Studies on the catalytic mechanism of H2-forming methylenetetrahydromethanopterin dehydrogenase: para-ortho H2 conversion rates in H2O and D2O

H₂–forming N ⁵,N ¹⁰ –methylenetetrahydromethanopterin dehydrogenase is a novel type of hydrogenase that contains neither nickel nor iron-sulfur clusters. Evidence has been presented that the reaction mechanism catalyzed by the enzyme is very similar to that of the formation of carbocations and H₂ from alkanes under superacidic conditions. We present here further results in support of this mechanism. It was found that the purified enzyme per se did not catalyze the conversion of para H₂ to ortho H₂, a reaction catalyzed by all other hydrogenases known to date. However, it catalyzed the conversion in the presence of the substrate N ⁵,N ¹⁰ –methenyltetrahydromethanopterin (CH≡H₄MPT⁺), indicating that for heterolytic cleavage of H₂ the enzyme-CH≡H₄MPT⁺ complex is required. In D₂O, the formation of HD and D₂ from H₂ rather than a para–ortho H₂ conversion was observed, indicating that after heterolytic cleavage of H₂ the dissociation of the proton from the enzyme-substrate complex is fast relative to the re-formation of free H₂.     

Stability of α-chymotrypsin conjugated with poly (ethylene glycols) and proxanols at high temperature and in watercosolvent mixtures

Summary -Chymotrypsin has been modified with poly(ethylene glycols) and proxanols, block-copolymers of poly(propylene oxide) and poly(ethylene oxide). These conjugates were several-fold more thermostable and showed high catalytic activity at elevated concentrations of water-miscible organic cosolvents (alcohols and dimethyl sulfoxide) which caused inactivation of free (non-modified) -chymotrypsin.     

A transposon insertion in the Arabidopsis SSR16 gene causes an embryo-defective lethal mutation

The SSR16 gene of Arabidopsis has been identified as a gene encoding a ribosomal protein S16 homolog through analysis of a transposon insertion mutation. The insertion mutation is lethal, arresting embryonic development at approximately the transition from the globular to the heart stage of embryonic development. Co-segregation of the mutant phenotype with the transposon-borne drug-resistance marker and loss of the inserted transposon concomitant with phenotypic reversion provided evidence that the transposon had caused the mutation. Sequences flanking the insertion site were amplified from DNA of viable heterozygotes by thermal asymmetric interlaced (TAIL) PCR. The amplified fragment flanking the 3' end of the inserted element was sequenced and found to be identical to an Arabidopsis expressed sequence tag (EST). The EST, in turn, contained a coding sequence homologous to the ribosomal protein S16 (RPS16) of bacteria such as Escherichia coli, Bacillus subtilis and Salmonella typhimurium , as well as Neurospora crassa mitochondria and higher plant plastids. Thus the gene identified by the embryo-defective lethal insertion mutation encodes an RPS16 homolog and has been designated the SSR16 gene.     

Size of genetic bottlenecks leading to virus fitness loss is determined by mean initial population fitness.

Genetic bottlenecks are important events in the genetic diversification of organisms and colonization of new ecological niches. Repeated bottlenecking of RNA viruses often leads to fitness losses due to the operation of Muller's ratchet. Herein we use vesicular stomatitis virus to determine the transmission population size which leads to fitness decreases of virus populations. Remarkably, the effective size of a genetic bottleneck associated with fitness loss is greater when the fitness of the parental population increases. For example, for starting virus populations with low fitness, population transfers of five-clone-to-five-clone passages resulted in a fitness increase. However, when a parental population with high fitness was transferred, 30-clone-to-30-clone passages were required simply to maintain fitness values.     

Extreme fitness differences in mammalian and insect hosts after continuous replication of vesicular stomatitis virus in sandfly cells.

Continuous, persistent replication of a wild-type strain of vesicular stomatitis virus in cultured sandfly cells for 10 months profoundly decreased virus replicative fitness in mammalian cells and greatly increased fitness in sandfly cells. After persistent infection of sandfly cells, fitness was over 2,000,000-fold greater than that in mammalian cells, indicating extreme selective differences in the environmental conditions provided by insect and mammalian cells. The sandfly-adapted virus also showed extremely low fitness in mouse brain cells (comparable to that in mammalian cell cultures). It also showed an attenuated phenotype, requiring a nearly millionfold higher intracranial dose than that of its parent clone to kill mice. A single passage of this adapted virus in BHK-21 cells at 37 degrees C restored fitness to near neutrality and also restored mouse neurovirulence. These results clearly illustrate the enormous capacity of RNA viruses to adapt to changing selective environments.     

A review of issues related to the quantification of bacteria from the phyllosphere

Abstract: Quantification of the size of epiphytic bacterial populations and characterization of their composition involves definition of a sampling strategy in time and space, the choice of methods for liberating the bacteria from the leaf surface and for recovering them for subsequent determination of the number of viable or culturable cells. This literature review focuses on some of the issues related to these choices. After briefly reviewing the different types of epiphytic colonizers we consider the biological, methodological, and statistical consequences of the choice of the sampling unit and of the spatial and temporal variability of population size and composition for epiphytic bacteria. The different methods available for the detection and enumeration of naturally occurring microorganisms in the phyllosphere are discussed. Advantages and drawbacks of each are described in this review designed as a 'hands-on' guide.     

Localization and neuroanatomy of catecholaminergic neurons in some rotifer species

We studied Dicranophorus sp., Platyias quadricornis (Ehrb.) and Rotaria tardigrada (Ehrb.). These rotifers, systematically distant from each other, show the same pattern of the catecholaminergic (CA-ergic) part of the nervous system. It is formed of a small (23–24), but steady number of neurons characteristic for each species. Three types of CA-ergic neurons are described. The sizes of neurons vary from to two to ten µm. The distribution of the brain neurons is correlated with body shape. Such a type of nervous system is topographically comparable to the concentrated orthogon of the flatworms.     

A Drosophila hsp70 gene contains long,antiparallel, coupled open reading frames (LAC ORFs) conserved in homologous loci

A clone isolated from a Drosophila auraria heat-shock cDNA library presents two long, antiparallel, coupled (LAC) open reading frames (ORFs). One strand ORF is 1,929 nucleotides long and exhibits great identity (87.5% at the nucleotide level and 94% at the amino acid level) with the hsp70 gene copies of D. melanogaster, while the second strand ORF, in antiparallel in-frame register arrangement, is 1,839 nucleotides long and exhibits 32% identity with a putative, recently identified, NAD⁺-dependent glutamate dehydrogenase (NAD⁺-GDH). The overlap of the two ORFs is 1,824 nucleotides long. Computational analysis shows that this LAC ORF arrangement is conserved in other hsp70 loci in a wide range of organisms, raising questions about possible evolutionary benefits of such a peculiar genomic organization.Correspondence to: Z.G. Scouras