Transactivation of gp130 in myeloma cells

Receptor transactivation, i.e., interaction between unrelated receptor systems, is a growing theme in cytokine and growth factor signaling. In this study we reveal for the first time the ability of IFN-alpha to transactivate gp130 in myeloma cells. An epidermal growth factor receptor/gp130 chimeric receptor previously shown by us to transactivate endogenous gp130, provided a complementary tool to study the underlying mechanisms of receptor cross-talk. Further analysis revealed that transactivation of gp130 by IFN-alpha did not require the extracellular or trans-membrane domain of gp130. Moreover, transactivation of gp130 was critically dependent upon Janus kinase activation by the initiating receptor and correlated with rapid and sustained Janus kinase 1 and tyrosine kinase (Tyk) 2 tyrosine phosphorylation. Finally, transactivation of gp130 may be a common theme in myeloma cells, perhaps providing a mechanism for enhanced or qualitatively distinct cellular responses to specific stimuli.     

Functional analysis of a carboxyl-terminal phosphorylation mutant of the bovine papillomavirus E1 protein

The papillomavirus E1 protein is essential for viral DNA replication, and phosphorylation of E1 appears to regulate protein function and DNA replication. Serine 584 of bovine papillomavirus E1 is in a conserved motif resembling a CK2 consensus site, and is phosphorylated by CK2 in vitro. Mutation of serine 584 to alanine eliminates replication of the viral genome in transient replication assays. Wild-type and mutant E1 proteins were expressed from recombinant baculoviruses and used to assess biochemical functions of the amino acid 584 substitution. Helicase enzyme activity, E1 binding to the viral E2 protein and to cellular DNA polymerase alpha-primase were all unaffected in the mutant protein. Binding of E1 to viral replication origin DNA sequences was reduced in the mutant, but not eliminated. The carboxyl-terminal region of the protein appears to play a role in regulating E1 function, and adds to a complex picture emerging for papillomavirus DNA replication control.     

SNAREs in opposing bilayers interact in a circular array to form conducting pores

The process of fusion at the nerve terminal is mediated via a specialized set of proteins in the synaptic vesicles and the presynaptic membrane. Three soluble N-ethylmaleimide-sensitive factor (NSF)-attachment protein receptors (SNAREs) have been implicated in membrane fusion. The structure and arrangement of these SNAREs associated with lipid bilayers were examined using atomic force microscopy. A bilayer electrophysiological setup allowed for measurements of membrane conductance and capacitance. Here we demonstrate that the interaction of these proteins to form a fusion pore is dependent on the presence of t-SNAREs and v-SNARE in opposing bilayers. Addition of purified recombinant v-SNARE to a t-SNARE-reconstituted lipid membrane increased only the size of the globular t-SNARE oligomer without influencing the electrical properties of the membrane. However when t-SNARE vesicles were added to a v-SNARE membrane, SNAREs assembles in a ring pattern and a stepwise increase in capacitance, and increase in conductance were observed. Thus, t- and v-SNAREs are required to reside in opposing bilayers to allow appropriate t-/v-SNARE interactions leading to membrane fusion.     

Direct interaction between SNAP-23 and L-type Ca2+ channel

During secretion, membrane-bound secretory vesicles dock and fuse at the base of porosomes in the cell plasma membrane. Among other proteins, the porosome is composed of SNAREs and Ca2+-channels. Ca2+-channels and SNAREs have been implicated in cell secretion. Several immunoprecipitation and binding studies suggest the physical interaction of the t-SNARE proteins, Syntaxin-1 and SNAP-25 with various Ca2+-channels. In this study, using yeast two-hybrid and immunoanalysis, we demonstrate for the first time, direct interaction of SNAP-23 and a L-type Ca2+-channel at the plasma membrane in pancreas.     

Structure, isolation, composition and reconstitution of the neuronal fusion pore

Neuronal communication is dependent on the fusion of 40-50 nm in diameter synaptic vesicles containing neurotransmitters, at the presynaptic membrane. Here we report for the first time at 5-8A resolution, the presence of 8-10 nm in diameter cup-shaped neuronal fusion pores or porosomes at the presynaptic membrane, where synaptic vesicles dock and fuse to release neurotransmitters. The structure, isolation, composition, and functional reconstitution of porosomes present at the nerve terminal are described. These findings reveal the molecular mechanism of neurotransmitter release at the presynaptic membrane of nerve terminals.     

Effect of age on lipid peroxides, lipofuscin and ascorbic acid contents of the lungs of male garden lizard

Oxidative damage was assessed through the estimation of lipid peroxides (LP) in the lungs of an ageing short-lived species of reptile, Calotes versicolor, commonly known as the garden lizard. Attempts were also made to trace its relationship with the age pigment, lipofuscin and the antioxidant ascorbic acid. While LP increased with advancing age the contents of both lipofuscin and ascorbic acid did not show appreciable change during maturation ( < 1-1 year old) but declined during senescence phase (1 to 2-4 year old). While the pattern of age associated changes in LP and ascorbic acid indicate similarity with the pattern observed in most of the mammals, the reduction of lipofuscin in older lizards is a significant departure from the common trend.     

Nitrite toxicity in Indian major carps: sublethal effect on selected enzymes in fingerlings of Catla catla, Labeo rohita and Cirrhinus mrigala

The effects of 96-h sublethal exposure of nitrite (1, 2, 4, 8 and 10.4 mg l(-1)) on selected enzymatic activities in serum and tissues of fingerlings of catla (Catla catla), rohu (Labeo rohita) and mrigal (Cirrhinus mrigala) were studied for the first time in these species. All three species responded almost identically to nitrite exposure. With increasing nitrite concentration, reduction in activities was observed in acetylcholinesterase (AChE) in brain and liver; alkaline phosphatase (ALP) in serum, brain and gill; and acid phosphatase (ACP) in gill, while progressive increase in alanine aminotransferase (ALAT) and aspartate aminotransferase (ASAT) activities in brain, gill and serum, and ACP activity in serum and brain was observed. Lactate dehydrogenase (LDH) activity increased in gill, liver, kidney, brain and serum of all three species with increasing nitrite concentration up to 8 mg l(-1) followed by reduction at 10.4 mg l(-1). The study revealed nitrite stress causing alteration in activities of all measured tissue and serum enzymes in the fingerlings, and so stresses the need for proper management of this particular nutrient in water during carp culture.     

Discovery of the Porosome: revealing the molecular mechanism of secretion and membrane fusion in cells

Secretion and membrane fusion are fundamental cellular processes involved in the physiology of health and disease. Studies within the past decade reveal the molecular mechanism of secretion and membrane fusion in cells. Studies reveal that membrane-bound secretory vesicles dock and fuse at porosomes, which are specialized plasma membrane structures. Swelling of secretory vesicles result in a build-up of intravesicular pressure, which allows expulsion of vesicular contents. The discovery of the porosome, its isolation, its structure and dynamics at nm resolution and in real time, its biochemical composition and functional reconstitution, are discussed. The molecular mechanism of secretory vesicle fusion at the base of porosomes, and vesicle swelling, have been resolved. With these findings a new understanding of cell secretion has emerged and confirmed by a number of laboratories.