Reduction of the Edited Domain of the Mitochondrial A6 Gene for ATPase Subunit 6 in Trypanosomatidae

The sequence of mitochondrial A6 (MURF4) was compared for several trypanosomatids in order to assess the reduction of the edited domain (ED). The association between the ED reduction and the phylogenetic position of a species proved to be less tight than believed earlier. Compared with digenetic species, monogenetic ones more often displayed ED reduction and had smaller ED.     

Endosymbiosis in protozoa of the Trypanosomatidae family

A small number of trypanosomatids present bacterium endosymbionts in the cytoplasm, which divide synchronously with the host cell. Crithidia oncopleti, Crithidia deanei. Crithidia desouzai, Blastocrithidia culicis and Herpetomonas roitmani are the best characterized species. The endosymbiont is surrounded by two membranes separated from each other by an electron-lucent space. The presence of the endosymbiont led to the appearance of morphological changes which include the lack of the paraflagellar rod associated to the axoneme, the morphology of the kinetoplast and the association of the sub-pellicular microtubules with portions of the protozoan plasma membrane. Aposymbiotic strains could be obtained by antibiotic treatment, opening the possibility to make comparative analysis of endosymbiont-containing an endosymbiont-free populations of the same species. It is clear that metabolic cycles are established between the prokaryiont and the host cell. The results obtained show that endosymbiont-containing species of trypanosomatids constitute an excellent model to study basic processes on the endosymbiont-host cell relationship and the origin of new organelles.     

Influence of growth conditions on the production of extracellular proteolytic enzymes in Paenibacillus peoriae NRRL BD-62 and Paenibacillus polymyxa SCE2

AIMS: To analyse the extracellular protease profile of two Paenibacillus species, Paenibacillus peoriae and Paenibacillus polymyxa, as well as how different growth media influenced its expression. METHODS AND RESULTS: Both bacteria were cultured in five media [Luria-Bertani broth, glucose broth, thiamine/biotin/nitrogen broth (TBN), trypticase soy broth and a defined medium] for 48 h at 32 degrees C. Our results showed a heterogeneous protease secretion pattern whose expression was dependent on medium composition. However, TBN induced the most quantitative and qualitative protease production on both Paenibacillus. The proteases were detected in neutral-alkaline pH range, being totally inhibited by 1,10-phenanthroline, a zinc-metalloprotease inhibitor. We also analysed the protease expression during the growth and, at least to P. peoriae, the most elevated protease activity was measured at 96 h, in which the highest number of spores and a low concentration of viable cells were observed. CONCLUSIONS: The results presented add P. peoriae and P. polymyxa to the list of neutral-alkaline extracellular protease producers. SIGNIFICANCE AND IMPACT OF THE STUDY: Paenibacillus species are ubiquitous in nature, are capable to form resistant spores and to produce several hydrolytic enzymes, including proteases. However, only few data concerning the production of these enzymes are available. Proteases produced by Paenibacillus strains may represent new sources for biotechnological use.     

Protease Inhibitors in Improvement of Plant Resistance to Pathogens and Insects

This review concerns the possibility of using plant inhibitors of proteolytic enzymes to improve plant resistance to insects and phytopathogens. The main argument in favor of this approach is that protease inhibitors are widespread in plant tissues and highly active with respect to various proteases of insects, bacteria, and fungi. Genetic engineering yields promising results in the field, as recent studies demonstrate. The main drawbacks of the approach and ways to improve its efficiency are discussed. Still, the approach has several advantages over the standard methods and is ecologically safe.__________Translated from Molekulyarnaya Biologiya, Vol. 39, No. 4, 2005, pp. 702–708.Original Russian Text Copyright © 2005 by Dunaevsky, Elpidina, Vinokurov, Belozersky.     

Infection rates and pathogenicity of trypanosomatid gut parasites in the water strider Gerris odontogaster (Zett.) (Heteroptera: Gerridae)

Summary Trypanosomatid flagellates are common protozoan gut parasites of a wide range of insect species. Water striders (Gerridae) harbour the trypanosomatid Blastocrithidia gerridis. Three different populations of the water strider Gerris odontogaster in northern Sweden were sampled to assess the infection rate dynamics of trypanosomatids. The initially very low infection rates (0%–15%) early in the season were followed by a rapid increase during the reproductive period of the water striders, reaching very high levels (80%–90%). The pathogenic effects of trypanosomatids on G. odontogaster adults were studied in laboratory experiments. The parasites caused a general reduction of host vigour. Male skating endurance was negatively correlated with the intensity of the trypanosomatid infection. However, infection of trypanosomatids increased the mortality among adults only when the water striders were subjected to food stress. The trypanosomatids did not reduce the fecundity of females provided with food. We suggest that trypanosomatid gut parasites may be an important mortality factor in water strider populations. Since the pathogenicity of the parasites is enhanced by stress, parasitism by trypanosomatids may contribute to the regulation of host populations.     

The structure-function relationship in the clostripain family of peptidases.

In this study we investigate the active-site structure and the catalytic mechanism of clostripain by using a combination of three separate techniques: affinity labelling, site-directed mutagenesis and molecular modelling. A benzamidinyl-diazo dichlorotriazine dye (BDD) was shown to act as an efficient active site-directed affinity label for Clostridium histolyticum clostripain. The enzyme, upon incubation with BDD in 0.1 m Hepes/NaOH buffer pH 7.6, exhibits a time-dependent loss of activity. The rate of inactivation exhibits a nonlinear dependence on the BDD concentration, which can be described by reversible binding of dye to the enzyme prior to the irreversible reaction. The dissociation constant of the reversible formation of an enzyme-BDD complex is KD = 74.6 +/- 2.1 micro m and the maximal rate constant of inactivation is k3 = 0.21 x min(-1). Effective protection against inactivation by BDD is provided by the substrate N-benzoyl-L-arginine ethyl ester (BAEE). Cleavage of BDD-modified enzyme with trypsin and subsequent separation of peptides by reverse-phase HPLC gave only one modified peptide. Amino acid sequencing of the modified tryptic peptide revealed the target site of BDD reaction to be His176. Site-directed mutagenesis was used to study further the functional role of His176. The mutant His176Ala enzyme exhibited zero activity against BAEE. Together with previous data, these results confirm that a catalytic dyad of His176 and Cys231 is responsible for cysteine peptidase activity in the C11 peptidase family. A molecular model of the catalytic domain of clostripain was constructed using a manually extended fold recognition-derived alignment with caspases. A rigorous iterative modelling scheme resulted in an objectively sound model which points to Asp229 as responsible for defining the strong substrate specificity for Arg at the P1 position. Two possible binding sites for the calcium required for auto-activation could be located. Database searches show that clostripain homologues are not confined to bacterial lineages and reveal an intriguing variety of domain architectures.     

New species of rotifer from India belonging to the family Brachionidae

During the course of an extensive survey of the ecology and systematics of the rotifers from Andhra Pradesh, India, a rotifer species new to science, having characters related to the genera Euchlanis, Dipeuchlanis and Squatinella, has been encountered and described as Pseudoeuchlanis longipedis gen. et sp. nov.     

Comparative molecular model building of two serine proteinases from cytotoxic T lymphocytes

Two genes that are expressed when precursor cytotoxic T lymphocytes are transformed to T killer cells have been cloned and sequenced. The derived amino acid sequences, coding for cytotoxic cell protease 1 (CCP1) and Hannuka factor (HF) are highly homologous to members of the serine proteinase family. Comparative molecular model building using the known three-dimensional structures and the derived amino acid sequences of the lymphocyte enzymes has provided useful structural information, especially in predicting the conformations of the substrate binding sites. In applying this modelling procedure, we used the X-ray structures of four serine proteinases to provide a structurally based sequence alignment: alpha-chymotrypsin (CHT), bovine trypsin (BT), Streptomyces griseus trypsin (SGT), and rat mast cell protease 2 (RMCP2). The root mean square differences in alpha-carbon atom positions among these four structures when compared in a pairwise fashion range from 0.79 to 0.97 A for structurally equivalent residues. The sequences of the two lymphocyte enzymes were then aligned to these proteinases using chemical criteria and the superimposed X-ray structures as guides. The alignment showed that the sequence of CCP1 was most similar to RMCP2, whereas HF has regions of homology with both RMCP2 and BT. With RMCP2 as a template for CCP1 and the two enzymes RMCP2 and BT as templates for HF, the molecular models were constructed. Intramolecular steric clashes that resulted from the replacement of amino acid side chains of the templates by the aligned residues of CCP1 and HF were relieved by adjustment of the side chain conformational angles in an interactive computer graphics device. This process was followed by energy minimization of the enzyme model to optimize the stereochemical geometry and to relieve any remaining unacceptably close nonbonded contacts. The resulting model of CCP1 has an arginine residue at position 226 in the specificity pocket, thereby predicting a substrate preference for P1 aspartate or glutamate residues. The model also predicts favorable binding for a small hydrophobic residue at the P2 position of the substrate. The primary specificity pocket of HF resembles that of BT and therefore predicts a lysine or arginine preference for the P1 residue. The arginine at position 99 in the model of HF suggests a preference for aspartate or glutamate side chains in the P2 position of the substrate. Both CCP1 and HF have a free cysteine in the segment of polypeptide 88 to 93.(ABSTRACT TRUNCATED AT 400 WORDS)     

Proteolytic activity in <Emphasis Type="Italic">Microcystis aeruginosa</Emphasis> PCC7806 is inhibited by a trypsin-inhibitory cyanobacterial peptide with a partial structure of microviridin

This paper describes the characterization of proteases in Microcystis aeruginosa PCC7806 cells being inhibited by a metabolite produced by another Microcystis strain, Microcystis Ku1. With casein and oligopeptide substrates and specific inhibitors we detected activity similar to bacterial serine endoproteases. Substrate SDS-polyacrylamide gel electrophoresis revealed the presence of nine bands of proteases (ca. 35∼125 kDa). The cyanobacterial enzymes were insensitive to endogenous trypsin-inhibitory metabolites. Microcystis Ku1 produced a metabolite, tentatively characterized as microviridin, inhibiting both cyanobacterial proteases and trypsin at an estimated IC₅₀ of, respectively, 2.2 and 9.0 μg mL⁻¹. On activity gels, inhibitors specific to animal trypsin and elastase and the putative microviridin led to an inactivation of the proteases associated with the 88 and 110 kDa bands. We hypothesize that in Microcystis populations there is a “cross-talk” between the inhibitors and the proteases, and only the colonies of identical chemotypes can possibly aggregate to form blooms.     

Conserved amino acid sequences in the bacterial sialyltransferases belonging to Glycosyltransferase family 80

Sialyltransferases are key enzymes for the biosynthesis of sialyl-glycoproteins and sialyl-lipids and the genes encoding sialyltransferases have been cloned from mammalian and bacterial source. In the mammalian sialyltransferase, existence of three conserved regions, named sialyl motifs, has been demonstrated. On the other hand, two short motifs, named D/E-D/E-G motif and HP motif, have been reported in the bacterial sialyltransferases very recently. From the results of multiple alignments among the sialyltransferases belonging to Glycosyltransferase family 80 and crystal structures of two reported sialyltransferases, it is clearly demonstrated that the third conserved-functional motif exists in the bacterial sialyltransferases that have been classified into Glycosyltransferase family 80 in this study.     

Characterization of a Bifidobacterium longum BORI dipeptidase belonging to the U34 family

A dipeptidase was purified from a cell extract of Bifidobacterium longum BORI by ammonium sulfate precipitation and chromatography on DEAE-cellulose and Q-Sepharose columns. The purified dipeptidase had a molecular mass of about 49 kDa and was optimally active at pH 8.0 and 50 degrees C. The enzyme was a strict dipeptidase, being capable of hydrolyzing a range of dipeptides but not tri- and tetrapeptides, p-nitroanilide derivatives of amino acids, or N- or C-terminus-blocked dipeptides. A search of the amino acid sequence of an internal tryptic fragment against protein sequences deduced from the total genome sequence of B. longum NCC2705 revealed that it was identical to an internal sequence of the dipeptidase gene (pepD), which comprised 1,602 nucleotides encoding 533 amino acids with a molecular mass of 60 kDa, and thereby differed considerably from the 49-kDa mass of the purified dipeptidase. To understand this discrepancy, pepD was cloned into an Escherichia coli expression vector (pBAD-TOPO derivative) to generate the recombinant plasmids pBAD-pepD and pBAD-pepD-His (note that His in the plasmid designation stands for a polyhistidine coding region). Both plasmids were successfully expressed in E. coli, and the recombinant protein PepD-His was purified using nickel-chelating affinity chromatography and reconfirmed by internal amino acid sequencing. The PepD sequence was highly homologous to those of the U34 family of peptidases, suggesting that the B. longum BORI dipeptidase is a type of cysteine-type N-terminal nucleophile hydrolase and has a beta-hairpin motif similar to that of penicillin V acylase, which is activated by autoproteolytic processing.     

Characterization of a Mycobacterium tuberculosis insertion sequence belonging to the IS3 family

A repetitive element (IS986), previously isolated from Mycobacterium tuberculosis and shown to detect multiple restriction fragment-length polymorphisms (RFLPs), has been sequenced. It consists of a potential insertion sequence of 1358bp, with 30-bp inverted repeat ends. IS986 has four potentially significant open reading frames (ORFs): ORFa1, ORFa2 and ORFb on one strand and ORFc on the complementary strand. The sequences of the potential translated products identify IS986 as a member of the IS3 family, with an apparent frameshift between ORFa1 and ORFa2. IS986 has potential as a highly specific probe for detection and typing of M. tuberculosis, as well as for transposon mutagenesis of mycobacteria. The sequence of IS986 is virtually identical to that of another recently described element, IS6110 (Thierry et al., 1990).     

Mass spectrometry in the biology of RNA and its modifications

Many powerful analytical techniques for investigation of nucleic acids exist in the average modern molecular biology lab. The current review will focus on questions in RNA biology that have been answered by the use of mass spectrometry, which means that new biological information is the purpose and outcome of most of the studies we refer to. The review begins with a brief account of the subject "MS in the biology of RNA" and an overview of the prevalent RNA modifications identified to date. Fundamental considerations about mass spectrometric analysis of RNA are presented with the aim of detailing the analytical possibilities and challenges relating to the unique chemical nature of nucleic acids. The main biological topics covered are RNA modifications and the enzymes that perform the modifications. Modifications of RNA are essential in biology, and it is a field where mass spectrometry clearly adds knowledge of biological importance compared to traditional methods used in nucleic acid research. The biological applications are divided into analyses exclusively performed at the building block (mainly nucleoside) level and investigations involving mass spectrometry at the oligonucleotide level. We conclude the review discussing aspects of RNA identification and quantifications, which are upcoming fields for MS in RNA research. This article is part of a Special Section entitled: Understanding genome regulation and genetic diversity by mass spectrometry.     

Trypsin and chymotrypsin-Iike enzymes of the sandfly Phlebotomus papatasi infected with Leishmania and their possible role in vector competence

Phlebotomus papatasi (Scopoli) is susceptible to infection with Leishmania major Yakimov & Schokov and resistant to L. donovani Laveran & Mesnil. The possibility that susceptibility depends on midgut levels of trypsin and chymotrypsin-like (esterolytic) enzymes was investigated. Infection with L. major reduced the trypsin-like activity to 93.5% and 86% of the control value at 20 and 30 h post feeding and increased it to 106% at 52 h. Infection with L. donovani reduced trypsin-like activity to 64% and 73% of the control value at 30 and 52 h post feeding. The overall amount of trypsin and chymotrypsin-like enzymes in L. major infections was reduced to 50% and 34% of the control value at 20 and 30 h post feeding and increased to 184% at 52 h. Only one of the enzymes separated by gel electrophoresis was lower throughout, i.e. peak D. Overall, the midgut enzyme level with L. donovani infection was 86% of the control value at 30 h post feeding and 105% at 52 h; their relative amounts changed throughout. Soybean trypsin inhibitor enabled L. donovani to survive and multiply in P. papatasi. It is suggested that a specific component of the trypsin-like activity prevents the survival of L. donovani in P. papatasi and that modulation of this factor enables L. major to survive.     

Detection and identification of bacterial endosymbionts in arbuscular mycorrhizal fungi belonging to the family Gigasporaceae

Intracellular bacteria have been found previously in one isolate of the arbuscular mycorrhizal (AM) fungus Gigaspora margarita BEG 34. In this study, we extended our investigation to 11 fungal isolates obtained from different geographic areas and belonging to six different species of the family Gigasporaceae. With the exception of Gigaspora rosea, isolates of all of the AM species harbored bacteria, and their DNA could be PCR amplified with universal bacterial primers. Primers specific for the endosymbiotic bacteria of BEG 34 could also amplify spore DNA from four species. These specific primers were successfully used as probes for in situ hybridization of endobacteria in G. margarita spores. Neighbor-joining analysis of the 16S ribosomal DNA sequences obtained from isolates of Scutellospora persica, Scutellospora castanea, and G. margarita revealed a single, strongly supported branch nested in the genus Burkholderia.     

A modified CTAB DNA extraction procedure for plants belonging to the family proteaceae

This paper describes rapid and efficient DNA extraction methods for mature leaves, resting buds and seedling leaves of genera in the family Proteaceae. The procedures combine and modify previously published techniques. The DNA can be digested by restriction endonucleases and is suitable for subsequent PCR amplification.     

Comparison of nucleotide sequences of mRNAs belonging to the mouse H-2 multigene family

The complete nucleotide sequences of three cDNAs coding for the C-terminal part of mouse histocompatibility (H-2) antigens, and for the 3' non coding regions of these clones have been determined. Comparison of the sequence indicates a large homology throughout the coding and non-coding regions and suggests the existence of a genetic mechanism which homogenizes nucleotide sequences among genes of the H-2 multigene family.