SYNTHETIC ACTIVITIES DURING SPERMATOGENESIS IN THE LOCUST

Isolated testes of the locust Schistocerca gregaria were immersed in solutions of tritiated thymidine, cytidine, uridine, or arginine for short periods to study nucleic acid and protein synthesis during spermatogenesis. DNA synthesis in this tissue is completed prior to initiation of meiosis. Protein synthesis continues throughout the whole meiotic cycle as well as during spermatid development. Meiotic cells, except those in metaphase through early telophase, and early spermatids are also actively synthesizing RNA. The heteropycnotic X-chromosome does not produce RNA at any stage of spermatogenesis. The rates of protein and particularly RNA synthesis decrease as chromosome condensation progresses. Depression of RNA synthesis, however, is not always accompanied by cytologically detectable condensation of chromatin, since very little or no RNA is synthesized in spermatids in which chromatin condensation has barely begun.     

Novel excitatory Conus peptides define a new conotoxin superfamily

A new class of Conus peptides, the I-superfamily of conotoxins, has been characterized using biochemical, electrophysiological and molecular genetic methods. Peptides in this superfamily have a novel pattern of eight Cys residues. Five peptides that elicited excitatory symptomatology, r11a, r11b, r11c, r11d and r11e, were purified from Conus radiatus venom; four were tested on amphibian peripheral axons and shown to elicit repetitive action potentials, consistent with being members of the 'lightning-strike cabal' of toxins that effect instant immobilization of fish prey. A parallel analysis of Conus cDNA clones revealed a new class of conotoxin genes that was particularly enriched (with 18 identified paralogues) in a Conus radiatus venom duct library; several C. radiatus clones encoded the excitatory peptides directly characterized from venom. The remarkable diversity of related I-superfamily peptides within a single Conus species is unprecedented. When combined with the excitatory effects observed on peripheral circuitry, this unexpected diversity suggests a corresponding molecular complexity of the targeted signaling components in peripheral axons; the I-conotoxin superfamily should provide a rich lode of pharmacological tools for dissecting and understanding these. Thus, the I-superfamily conotoxins promise to provide a significant new technology platform for dissecting the molecular components of axons.     

Moving as a reason for pet relinquishment: a closer look

One of the most frequently given reasons for relinquishing a companion animal to an animal shelter is that the person or family is moving. Telephone interviews conducted with 57 caregivers who relinquished animal companions to a shelter in the midwestern United States covered details of the move, characteristics both of the caregivers and the animals, and efforts to avoid relinquishment. A human-nonhuman animal bonding scale also was administered. Although some participants had additional reasons for relinquishment, the majority had given up their pets solely because they were moving. Most had relatively low income, were moving for employment reasons, and were renting their homes. Landlord restrictions were an important factor in relinquishment. High scores on the bonding scale and spontaneous expressions of discomfort and sorrow suggest that external pressures overrode attachment to the animal and the pain of relinquishment.     

Thrombin Specificity: Further Evidence for the Importance of the β-Insertion Loop and Trp96. Implications of the Hydrophobic Interaction Between Trp96 and Pro60B Pro60C for the Activity of Thrombin

A number of thrombin mutants have been constructed to investigate the role of Trp⁹⁶ and the β-insertion loop for the specificity of thrombin. Thrombin(60D) consists of the replacement of the β-insertion loop (14 amino acid residues from 59 to 63, including a 9-residue insertion at position 60) with the corresponding four residues in trypsin, Tyr-Lys-Ser-Gly; thrombin(GGG) is a smaller loop mutation in which the residues Tyr^60APro^60BPro^60CTrp^60D Asp^60ELys^60F of the β-insertion loop were replaced by Gly-Gly-Gly; thrombin(96S) consists of a point mutation Trp⁹⁶→Ser; and thrombin(GGG/96S) is the double mutant incorporating both changes. Thrombin(96S) clots fibrinogen ~3 times more slowly than thrombin, with the two β-insertion loop mutants, thrombin(GGG) and thrombin(GGG/96S), reacting ~3000- and 1300-fold more slowly, respectively. The specificity constant k _cat/K _m for the cleavage of fibrinopeptide A and fibrinopeptide B by thrombin(96S) was 2.6 and 0.35 μM^?1 s^?1 respectively, compared to 10 and 2.5 μM^?1 s^?1 for wild-type recombinant thrombin, respectively. Kinetic constants were determined for the hydrolysis of H-D-phenylalanyl-L-pipecolyl-L-arginine-p-nitroaniline. The Michaelis constant K _m increased ~6-fold for thrombin(96S) and >200-fold for thrombin(GGG) and thrombin(GGG/96S) when compared to wild-type recombinant thrombin, while the catalytic constant k _cat remained approximately the same. All mutants were more susceptible to inhibition by BPTI than wild-type recombinant thrombin. Clearly, the β-insertion loop is important for thrombin activity. But the mutation of Trp⁹⁶→Ser can compensate somewhat for the loss of binding at the β-insertion loop. The deletion of the hydrophobic interaction between Trp⁹⁶ and Pro^60BPro^60C appears to decrease the stability of the β-insertion loop, thereby causing a decrease in binding efficiency.     

Autophagy induced by a sulphamoylated estrone analogue contributes to its cytotoxic effect on breast cancer cells

Background

Autophagy can either be protective and confer survival to stressed cells, or it can contribute to cell death. The antimitotic drug 2-ethyl-3-O-sulpamoyl-estra-1,3,5(10),15-tetraen-17-ol (ESE-15-ol) is an in silico-designed 17-β-estradiol analogue that induces both autophagy and apoptosis in cancer cells. The aim of the study was to determine the role of autophagy in ESE-15-ol-exposed human adenocarcinoma breast cancer cells; knowledge that will contribute to future clinical applications of this novel antimitotic compound. By inhibiting autophagy and determining the cytotoxic effects of ESE-15-ol-exposure, deductions could be made as to whether the process may confer resistance to the drug, or alternatively, contribute to the cell death process.

Methods and results

Spectophometrical analysis via crystal violet staining was used to perform cytotoxicity studies. Morphology studies were done using microscopic techniques namely polarization-optical transmitted light differential interference light microscopy, fluorescent microscopy using monodansylcadaverine staining and transmission electron microscopy. Flow cytometry was used to quantify the autophagy inhibition and assess cell viability. Results obtained indicated that 3-methyladenine inhibited autophagy and increased cell survival in both MCF-7 and MDA-MB-231 cell lines.

Conclusion

This in vitro study inferred that autophagy inhibition with 3-methyladenine does not confer increased effectiveness of ESE-15-ol in inducing cell death. Thus it may be concluded that the autophagic process induced by ESE-15-ol exposure in MCF-7 and MDA-MB-231 cells plays a more significant role in cell death than conferring survival.

     

The psychological outcome of constitutional delay of growth and puberty

Does being of short stature (SS) matter? Growth studies are important because endocrinologists need to be able to give not only diagnostic but also prognostic indication to those of SS, and give advice for or against treatment. Studies on body height may give significant insight into the behavioural-hypothalamic-pituitary axis underlying the surface effect on social behaviour. This research presents adult follow-up studies of 49 males who attended at a growth clinic as children and who were diagnosed as having the condition of maturational delay short stature (MDSS). From the psychological perspective, the elements of diagnosis and prognosis, attitudinal influences both social and individual, treatment, and psychological issues which could have bearing on SS are drawn together. Where there is a poor psychosocial outcome finding, the MDSS patients seem to be more like those with growth hormone (GH) deficiency. With GH deficiency (GHD), a poor psychosocial outcome of SS has been demonstrated with greater certainty. The current situation is that there remains divergence between previous SS outcome studies. Differing research SS psychosocial outcomes are analysed and a methodological explanation of past divergent outcomes is presented, being: (1) differences in sensitivity between psychological assessment tools; (2) Invalidation by confounding the psychological with sociological outcomes, and (3) confounding of results by failure to control for psychological states. An innovative new approach of changing the methodology of past SS research from the biological to the psychological perspective is employed: previous SS research has appointed height status as the independent variable, with psychosocial outcome as the dependent variable. The innovative approach in this research is to reverse that order and psychological status has been made the independent variable for both the MDSS and for a predetermined psychologically and physically healthy (PPH) comparison group of males. The results of the follow-up studies in this research indicate that there is an association between psychological distress and attained height which supports other studies where the findings indicate a poor psychological outcome associated with SS. It can therefore be concluded that the psychological status of children should be determined when they attend a growth clinic with concerns of SS.