Heterogeneity in buffalo lutropin.

Lutropin (LH-1) from water buffaloes has been shown to exhibit microheterogeneity in the N-terminal amino-acid sequence of its alpha-subunit. The beta-subunit did not exhibit such microheterogeneity. Another protocol of purification yielded a preparation of buffalo LH (bu LH-2) different from the buffalo LH-1 in certain physico-chemical properties like ease of dissociation into subunits, sugar composition, isoelectric point, and elution profile on S-200. Data appear to indicate the presence of more than one form of buffalo lutropin.     

Isubgol as an Alternative Gelling Agent for Microbial Culture Media

Isubgol, the mucilaginous husk derived from the seeds of Plantago ovata, has been successfully used as a gelling agent for microbial culture media. As illustrative examples, fast growing symbiotic bacterium, Rhizobium meliloti and saprophytic fungi, Aspergillus flavus and Penicillium chrysogenum were cultured on media gelled with either Isubgol or agar. All the three microbes employed in the study exhibited normal growth when cultured on their respective media gelled with Isubgol. Rather, Isubgol gelled medium appears to promote the growth of bacterial cultures as the colonies on this medium were denser than the corresponding ones on the medium gelled with agar. Likewise, on Isubgol gelled medium, sporulation in both the fungi took place earlier than on the medium gelled with agar, thus indicating the promotive influence of the former gelling agent.     

Effect of LH-RH on the release of sulfated lutropin: a potential in vitro bioassay for LH-RH

Sheep pituitary cells prelabelled with radioactive [35S] sulfate (35SO4(2-)) were incubated with different concentrations of LH-RH and the release of LH (lutropin) into the medium was monitored in terms of immunoprecipitable [35S] sulfated LH radioactivity and estimation of LH in the same sample by radioimmunoassay. A dose dependent response was obtained with a maximum of a 16 fold increase in immunoprecipitable 35SO4(2-) -labelled LH radioactivity in the medium which was confirmed by radioimmunoassay. Similar results were also obtained for Buserelin, a well known superactive analogue of LH-RH. However, the half maximal response for Buserelin was obtained at 3-5 nM in comparison to 80.5 nM for LH-RH. After the maximal response to LH-RH as well as Buserelin, a further increase in the concentrations caused a decrease in the release of immunoprecipitable [35S]-sulfate labelled LH into the medium. Differential labelling of stored and newly synthesized LH with radioactive [35S] sulfate and [3H]-labelled leucine revealed that there was a dose dependent increase in the [35S] sulfate labelled LH into the medium whereas the release of [3H]-leucine labelled newly synthesized LH did not show a parallel increase either at different concentrations of LH-RH or at different time intervals. The above observations strongly suggest the possibility of sulfation of LH being the potential signal indicating the storage of LH in sheep pituitary cells. Another important observation in our study was that the dose dependent response of LH-RH in the form of release of [35S]-sulfate labelled LH, which was monitored by immunoprecipitation with specific LH antiserum, can be used in an in vitro bioassay for LH-RH. We believe that a new cheap and sensitive in vitro bioassay could be developed on the basis of this observation.     

Supervillin binding to myosin II and synergism with anillin are required for cytokinesis

Cytokinesis, the process by which cytoplasm is apportioned between dividing daughter cells, requires coordination of myosin II function, membrane trafficking, and central spindle organization. Most known regulators act during late cytokinesis; a few, including the myosin II–binding proteins anillin and supervillin, act earlier. Anillin''s role in scaffolding the membrane cortex with the central spindle is well established, but the mechanism of supervillin action is relatively uncharacterized. We show here that two regions within supervillin affect cell division: residues 831–1281, which bind central spindle proteins, and residues 1–170, which bind the myosin II heavy chain (MHC) and the long form of myosin light-chain kinase. MHC binding is required to rescue supervillin deficiency, and mutagenesis of this site creates a dominant-negative phenotype. Supervillin concentrates activated and total myosin II at the furrow, and simultaneous knockdown of supervillin and anillin additively increases cell division failure. Knockdown of either protein causes mislocalization of the other, and endogenous anillin increases upon supervillin knockdown. Proteomic identification of interaction partners recovered using a high-affinity green fluorescent protein nanobody suggests that supervillin and anillin regulate the myosin II and actin cortical cytoskeletons through separate pathways. We conclude that supervillin and anillin play complementary roles during vertebrate cytokinesis.     

Carboxyl terminal domain basic amino acids of mycobacterial topoisomerase I bind DNA to promote strand passage

Bacterial DNA topoisomerase I (topoI) carries out relaxation of negatively supercoiled DNA through a series of orchestrated steps, DNA binding, cleavage, strand passage and religation. The N-terminal domain (NTD) of the type IA topoisomerases harbor DNA cleavage and religation activities, but the carboxyl terminal domain (CTD) is highly diverse. Most of these enzymes contain a varied number of Zn²⁺ finger motifs in the CTD. The Zn²⁺ finger motifs were found to be essential in Escherichia coli topoI but dispensable in the Thermotoga maritima enzyme. Although, the CTD of mycobacterial topoI lacks Zn²⁺ fingers, it is indispensable for the DNA relaxation activity of the enzyme. The divergent CTD harbors three stretches of basic amino acids needed for the strand passage step of the reaction as demonstrated by a new assay. We also show that the basic amino acids constitute an independent DNA-binding site apart from the NTD and assist the simultaneous binding of two molecules of DNA to the enzyme, as required during the catalytic step. Although the NTD binds to DNA in a site-specific fashion to carry out DNA cleavage and religation, the basic residues in CTD bind to non-scissile DNA in a sequence-independent manner to promote the crucial strand passage step during DNA relaxation. The loss of Zn²⁺ fingers from the mycobacterial topoI could be associated with Zn²⁺ export and homeostasis.