Magnesium ion effects on microtubule nucleation in vitro

Much interest has currently been attached to the length distribution of microtubules polymerized in vitro and the related question of their possible 'dynamic instability'. Fundamental to this question is the mechanism of microtubule nucleation, which controls the rates of assembly and disassembly of microtubule protein in vitro. These kinetics are affected by a number of factors, including both the guanine nucleotides, GTP and GDP, and magnesium ion. Mg2+ exerts complex effects, as indicated by the existence of an optimal Mg2+ concentration for the maximum assembly rate of microtubule protein, and we investigate these effects in this report. At [Mg2+] greater than 0.5 mM, the characteristic lag-phase is substantially increased and the rate of assembly is greatly reduced without affecting the critical concentration significantly. We show that increasing [Mg2+] has two effects on the assembly process: nucleation is less efficient and the intrinsic rate constant for the elongation reaction is reduced. Lowering [Mg2+] (less than 0.5 mM) also inhibits nucleation. These effects of varying [Mg2+] can be explained predominantly in terms of enhanced stability of the microtubule-associated protein-containing oligomeric species present in the microtubule protein preparation. [Mg2+] is thus found to be a further important factor in microtubule nucleation, and hence, in determining length distributions in assembling microtubules.     

Seminiferous tubule involution in elderly men

The observation of different types of seminiferous tubules (from tubules with normal spermatogenesis to sclerosed tubules) in aging human testes points to the progressive stages of tubular involution in elderly men. The tubules with hypospermatogonesis (reduced number of elongated spermatids) show numerous morphological anomalies in the germ cells, including multinucleated cells. Abnormal germ cells degenerate, causing Steroli cell vacuolation. These vacuoles correspond to dilations of the extracellular spaces resulting from the premature exfoliation of germ cells. Degenerating cells that are phagocytized by Sertoli cells lead to an accumulation of lipid droplets in the Sertoli cell cytoplasm. The loss of germ cells begins with spermatids, but progressively affects the preceding germ cell types, and tubules with maturation arrested at the level of spermatocytes or spermatogonia are observed. Simultaneously, an enlargement of the tunica propria occurs. This leads to the formation of sclerosed tubules, some of which display a low seminiferous epithelium consisting of a few cells--including lipid-loaded Sertoli cells and both Ap and Ad spermatogonia--and others, showing complete sclerosis, are devoid of seminiferous epithelium. The development of tubular involution is similar to that reported after experimental ischemia, which also seems to cause nonspecific effects on the testis such as multinucleate cells, vacuoles, and increased lipids in Sertoli cells.     

Human retroviral sequences on the Y chromosome.

Novel endogenous human retroviral sequences were cloned by low-stringency hybridization, using the pol gene of endogenous human retrovirus 51-1. One clone, lambda NP-2, contained gag, pol, env, and long terminal repeat sequences related to the corresponding portions of clone 51-1 and the closely related full-length endogenous human retrovirus 4-1. The sequence of the env gene of NP-2 was 73% homologous to that of 4-1. Genomic Southern blots of male and female DNAs showed that NP-2 is located on the Y chromosome and that the Y chromosome also contains one other sequence closely related to the env and 3' flanking regions of NP-2. Conservation of flanking DNA suggests that the second Y chromosome copy of the NP-2 env sequence arose by gene duplication rather than provirus insertion.     

Regulatory properties of brain glutamate decarboxylase

1. Glutamate decarboxylase is a focal point for controlling gamma-aminobutyric acid (GABA) synthesis in brain. Several factors that appear to be important in the regulation of GABA synthesis have been identified by relating studies of purified glutamate decarboxylase to conditions in vivo. 2. The interaction of glutamate decarboxylase with its cofactor, pyridoxal 5'-phosphate, is a regulated process and appears to be one of the major means of controlling enzyme activity. The enzyme is present in brain predominantly as apoenzyme (inactive enzyme without bound cofactor). Studies with purified enzyme indicate that the relative amounts of apo- and holoenzyme are determined by the balance in a cycle that continuously interconverts the two. 3. The cycle that interconverts apo- and holoenzyme is part of the normal catalytic mechanism of the enzyme and is strongly affected by several probable regulatory compounds including pyridoxal 5'-phosphate, ATP, inorganic phosphate, and the amino acids glutamate, GABA, and aspartate. ATP and the amino acids promote apoenzyme formation and pyridoxal 5'-phosphate and inorganic phosphate promote holoenzyme formation. 4. Numerous studies indicate that brain contains multiple molecular forms of glutamate decarboxylase. Multiple forms that differ markedly in kinetic properties including their interactions with the cofactor have been isolated and characterized. The kinetic differences among the forms suggest that they play a significant role in the regulation of GABA synthesis.     

Synthesis of methyl 2-acetamido-2-deoxy-3-O-(β-d-galactopyranosyl)-α-d-galactopyranoside from a corresponding thioglycoside derivative

The title disaccharide glycoside was synthesized by halide ion-promoted glycosidation, using methanol and the disaccharide bromide derived from methyl 2-azido-3-O-(2,3,4,6-tetra-O-benzoyl--d-galactopyranosyl)-4,6-O-benzylidene-2-deoxy-1-thio--d-galactopyranoside. This derivative in turn was prepared by silver triflate-promoted condensation of monosaccharide derivatives.     

Synthesis of 3-O-β-d-xylopyranosyl-l-serine (xylosylserine) andO-β-d-galactopyranosyl-(1-4)-O-β-d-xylopyranosyl-l-serine (galactosylxylosylserine) and use of the synthetic products for detection of galactosyltransferase I activity in rat liver

3-O-β-d-Xylopyranosyl-l-serine (xylosylserine) was synthesized by the following three-step procedure: 1) 2,3,4-tri-O-benzoyl-α-d-xylopyranosyl bromide (benzobromoxylose) was condensed withN-carbobenzoxy-l-serine benzyl ester using the silver triflate-collidine complex as promoter; 2) theN-carbobenzoxy and benzyl ester groups in the resultant glycoside were cleaved by transfer hydrogenation with palladium black as catalyst and ammonium formate as hydrogen donor; and 3) the benzoyl groups were removed with methanolic ammonia. Xylosylserine was obtained in an overall yield of 70%. O-β-d-Galactopyranosyl-(1-4)-O-β-d-xylopyranosyl-(1-3)-l-serine (galactosylxylosylserine) was also synthesized by this methodology and was characterized by 2-dimensional (2D) NMR spectroscopy techniques. The two serine glycosides (xylosylserine and galactosylxylosylserine) were used in detection and partial purification of galactosyltransferase I (UDP-d-galactose:d-xylose galactosyltransferase) from adult rat liver.     

Carbon and nitrogen metabolism in ectomycorrhizal fungi and ectomycorrhizas

The literature concerning the metabolism of carbon and nitrogen compounds in ectomycorrhizal associations of trees is reviewed. The absorption and translocation of mineral ions by the mycelia require an energy source and a reductant which are both supplied by respiratory catabolism of carbohydrates produced by the host plant. Photosynthates are also required to generate the carbon skeletons for amino acid and carbohydrate syntheses during the growth of the mycelia. Competition for photosynthates occurs between the fungal cells and the various vegetative sinks in the host tree. The nature of carbon compounds involved in these processes, their routes of metabolism, the mechanisms of control and the partitioning of metabolites between the various sites of utilization are only poorly understood. Both ascomycetous and basidiomycetous ectomycorrhizal fungi synthesize and some, if not all, accumulate mannitol, trehalose and triglycerides. The fungal strains employ the Embden--Meyerhof pathway of glucose catabolism and the key enzymes of the pentose phosphate pathway (6-phosphogluconate dehydrogenase, glucose-6-phosphate dehydrogenase, transaldolase and transketolase). Anaplerotic CO2 fixation, via pyruvate carboxylase and/or phosphoenolpyruvate carboxykinase, provides high pools of amino acids. This process could be important in the recapture and assimilation of respired CO2 in the rhizosphere. The ectomycorrhizas are thought to contain the Embden--Meyerhof pathway, the pentose phosphate pathway and the tricarboxylic acid cycle, which provide the carbon skeletons for the assimilation of ammonia into amino acids. The main route of assimilation of ammonia appears to be through the glutamine synthetase-glutamate synthase cycle in the ectomycorrhizas. Glutamate dehydrogenase plays a minor role in this process. Glutamate dehydrogenase and glutamine synthetase are present in free-living ectomycorrhizal fungi and they participate in the assimilation of ammonia and the synthesis of amino acids through the glutamate dehydrogenase/glutamine synthetase sequence. In both in vitro cultures of fungi and ectomycorrhizas, the assimilated nitrogen accumulates in glutamine. Glutamine, but also ammonia, are thought to be exported from the fungal tissues to the host cells. Studies on the metabolism of ectomycorrhizas and ectomycorrhizal fungi have focused on the metabolic pathways and compounds which accumulate in the symbiotic tissues. Studies on regulation of the overall process, and the control of enzyme activity in particular, are still fragmentary.(ABSTRACT TRUNCATED AT 400 WORDS)     

Convergence to equilibrium in a genetic model with differential viability between the sexes

A single locus, diallelic selection model with female and male viability differences is studied. If the variables are ratios of allele frequencies in each sex, a 2-dimensional difference equation describes the model. Because of the strong monotonicity of the resulting map, every initial genotypic structure converges to an equilibrium structure assuming that no equilibrium has eigenvalues on the unit circle.Partially supported by funds provided by a Science and Education Grant to the USDA-Forest Service, Southeastern Forest Experiment Station, Population Genetics of Forest Trees Research Unit, Raleigh, USASupported by a grant from the Max Kade Foundation, New York, USA     

Purification and separation of two soluble fucosyltransferase activities of small intestinal mucosa

1. Rat small intestinal soluble fucosyltransferase is purified more than 2000-fold using chromatographic procedures with DEAE-cellulose, CM-cellulose, GDP-Sepharose and Concanavalin A-Sepharose. 2. Chromatography on Sephadex G15 of the final enzymatic fraction clearly separates two activities: a first peak incorporates fucose on asialoserotransferrin and a second peak on asialofetuin. 3. The use of small saccharidic acceptors (phenylgalactose, lactose, lacto-N-fucopentaose I) and the analysis of fucosylated asialoglycoproteins indicate that the first activity corresponds to an alpha-(3/4)-fucosyltransferase and the second one to an alpha-(1-2)-fucosyltransferase. 4. Protein analysis by polyacrylamide gel electrophoresis in the presence of SDS for each enzyme shows two bands corresponding to a mol. wt of about 65,000 and 70,000. The two enzymes have the same sensitivity to the action of N-ethylmaleimide.