Structural basis for allosteric regulation of the monomeric allosteric enzyme human glucokinase

Glucokinase is a monomeric enzyme that displays a low affinity for glucose and a sigmoidal saturation curve for its substrate, two properties that are important for its playing the role of a glucose sensor in pancreas and liver. The molecular basis for these two properties is not well understood. Herein we report the crystal structures of glucokinase in its active and inactive forms, which demonstrate that global conformational change, including domain reorganization, is induced by glucose binding. This suggests that the positive cooperativity of monomeric glucokinase obeys the "mnemonical mechanism" rather than the well-known concerted model. These structures also revealed an allosteric site through which small molecules may modulate the kinetic properties of the enzyme. This finding provided the mechanistic basis for activation of glucokinase as a potential therapeutic approach for treating type 2 diabetes mellitus.     

Hydrolysis of newspaper polysaccharides under sulfate reducing and methane producing conditions

The initial decomposition rates of cellulose and hemicellulose were measured using toluene to specifically inhibit the microbial uptake of hydrolysis products during the degradation of newspaper under sulfate reducing and methane producing conditions. The amount of glucose and xylose accumulation in the first 2 weeks of incubation period was higher in the sulfate reducing condition compared to the methane producing condition. It was estimated that 28 and 6% of initially loaded cellulose in the sulfate reducing condition and the methane producing condition was hydrolyzed, respectively. Accordingly, the newspaper-cellulose hydrolysis rate constant was estimated to be 6.7 times higher in sulfate reducing condition than in methane producing condition. Based on the glucose accumulation patterns, when sulfate reducing bacteria (SRB) were inhibited by anthraquinone and molybdate (Na₂MoO₄), it may be suggested that SRB might have contributed to the hydrolysis of cellulose, while their effect on the hydrolysis of hemicellulose could not be elucidated.     

Absence of extensive recombination between inter- and intraspecies mitochondrial DNA in mammalian cells

Recombination of mammalian mitochondrial DNA (mtDNA) was examined using mouse X rat somatic cell hybrid clones and rat cybrid clones. The mouse X rat hybrids were isolated by fusion of chloramphenicol-sensitive (CAPs) mouse and CAP-resistant (CAPr) rat cells. The rat cybrids were isolated by fusion of rat cells with type B mtDNA and enucleated cells with type A mtDNA. Genetic and physical analyses showed that the mtDNAs of the hybrids and cybrids were simple mixtures of the two parental mtDNAs except in the following two cases: One was subclone H2-9 of mouse X rat hybrids, which was CAPr even though mtDNA from the CAPs mouse parent was predominantly retained. The other was rat cybrid subclones, Y12-24 and -61, which showed specific loss of one Hinf I fragment of type B mtDNA, B10. These observations suggest that, in contrast to the case with plant mtDNA, recombination of mammalian mtDNA occurs rarely, if at all.     

Application of high-performance capillary electrophoresis to analysis of carbohydrates, especially those in glycoproteins

Methods for the component monosaccharide analysis and oligosaccharide mapping for glycoprotein research, based on HPCE of reductively pyridylaminated (PA) derivatives, are described. the component monosaccharides released from glycoproteins by acid hydrolysis are converted to PA derivatives and analyzed by HPCE as borate complexes. They can be quantified in the picomole range (introduced amount) with high reproducibility. The oligosaccharides released by hydrazinolysis are similarly converted to PA derivatives. Two-dimensional mapping of the relative mobilities of these derivatives, obtained in an acidic phosphate buffer and an alkaline borate buffer, ensures reliable identification of the oligosaccharides.     

The mechanism of block of glutamate synapses by dipicolinic acid

The effect of dipicolinic acid (2,6-pyridine dicarboxylic acid) on the mealworm neuromuscular junction was studied using conventional microelectrode recording techniques. Dipicolinic acid (10^?5-10^?3 M) added to the bathing solution reversibly blocked neuromuscular transmission. The depolarization in response to iontophoretically applied L-glutamate (glutamate potential) was not affected by dipicolinic acid even when the neurally evoked excitatory postsynaptic potential (EPSP) was totally abolished. Focal extracellular recordings from single synaptic sites revealed that in the presence of 1 x 10^?4 M dipicolinic acid the presynaptic spike was unchanged, but the quantal content for evoked transmitter release was reduced. The calcium-dependent action potential elicited by direct stimulation of the muscle fiber was not impaired by dipicolinic acid. These results suggest that dipicolinic acid interferes with the transmitter-releasing mechanism from the presynaptic terminal.     

New Brachiopod Species <Emphasis Type="Italic">Leurosina katasumiensis</Emphasis> (Chonetida) from the Kungurian Katasumi Limestone of the Kusu Area,Central Japan

A new brachiopod species, Leurosina katasumiensis sp. nov. (order Chonetida, family Rugosochonetidae, subfamily Rugosochonetinae), is described from the Kungurian Stage (Lower Permian, Nabeyama Formation) of central Japan.     

Comparison of analytical methods for profiling <Emphasis Type="Italic">N-</Emphasis> and <Emphasis Type="Italic">O-</Emphasis>linked glycans from cultured cell lines

The Human Disease Glycomics/Proteome Initiative (HGPI) is an activity in the Human Proteome Organization (HUPO) supported by leading researchers from international institutes and aims at development of disease-related glycomics/glycoproteomics analysis techniques. Since 2004, the initiative has conducted three pilot studies. The first two were N- and O-glycan analyses of purified transferrin and immunoglobulin-G and assessed the most appropriate analytical approach employed at the time. This paper describes the third study, which was conducted to compare different approaches for quantitation of N- and O-linked glycans attached to proteins in crude biological samples. The preliminary analysis on cell pellets resulted in wildly varied glycan profiles, which was probably the consequence of variations in the pre-processing sample preparation methodologies. However, the reproducibility of the data was not improved dramatically in the subsequent analysis on cell lysate fractions prepared in a specified method by one lab. The study demonstrated the difficulty of carrying out a complete analysis of the glycome in crude samples by any single technology and the importance of rigorous optimization of the course of analysis from preprocessing to data interpretation. It suggests that another collaborative study employing the latest technologies in this rapidly evolving field will help to realize the requirements of carrying out the large-scale analysis of glycoproteins in complex cell samples.     

Evidence for a presynaptic action of chlordimeform at the insect neuromuscular junction

The mechanism of action of chlordimeform on the mealworm nerve-muscle preparation was studied with microelectrodes. Chlordimeform affected neither the mean amplitude nor the frequency of spontaneous miniature excitatory postsynaptic potentials. Extracellular focal recordings show that in the presence of 0.8 mM chlordimeform the presynaptic spike is almost unchanged, but the quantal content for evoked transmitter release is reduced. It is suggested that chlordimeform decreases the influx of calcium at the presynaptic terminal during the active phase of the nerve terminal action potential, thereby inhibiting evoked transmitter release.     

A quantitative description of the dynamics of excitation and inhibition in the eye of Limulus

By means of intracellular microelectrode techniques, we have measured the dynamics of the several processes which translate light stimulation into spike activity in the Limulus eye. The transductions from light to voltage and from voltage to spike rate, and the lateral inhibitory transduction from spike rate to voltage, we have characterized by transfer functions. We have checked the appropriateness of treating the eye as a system of linear transducers under our experimental conditions. The response of the eye to a large spot of light undergoing sine flicker has been correctly predicted.