Changes in leaching properties of chick skeletal muscles on denervation

The leaching of electrolytes in normal and denervated chick gastrocnemius muscle has been studied by recording electroconductivity changes in donor-solvent (muscle-water) system at 11 intervals leading to a total immersion of 5 hr 15 min, at days 1,5,10,21 and 28, post-hatching. The results show age related changes in the permeability properties of normal muscle membrane system. The loss of neural control induces a blocking of electrolytes 10 days post-denervation. The possible nature of blocking mechanism has been related to the non-availability of neuro-trophic factors in the tissue.     

Calmodulin-stimulated cyclic nucleotide phosphodiesterases in plasma membranes of bovine epididymal spermatozoa

Cyclic nucleotide phosphodiesterase in the plasma membranes of bovine epididymal spermatozoa was stimulated by added Ca2+ and calmodulin. The rate of hydrolysis and responsiveness toward calmodulin was greater for cAMP than for cGMP. The kinetic analysis of the activity revealed two forms of phosphodiesterase with apparent Km values of 7.5 and 95 microM for cAMP. Calmodulin stimulated both of the activities by increasing the Vmax without affecting the Km's. The activity response with respect to Ca2+ concentration appears to be biphasic in both the absence and presence of added calmodulin. Trifluoperazine inhibited the Ca2+- and calmodulin-sensitive enzyme activity in a dose-dependent manner. The calmodulin-stimulated phosphodiesterase activity in the sperm plasma membranes can be solubilized and absorbed to a Calmodulin-Sepharose affinity column in the presence of Ca2+.     

Isolation of a constitutively expressed enzyme for hydrolysis of carbaryl in Pseudomonas aeruginosa.

A hydrolase constitutively expressed in Pseudomonas aeruginosa which converts carbaryl to 1-naphthol was purified 1,767-fold by using a combination of anion-exchange, hydroxylapatite, gel filtration, and hydrophobic interaction chromatography techniques. The presence of Triton X-100 in buffers was necessary for deaggregation and purification of the hydrolase. This is the first membrane-bound hydrolase involved in the hydrolysis of any methylcarbamate pesticide purified from a bacterial source to date. The enzyme exhibited a unique specificity of hydrolyzing only carbaryl (1-naphthyl N-methylcarbamate) but not any other methylcarbamates. The purified enzyme was a monomer with a molecular mass of 65,000 Da. The pH and temperature optima for the enzyme activity were 8.5 and 45 degrees C, respectively. No cofactor requirement for the hydrolase activity could be demonstrated, and none of the divalent cations studied affected the activity of the enzyme. Also, the enzyme activity was not affected by the thiols: dithioerythritol, dithiothreitol, and 2-mercaptoethanol. The Km and Vmax values for carbaryl were 9 microM and 7.9 mumol/min/mg of protein, respectively.     

The barley 60 kDa jasmonate-induced protein (JIP60) is a novel ribosome-inactivating protein

The N-terminal region of a 60 kDa, jasmonate-induced protein of barley leaves (JIP60) is shown to be homologous to the catalytic domains of plant ribosome-inactivating proteins (RIP). Western blotting of leaf extracts and in vitro reconstitution experiments indicate that JIP60 is synthesized as a precursor which is processed in vivo. This is in keeping with in vitro translation experiments indicating that a deletion derivative of the N-terminal region, but not the putative precursor, strongly inhibits protein synthesis on reticulocyte ribosomes. The inhibition of ribosome function is associated with depurination of 26S rRNA, characteristic of plant RIPs. This indicates that JIP60 is a novel ribosome-inactivating protein requiring at least two processing events for full activation. JIP60 derivatives do not significantly inhibit in vitro protein synthesis on wheat germ ribosomes. These and other results suggest that JIP60 may be involved in plant defence.     

Expression and mutagenesis of human poly(ADP-ribose) polymerase as a ubiquitin fusion protein from Escherichia coli

The cDNA of human poly(ADP-ribose) polymerase (pADPRP), encoding the entire protein, was subcloned into the Escherichia coli expression plasmid pYUb. In this expression system, the carboxyl terminus of ubiquitin is fused to the amino terminus of a target protein, in this case pADPRP, stabilizing the accumulation of the cloned gene product. Following induction of the transformed cells, the sonicated extract contained a unique protein immunoreactive with both pADPRP and ubiquitin antibodies and corresponding to the predicted mobility of the fusion protein in SDS-PAGE. Fusion of ubiquitin to pADPRP increased the yield of pADPRP approximately 10-fold compared to that of the unfused enzyme. The resulting recombinant fusion protein had catalytic properties which were nearly identical to those of native pADPRP obtained from mammalian tissues. These properties included specific activity, Km for NAD, response to DNA strand breaks, response to Mg2+, inhibition by 3-aminobenzamide, and activity in activity gel analysis. An initial analysis by deletion mutagenesis of pADPRP's functional domains revealed that deletions in the NAD binding domain eliminated all activity; however, partial polymerase activity resulted from deletion in the DNA binding or automodification domains. The activities were not enhanced by breaks in DNA. We further report a colony filter screening procedure designed to identify functional polymerase molecules which will facilitate structure/function studies of the polymerase.     

Escherichia coli nitrate reductase subunit A: its role as the catalytic site and evidence for its modification.

Subunits A and B were isolated from purified nitrate reductase by preparative electrophoresis in low levels of sodium dodecyl sulfate. Nonheme iron and low levels of molybdenum were associated with isolated subunit A but not with isolated subunit B. After dialysis against a source of molybdenum cofactor, subunit A regained tightly bound molybdenum and concomitantly regained enzyme activity and reactivity with anti-nitrate reductase antiserum. Subunit B neither bound cofactor nor regained activity or reactivity with antiserum. These data indicate that subunit A contains the active site of the enzyme. Subunit A was also found to be modified posttranslationally in a similar fashion as is subunit B. This was determined by comparison of partial proteolytic digests and amino acid analyses of A subunits from precursor and membrane-bound forms of nitrate reductase.     

Optoelectronic properties of naphtho[2, 1-b:6, 5-b′]difuran derivatives for photovoltaic application: a computational study

Some important optoelectronic properties of naphtho[2,1-b:6,5-b′]difuran (DPNDF) and its two derivatives have been limelighted by applying the density functional theory (DFT). Due to their low cost, high stability and earth abundance, the DPNDF and its derivatives are considered as potential organic semiconductor materials for their optoelectronics applications. Highly proficient derivatives are obtained systematically by attaching –CN (cyanide) to DPNDF at various sites. Our calculations indicate that DPNDF has a wide and direct band gap with an energy gap of 3.157 eV. Whereas the band gaps of its derivatives are found to be decreased by 88 meV for derivative “a” and 300 meV for derivative “b” as a consequence of p orbitals present in C and N atoms in derivative structures. The narrowing of the energy gap and density of states for the derivatives of DPNDF in the present investigation suggest that energy gap can be engineered for desirable optoelectronic applications via derivatives designing. Furthermore, their obtained results for optical parameters such as the dielectric and conductivity functions, reflectivity, refractive index, and the extinction coefficients endorses their aptness for optoelectronic applications.

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Graphical Abstract Real part of dielectric function for derivative “b”

     

Achievement of high cell density and high antibody productivity by a controlled-fed perfusion bioreactor process

Controlled feeding of nutrient supplements to a cell culture to enhance monoclonal antibody productivity has been practiced widely in high-yield, fed-batch processes. In this study, a similar feeding concept has been applied to a perfused culture and evaluated for the effects on bioreactor productivity and product quality. Our experimental results show that, by using such a "controlled-fed perfusion" approach, the volumetric antibody productivity (antibody per liter per day) was significantly increased by nearly twofold over the perfusion process, and surpassed fed-batch and batch processes by almost tenfold. The substantial boost in the overall productivity is attributable primarily to the combined effects of increased cell density as well as reduced product dilution. Both were achieved through careful nutrient supplementation in conjunction with metabolite minimization. As the manufacturing process evolved from roller bottles to the controlled-fed perfusion bioreactor system, the immunoreactivity and the cDNA sequences of the antibody were well preserved. However, the product glycosylation distribution patterns did alter. The controlled-feed perfusion process demonstrated a unique encompassment of the advantages of fed-batch and perfusion methods; that is, high product concentration with high volume throughput. Therefore, it may be very suitable for large-scale production of monoclonal antibodies.     

Molecular analysis of system N suggests novel physiological roles in nitrogen metabolism and synaptic transmission

The amino acid glutamine has a central role in nitrogen metabolism. Although the molecular mechanisms responsible for its transport across cell membranes remain poorly understood, classical amino acid transport system N appears particularly important. Using intracellular pH measurements, we have now identified an orphan protein related to a vesicular neurotransmitter transporter as system N. Functional analysis shows that this protein (SN1) involves H+ exchange as well as Na+ cotransport and, under physiological conditions, mediates glutamine efflux as well as uptake. Together with the pattern of SN1 expression, these unusual properties suggest novel physiological roles for system N in nitrogen metabolism and synaptic transmission.