GPI-anchored protein organization and dynamics at the cell surface

The surface of eukaryotic cells is a multi-component fluid bilayer in which glycosylphosphatidylinositol (GPI)-anchored proteins are an abundant constituent. In this review, we discuss the complex nature of the organization and dynamics of GPI-anchored proteins at multiple spatial and temporal scales. Different biophysical techniques have been utilized for understanding this organization, including fluorescence correlation spectroscopy, fluorescence recovery after photobleaching, single particle tracking, and a number of super resolution methods. Major insights into the organization and dynamics have also come from exploring the short-range interactions of GPI-anchored proteins by fluorescence (or Förster) resonance energy transfer microscopy. Based on the nanometer to micron scale organization, at the microsecond to the second time scale dynamics, a picture of the membrane bilayer emerges where the lipid bilayer appears inextricably intertwined with the underlying dynamic cytoskeleton. These observations have prompted a revision of the current models of plasma membrane organization, and suggest an active actin-membrane composite.     

DNA polymerase-delta from the silk glands of Bombyx mori.

The silk gland of Bombyx mori is a terminally differentiated tissue in which DNA replication continues without cell or nuclear division during larval development. DNA polymerase-delta activity increases in the posterior and middle silk glands during the development period, reaching maximal levels in the middle of the fifth instar larvae. The enzyme has been purified to homogeneity by a series of column chromatographic and affinity purification steps. It is a multimer comprising of three heterogeneous subunits, M(r) 170,000, 70,000, and 42,000. An auxiliary protein from B. mori silk glands, analogous to the proliferating cell nuclear antigen, enhances the processivity of the enzyme and stimulates catalytic activity by 3-fold. This auxiliary protein has also been purified to homogeneity. It is a dimer comprised of a single type M(r) 40,000 subunit. Polymerase-delta possesses an intrinsic 3'----5' exonuclease activity which participates in proofreading by mismatch repair during DNA synthesis and is devoid of any primase activity. DNA polymerase-delta activity could be further distinguished from polymerase-alpha from the same tissue based on its sensitivity to various inhibitors and polyclonal antibodies to the individual enzymes. Like DNA polymerase-alpha, polymerase-delta is also tightly associated with the nuclear matrix. The polymerase alpha-primase complex could be readily separated from polymerase-delta (exonuclease) in the purification protocol adopted. DNA polymerase-delta from B. mori silk glands resembles the mammalian delta-polymerases. Considering that both DNA polymerase-delta and -alpha are present in nearly equal amounts in this highly replicative tissue and their close association with the nuclear matrix, the involvement of both the enzymes in the chromosomal endoreplication process in B. mori is strongly implicated.     

Arginyl-tRNA synthetase from Mycobacterium smegmatis SN2: purification and kinetic mechanism

Arginyl-tRNA synthetase [L-Arg: tRNAArg ligase (AMP forming) EC 6.1.1.19] has been purified to homogeneity from Mycobacterium smegmatis SN2. The enzyme is a monomer of molecular weight 56,000. The kinetic patterns obtained by initial velocity and product inhibition studies are consistent with a rapid equilibrium random ter ter mechanism. Polyamines stimulated the formation of arginyl-tRNA, the stimulation being more significant at sub-optimal Mg2+ concentrations. Initial velocity studies performed in the presence of sub-optimal Mg2+ and spermine also indicated that the kinetic mechanism remained sequential random. Various attempts to reveal the formation of enzyme-bound arginyl-adenylate provided no evidence for its existence. The reverse reaction, i.e., the deacylation of arginyl-tRNA, required both AMP and PPi. This observation is consistent with the mechanism proposed.     

Seasonal growth and reproduction in a highly fecund brachyuran crab, Metopograpsus messor (Forskal) (Grapsidae)

The annual cycle of a Metopograpsus population (Muzhupilangad estuary) had three distinct periods: (1) growth-reproduction (January–May), when crabs were involved in moult and reproduction; (2) inactive period (June–July), and (3) reproductive period (August–December). Usually, spawning was immediately followed by another vitellogenic cycle, paralleled by the embryogenesis of prehatch eggs in the brood. Moulting was seemingly an annual event. In the programming of moult and reproduction, the species deviated from the common brachyuran pattern, inasmuch as the postmoult females engaged in active vitellogenesis. The synchrony in the stages of maturation and spawning, and the precision with which the physiological events are programmed, make this highly fecund species an ideal model for an integrated study of the physiology of growth and reproduction.     

II. Novel HCV NS5B polymerase inhibitors: discovery of indole C2 acyl sulfonamides

Development of SAR at the C2 position of indole lead 1, a palm site inhibitor of HCV NS5B polymerase (NS5B IC(50)=0.053μM, replicon EC(50)=4.8μM), is described. Initial screening identified an acyl sulfonamide moiety as an isostere for the C2 carboxylic acid group. Further SAR investigation resulted in identification of acyl sufonamide analog 7q (NS5B IC(50)=0.039μM, replicon EC(50)=0.011μM) with >100-fold improved replicon activity.     

Biofuels: opportunities and challenges in India

Energy plays a vital role in the economic growth of any country. Current energy supplies in the world are unsustainable from environmental, economic, and societal standpoints. All over the world, governments have initiated the use of alternative sources of energy for ensuring energy security, generating employment, and mitigating CO₂ emissions. Biofuels have emerged as an ideal choice to meet these requirements. Huge investments in research and subsidies for production are the rule in most of the developed countries. India started its biofuel initiative in 2003. This initiative differs from other nations’ in its choice of raw material for biofuel production—molasses for bioethanol and nonedible oil for biodiesel. Cyclicality of sugar, molasses, and ethanol production resulted in a fuel ethanol program which suffered from inconsistent production and supply. The restrictive policies, availability of molasses, and cost hampered the fuel ethanol program. Inconsistent policies, availability of land, choice of nonnative crops, yield, and market price have been major impediments for biodiesel implementation. However, a coherent, consistent, and committed policy with long-term vision can sustain India’s biofuel effort. This will provide energy security, economic growth, and prosperity and ensure a higher quality of life for India.