Effects of ultraviolet (254nm) irradiation on egg hatching and adult emergence of the flour beetles, Tribolium castaneum, T. confusum and the almond moth, Cadra cautella.

The eggs of the stored grain pests, Tribolium castaneum (Herbst), T. confusum (Duval) (Coleoptera: Tenebrionidae) and Cadra cautella (Walker) (Lepidoptera; Pyralidae) belonging to three age groups, 1, 2, and 3 days-old, were exposed to ultraviolet (UV) radiation with 254nm wavelength (UV-C) for different durations to determine irradiation effects on egg-hatching and adult emergence. An increase in time of exposure to UV-rays caused a gradual decrease in the percentage of hatching of eggs in all age groups of eggs. No hatching occurred after 24 minutes of exposure in 2 and 3 day-old eggs of T. confusum. C. cautella eggs were less sensitive to UV-rays than were T. castaneum and T. confusum eggs. All the exposure periods significantly reduced the eclosion of adults in all the experimental insects. No adults emerged when 3 day-old eggs of T. castaneum were irradiated for 16 or 24 minutes, or from 2 and 3 day-old eggs T. confusum irradiated for 16 or 24 minutes.

The centrosome in vertebrates: more than a microtubule-organizing center.

The somatic cells of all higher animals contain a single minute organelle called the centrosome. For years, the functions of the centrosome were thought to revolve around its ability to nucleate and organize the various microtubule arrays seen in interphase and mitosis. But the centrosome is more than just a microtubule-organizing center. Recent work reveals that this organelle is essential for cell-cycle progression and that this requirement is independent of its ability to organize microtubules. Here, we review the various functions attributed to the centrosome and ask which are essential for the survival and reproduction of the cell, the organism, or both.

Tumor-specific proteolytic processing of cyclin E generates hyperactive lower-molecular-weight forms.

Cyclin E is a G(1) cyclin essential for S-phase entry and has a profound role in oncogenesis. Previously this laboratory found that cyclin E is overexpressed and present in lower-molecular-weight (LMW) isoforms in breast cancer cells and tumor tissues compared to normal cells and tissues. Such alteration of cyclin E is linked to poor patient outcome. Here we report that the LMW forms of cyclin E are hyperactive biochemically and they can more readily induce G(1)-to-S progression in transfected normal cells than the full-length form of the protein can. Through biochemical and mutational analyses we have identified two proteolytically sensitive sites in the amino terminus of human cyclin E that are cleaved to generate the LMW isoforms found in tumor cells. Not only are the LMW forms of cyclin E functional, as they phosphorylate substrates such as histone H1 and GST-Rb, but also their activities are higher than the full-length cyclin E. These nuclear localized LMW forms of cyclin E are also biologically functional, as their overexpression in normal cells increases the ability of these cells to enter S and G(2)/M. Lastly, we show that cyclin E is selectively cleaved in vitro by the elastase class of serine proteases to generate LMW forms similar to those observed in tumor cells. These studies suggest that the defective entry into and exit from S phase by tumor cells is in part due to the proteolytic processing of cyclin E, which generates hyperactive LMW isoforms whose activities have been modified from that of the full-length protein.

The pathogenicity of Bacillus thuringiensis ssp. kurstaki to gamma-irradiated Cadra cautella (Walker) (Lepidoptera: Phycitidae).

The present investigation deals with the effects of Bacillus thuringiensis ssp. kurstaki on various biological parameters of gamma-irradiated Cadra cautella. The pathogen, irradiation, and their combinations significantly affected the insects by increasing their mortality and developmental periods, reducing the pupal and adult survival (%), and the adult longevity and reproductive potential were also significantly reduced. It was observed that irradiation-pathogen combinations produced additive to synergistic effects on C. cautella.

Syk-dependent phosphorylation of microtubules in activated B-lymphocytes.

Syk is a protein-tyrosine kinase that is essential for B-lymphocyte development and B-cell signaling. Syk phosphorylates tubulin on tyrosine both in vitro and in intact lymphocytes. Here we show that (alpha)-tubulin present within the cytoskeletal microtubule network was phosphorylated in a Syk-dependent manner following the activation of B-cells by engagement of the B-cell antigen receptor or by treatment with the phosphotyrosine phosphatase inhibitor, pervanadate. Immunofluorescence staining of microtubule cytoskeletons and western blotting studies with antibodies to phosphotyrosine confirmed the phosphorylation of polymerized tubulin in Syk-expressing, but not Syk-deficient, cells. At low concentrations of pervanadate, centrosomes appeared to be preferentially tyrosine-phosphorylated. Tubulin phosphorylated to a high stoichiometry on tyrosine assembled into microtubules in vitro, and preassembled microtubules were also phosphorylated by Syk kinase in vitro. Thus, Syk has the capacity to interact with microtubule networks within the B-lymphocyte and catalyzes the phosphorylation of the (alpha)-tubulin subunit. Syk-dependent phosphorylation of microtubules may affect the ability of the microtubule cytoskeleton to serve as a platform upon which signaling complexes are assembled.

Purification of microtubule proteins from Xenopus egg extracts: identification of a 230K MAP4-like protein.

We describe the purification of microtubule proteins from Xenopus egg extracts by temperature-dependent assembly and disassembly in the presence of dimethyl sulfoxide and identify a number of presumptive microtubule-associated proteins (MAPs). One of these proteins has a molecular weight of 230 kDa and is immunologically related to HeLa MAP4. We show that this MAP is heat stable and phosphorylated, and that it promotes elongation of microtubules from axonemes.

cdc2 kinase-induced destabilization of MAP2-coated microtubules in Xenopus egg extracts.

During the interphase to metaphase transition, microtubules are destabilized by a cdc2 kinase-dependent phosphorylation event. This destabilization is due to a dramatic increase in the rate at which each growing microtubule starts to shrink (catastrophe rate). In principle, this could be brought about by lowering the affinity of stabilizing MAPs for the microtubule wall, by activating a factor that would actively increase the catastrophe rate or by an alteration of both parameters. Here we examine the stabilizing effect of bovine brain MAP2 on microtubules assembled in interphase Xenopus egg extracts. We show that this MAP strongly stabilizes microtubules assembled in the extracts against nocodazole-induced depolymerization. However, it does not protect them from the cdc2 kinase-induced shortening and destabilization. Moreover, the steady-state length of centrosome-nucleated microtubules in cdc2-treated extracts containing MAP2 is similar to that found in extracts lacking exogenous MAP2. We also show that although exogenous MAP2 is phosphorylated by cdc2 kinase in the extract, this is not the cause of microtubule destabilization. These results indicate that increased microtubule dynamics during mitosis is due to the activation of a factor that can function independently of the presence of active, stabilizing factors.

Assessment of immune responses to H-Y antigen in naturally inseminated and sperm-injected mice using cell-mediated cytotoxicity assays.

An in vitro cell-mediated cytotoxicity assay has been used to assess immunity to the male-specific H-Y antigen in female mice after either insemination with male cells at syngeneic natural mating or after injection with syngeneic sperm cell suspensions. Lymphocytes showing specific cytotoxicity for the H-Y antigen could be recovered from both spleen and lymph nodes of female mice injected with sperm. However, insemination of male cells at natural mating did not apparently prime cytotoxic cells against the H-Y antigen in either the spleen or para-aortic lymph nodes draining the uterus of female mice mated once or repeatedly (3-10 X) in the absence of pregnancy. These results are discussed in relation to the factors regulating the immune responsiveness of the female to inseminated antigens.

Increases in the numbers of immunoglobulin-secreting cells in lymph nodes responding to sperm and other stimuli: possible relationship to immunosuppression.

It has been reported that, in early pregnancy in mice, there is an increase in the number of immunoglobulin-secreting cells in the lymph nodes which drain the uterus. This paper describes the results of further investigations provoked by interest in these early changes. Increases in the numbers of immunoglobulin-secreting cells were observed in syngeneically, but scarcely or not at all in allogeneically, mated mice. Increases were not observed in surgically sterilized female mice inseminated by normal males. However, subcutaneous injection of sperm provoked massive increases in the numbers of immunoglobulin-secreting cells in the lymph nodes draining the injection site. The changes were compared with those provoked by the injection of spleen cells and LPS. The results are discussed in relation to the nature of the interactions provoking the increases in the number of immunoglobulin-secreting cells and their possible relationship to immunosuppression, and the relative immunological unresponsiveness which the female shows to the challenge of inseminated sperm.

Detection of anti-sperm activities of monoclonal antibodies to human sperm.

The anti-sperm activities of a series of monoclonal antibodies to human sperm have been compared using agglutination, immunofluorescence, ELISA and 'panning' assays. The antibodies fell into two categories, those that could be detected by agglutination but not immunofluorescence assays and those that could be detected by immunofluorescence but not agglutination. Antibodies positive in the agglutination assays were also positive in the 'panning' assay. None of the antibodies tested was positive in the ELISA assays. These results, and others, are discussed in relation to the problems associated with the detection of anti-sperm antibodies in sub-fertile human populations.

Detection of antibody-coated sperm by 'panning' procedures.

The use of 'panning' procedures to detect immunoglobulin on the sperm surface are described. Wells on plastic plates are coated with anti-immunoglobulin molecules by either 1-step or 2-step procedures and the sperm under test are then incubated in these wells for up to 1 h and the wells washed. Antibody-coated sperm remain attached in large numbers while control sperm are washed out. These procedures have the advantages that they are cheap, simple, do not involve sperm fixation and may be used with relatively dilute cell suspensions and with sperm of low motility. The potential applications of the procedures are discussed.

Failure of spleen cell migration assays to detect cell-mediated immunity to spermatozoa after natural mating in female mice.

We have re-examined whether immunity to spermatozoa can be detected by spleen cell migration assays in inseminated female mice. Unlike previous results, however, sperm suspensions inhibited the migration of splenic leucocytes obtained from mated as well as control mice. Small differences were observed between leucocytes from untreated virgin mice and leucocytes from mice immunized with spermatozoa in adjuvant. In these experiments lymph node cells draining the injection site were added to the spleen cells.