homologues in yeast and animal species play an important role in cell cycle regulation. These PPIases consist of an NH(2)-terminal WW domain that binds to specific phosphoserine- or phosphothreonine-proline motifs present in a subset of phosphoproteins and a COOH-terminal PPIase domain that specifically isomerizes the phosphorylated serine/threonine-proline peptide bonds. Here, we describe the isolation of MdPin1, a Pin1 homologue from the plant species apple (Malus domestica) and show that it has the same phosphorylation-specific substrate specificity and can be inhibited by juglone in vitro, as is the case for Pin1. A search in the plant expressed sequence tag data bases reveals that the Pin1-type PPIases are present in various plants, and there are multiple genes in one organism, such as soybean (Glycine max) and tomato (Lycopersicon esculentum). Furthermore, all these plant Pin1-type PPIases, including AtPin1 in Arabidopsis thaliana, do not have a WW domain, but all contain a four-amino acid insertion next to the phospho-specific recognition site of the active site. Interestingly, like Pin1, both MdPin1 and AtPin1 are able to rescue the lethal mitotic phenotype of a temperature-sensitive mutation in the Pin1 homologue ESS1/PTF1 gene in Saccharomyces cerevisiae. However, deleting the extra four amino acid residues abolished the ability of AtPin1 to rescue the yeast mutation under non-overexpression conditions, indicating that these extra amino acids may be important for mediating the substrate interaction of plant enzymes. Finally, expression of MdPin1 is tightly associated with cell division both during apple fruit development in vivo and during cell cultures in vitro. These results have demonstrated that phosphorylation-specific PPIases are highly conserved functionally in yeast, animal, and plant species. Furthermore, the experiments suggest that although plant Pin1-type enzymes do not have a WW domain, they may fulfill the same functions as Pin1 and its homologues do in other organisms.

EFFECTS OF FIRM PERFORMANCE, ORGANIZATIONAL SLACK, AND DEBT ON ENTRY TIMING: A STUDY OF TEN EMERGING PRODUCT MARKETS IN USA

As technologies change and market environments evolve, firms introduce new products to create or satisfy new market demands or to substitute for existing products. A key strategic decision for firms contemplating entry into emerging product markets is the timing of their entry. Firms may choose to enter earlier and hope to preempt critical strategic resources, but they take the risk of premature entry. On the other hand, firms may prefer to enter later and wait for technical and market uncertainties to subside, but they face the potential problem of being frozen out of a profitable market by participants who risked earlier entry (Lieberman and Montgomery 1988; Mitchell 1989, 1991; Lilien and Yoon 1990; Robinson et al . 1992). This study examines three organizational factors that are hypothesized to affect a firm's strategic decision regarding timing of entry into an emerging product market: performance, organizational slack, and debt position. We begin with an overview of entry-timing research followed by some background on research in performance, organizational slack, and debt. We then develop hypotheses concerned with these three organizational factors and entry timing. We test these hypotheses in a multiindustry sample of firms in USA, and control for the influence of industry and firm variables that could be expected to affect entry-timing decisions.

A role for p75 neurotrophin receptor in the control of apoptosis-driven hair follicle regression.

To examine the mechanisms that underlie the neurotrophin-induced, apoptosis-driven hair follicle involution (catagen), the expression and function of p75 neurotrophin receptor (p75NTR), which is implicated in apoptosis control, were studied during spontaneous catagen development in murine skin. By RT-PCR, high steady-state p75NTR mRNA skin levels were found during the anagen-catagen transition of the hair follicle. By immunohistochemistry, p75NTR alone was strongly expressed in TUNEL+/Bcl2- keratinocytes of the regressing outer root sheath, but both p75NTR and TrkB and/or TrkC were expressed by the nonregressing TUNEL-/Bcl2+ secondary hair germ keratinocytes. To determine whether p75NTR is functionally involved in catagen control, spontaneous catagen development was compared in vivo between p75NTR knockout (-/-) and wild-type mice. There was significant catagen retardation in p75NTR knockout mice as compared to wild-type controls (P<0.05). Instead, transgenic mice-overexpressing NGF (promoter: K14) showed substantial acceleration of catagen (P<0.001). Although NGF, brain-derived neurotrophic factor (BDNF), and neurotrophin 3 (NT-3) accelerated catagen in the organ-cultured skin of C57BL/6 mice, these neurotrophins failed to promote catagen development in the organ-cultured p75NTR null skin. These findings suggest that p75NTR signaling is involved in the control of kerotinocyte apoptosis during catagen and that pharmacological manipulation of p75NTR signaling may prove useful for the treatment of hair disorders that display premature entry into catagen.

Inhibiting Phosphodiesterase Activity to Maintain Germinal Vesicle Stage Arrest Inhibits DNA Strand Breaks in Germinal Vesicle Stage Mouse Oocytes

Objective: Oocyte maturation in vitro offers many advantages to women undergoing ART, but the success rate remains low. One possibility is premature entry into meiosis in culture. Inhibitors of phosphodiesterase block meiosis in mouse oocytes, presumably by elevating cAMP and activating protein kinase A. In contrast, inhibitors of DNA topoisomerase II (TOPO II), required for the DNA strand breaks and repair necessary for chromosome condensation and separation, lead to germinal vesicle (GV) breakdown and extrusion of the entire chromosomal complement (“chemical enucleation”) of mouse oocytes. These opposing effects imply that maintaining the GV may stabilize TOPO II activity. We have tested this possibility. Design: Mouse oocytes were cultured without and with the phosphodiesterase inhibitor, isobutylmethylxanthine (IBMX) to maintain GV arrest and examined for DNA strand breaks as evidence of TOPO II activity. Some experiments included the antibiotic, distamycin, which stimulates TOPO II activity by exposing AT-rich regions of DNA. Oocytes from postpubertal females either not stimulated or stimulated with gonadotropin were examined. Materials and Methods: Oocytes were fixed and subjected to a sensitive terminal-uridine-nucletide-end-labeling (TUNEL) assay to detect DNA strand breaks. Two assay conditions were employed, without and with exposure to 2N HCL to remove chromatin histones. The incorporation of biotinylated uridine was detected by streptavidin-bound horseradish peroxidase reacting with the azo-dye, AEC. The oocytes were mounted under coverslips and counterstained with Hoechst to visualize DNA. Results: Forty two percent of GV eggs not pre-treated with HCl from both classes of postpubertal females were TUNEL assay positive at the time of collection from ovarian follicles. The frequency increased to 90% if the oocytes were pre-treated with HCl, implying histone masking of some of the DNA strand breaks. The TUNEL assay staining corresponded to Hoechst staining patterns, implying involvement of all regions of the chromatin. In contrast, following 24 hours of culture in IBMX, the frequency of TUNEL assay positive GV stage eggs decreased to 15 to 17% without or with pre-treatment with HCl. Co-culture with distamycin resulted in a return to 88% TUNEL-positive germinal vesicles. Overnight culture without IBMX and/or following transfer to IBMX-free conditions resulted in the resumption of meiosis and TUNEL-positive metaphase chromosomes. Conclusions: DNA strand breaks are common in the chromatin of mouse oocytes. IBMX exposure eliminates the strand breaks, apparently by inhibiting the cleavage, but not the repair activity of TOPO II. The inhibition is reversible. This novel finding supports the efficacy of extending oocyte culture in the presence of IBMX. Supported by the Assisted Reproduction Foundation and the Urologic Research Fund.

Adenovirus-mediated E2F-1 gene transfer induces an apoptotic response in human gastric carcinoma cells that is enhanced by cyclin dependent kinase inhibitors.

E2F -1 is a transcription factor that regulates cell cycle progression into S-phase. Deregulation of E2F-1 activity has been associated with cellular commitment to apoptosis. Also critical in the regulation of S-phase are the actions of the cyclin dependent kinases, Cdk2 and cdc2. Inhibition of these cyclin dependent kinases has been similarly associated with disrupting orderly S-phase progression and causing subsequent apoptosis in certain cancer cells. In this study, we examine the ability of adenovirus-mediated E2F-1 overexpression to induce apoptosis in human gastric carcinoma cells. Furthermore, we investigate the effect of the cyclin dependent kinase inhibitors, olomoucine and roscovitine, on E2F-1-mediated apoptosis in human gastric carcinoma cells. AGS and SNU-1 gastric adenocarcinoma cells were infected with adenoviral vectors expressing E2F-1 (Ad5CMVE2F-1) or control viruses expressing beta-galactosidase (Ad5CMVLacZ) or lacking a transgene (Ad5). Gastric adenocarcinoma cells were then independently treated with roscovitine or olomoucine. Finally, gastric adenocarcinoma cells were infected with the various adenoviral vectors in combination with roscovitine or olomoucine. E2F-1 overexpression resulted in an 85% reduction in cell viability at 72 h compared to controls. Combining E2F-1 overexpression with roscovitine resulted in >99% reduction in cell viability by 72 h. Overexpression of E2F-1 resulted in premature S-phase entry and G2/M arrest at 24 h, followed by apoptosis by 72 h. Combining E2F-1 overexpression with roscovitine resulted in an earlier G2/M arrest, followed by a more complete, widespread apoptotic response by 24 h. Caspase 3/CPP32 activation and PARP cleavage in response to E2F-1 overexpression, alone and in combination with roscovitine, implicate the caspase cascade in E2F-1-mediated apoptosis of gastric cancer cells. Bax levels also increased in response to E2F-1 gene transfer, alone and in combination with roscovitine. E2F-1 overexpression induces widespread apoptosis in human gastric carcinoma cells. Combining E2F-1 overexpression with cyclin-dependent kinase inhibitors results in an enhanced apoptotic response, causing nearly complete gastric tumor cell death within 72 h. E2F-1 gene therapy in combination with cyclin dependent kinase inhibitors is a potentially active chemogene therapy strategy for the treatment of human gastric cancer.

Phacoemulsification in eyes with white cataract

Purpose To study the results of phacoemulsification in eyes with white cataract. Setting Aravind Eye Hospital, Madurai, India. Methods This retrospective study comprised 212 consecutive patients with white cataract: 192 mature (90.6%), 11 intumescent (5.2%), and 9 hypermature (4.2%). Patients had phacoemulsification and continuous curvilinear capsulorhexis. After the nucleus was removed by the divide and conquer or the phaco chop technique, a posterior chamber intraocular lens was implanted. Preoperative and intraoperative findings, as well as postoperative outcomes, were analyzed. Results Intraoperative complications included premature entry of the tunnel into the anterior chamber in 4 eyes (1.9%), incomplete capsulorhexis in 60 (28.3%), posterior capsular tear in 4 (1.9%), conversion to a manual nonphacoemulsification technique in 4 (1.9%), intraoperative miosis in 7 (3.3%), and iris chafing in 2 (0.9%). Mean phacoemulsification time was 2.03 minutes (range 0 to 5.8 minutes). A best corrected visual acuity of 6/9 or better was attained in 131 patients (61.8%) on the first postoperative day and in 199 patients (93.9%) at 1 month. Postoperative complications included moderate transient striate keratopathy with corneal edema in 12 eyes (5.7%) and iritis in 2 (0.9%). Conclusion Phacoemulsification was a safe and effective technique to remove white mature cataract in eyes in a developing country.

Gdt1, a new signal transduction component for negative regulation of the growth-differentiation transition in Dictyostelium discoideum.

Discoidin I expression was used as a marker to screen for mutants affected in the growth-differentiation transition (GDT) of Dictyostelium. By REMI mutagenesis we have isolated mutant 2-9, an overexpressor of discoidin I. It displays normal morphogenesis but shows premature entry into the developmental cycle. The disrupted gene was denominated gdt1. The mutant phenotype was reconstructed by disruptions in different parts of the gene, suggesting that all had a complete loss of function. gdt1 was expressed in growing cells; the levels of protein and mRNA appear to increase with cell density and rapidly decrease with the onset of development. gdt1 encodes a 175-kDa protein with four putative transmembrane domains. In the C terminus, the derived amino acid sequence displays some similarity to the catalytic domain of protein kinases. Mixing experiments demonstrate that the gdt1(-) phenotype is cell autonomous. Prestarvation factor is secreted at wild-type levels. The response to folate, a negative regulator of discoidin expression, was not impaired in gdt1 mutants. Cells that lack the G protein alpha2 display a loss of discoidin expression and do not aggregate. gdt1(-)/Galpha2(-) double mutants show no aggregation but strong discoidin expression. This suggests that gdt1 is a negative regulator of the GDT downstream of or in a parallel pathway to Galpha2.