The Detection of Cyanobacterial Cells by a Non-Fluorescent in situ Hybridization Method

Seven cyanobacterial strains (Anabaena macrospora NIER10016, Oscillatoria sp. NIER10042, Microcystis aeruginosa NIER10015, M. ichtyoblabe NIER10025 and NIER10040, M. novacekii NIER10029, and M. wesenbergii NIER10068) were tested by a nonfluorescent in situ hybridization method using two specific horseradish peroxidase–labeled oligonucleotide probes and two chromogenic substrates. This approach was shown to be appropriate for the analysis of natural samples.     

Taxonomic studies on the Mycobacterium tuberculosis series

Numerical classification of slowly growing mycobacteria, including 159 strains received as Mycobacterium tuberculosis, M. bovis, M. africanum, and M. microti, was carried out using 88 characters, and the following results were obtained. 1) All 159 strains received as M. tuberculosis, M. bovis, M. africanum, and M. microti formed one cluster, and no clear-cut differentiation among four species was achieved. These four species should be reduced to one species, M. tuberculosis. 2) Within the cluster, two subclusters appeared, with a number of strains located outside the subclusters. One subcluster was composed, except for two strains, of only M. tuberculosis strains, and another of M. bovis and M. africanum strains only. The subclusters were regarded as subspecies tuberculosis and subspecies bovis, respectively. M. africanum was regarded as a synonym of M. bovis, i.e., a niacin-positive variety of M. bovis (subsp. bovis). 3) An intermediate subcluster was observed outside the M. tuberculosis and M. bovis subclusters. This may be regarded as intermediate between the two subspecies, tuberculosis and bovis. 4) In the last two decades, the characteristics of M. tuberculosis strains isolated from patients seemed to be approaching those of M. bovis strains. The recently isolated strains showed stronger arylsulfatase activity and lower resistance to thiophene-2-carboxylic acid hydrazide than the strains isolated 16 to 19 years ago.     

Site of alkylation of the major ribonuclease from Aspergillus saitoi with iodoacetate

A base non-specific and adenylic acid preferential ribonuclease from Aspergillus saitoi (RNase M) was modified by [14C]iodoacetic acid. RNase M was inactivated with concomitant incorporation of about 1 mol equivalent of carboxymethyl group. Carboxymethylated RNase M (CM RNase M) thus obtained was reduced and carboxymethylated (RCM CM RNase M). From tryptic and chymotryptic digests of RCM CM RNase M, two carboxymethylated histidine-containing peptides labeled with radioactivity were isolated. The amino acid sequences of these two peptides were determined to be Thr-Ile-His-Gly-Leu-Trp-Pro-Asp-Asn-Cys-Asp-Gly-Ser-Tyr... and His-Gly-Thr-Cys-Ile-Asn-Thr-Ile-Asp-Pro-Ser-Cys-Tyr-Pro-Asp-Asp-Tyr-Ala. .... The distribution of the radioactivity on the former and latter peptides was 43% and 57%, respectively. The results indicated that two histidine residues are involved in the active site of RNase M, and the modification of either one of the two histidine residues inactivates RNase M. The CD spectrum of carboxymethylated RNase M indicated that some tryptophan residue(s) with a CD band at 287 nm is in the proximity of the active site histidine residues of RNase M.     

Flavonoid chemistry of the endemic species of Myrceugenia (Myrtaceae) of the Juan Fernandez Islands and relatives in continental South America

Twenty-one flavonoids were isolated from the leaves of two endemic species ofMyrceugenia on the Juan Fernandez Islands,M. fernandeziana andM. schulzei, from two related species from Brazil,M. campestris andM. rufescens, and from five species from continental Chile,M. colchaguensis, M. exsucca, M. lanceolata, M. pinifolia, andM. rufa. A phenetic analysis was used to evaluate chemical similarities.Myrceugenia campestris andM. rufescens appear most closely related to each other based on flavonoid profiles, and they are also different from the other seven species. The two endemic species in the Juan Fernandez Islands,M. fernandeziana andM. schulzei, group with the continental Chilean species. The former is most closely related toM. lanceolata, and the latter clusters withM. exsucca, although somewhat distantly. The results suggest that the two Juan Fernandez endemics are derived from two introductions from the Chilean continent and not from immigrants from the eastern side of the Andes.     

Inhibition of human blood coagulation factor Xa by alpha 2-macroglobulin

The inactivation of activated factor X (factor Xa) by alpha 2-macroglobulin (alpha 2M) was studied. The second-order rate constant for the reaction was 1.4 X 10(3) M-1 s-1. The binding ratio was found to be 2 mol of factor Xa/mol of alpha 2M. Interaction of factor Xa with alpha 2M resulted in the appearance of four thiol groups per molecule of alpha 2M. The apparent second-order rate constants for the appearance of thiol groups were dependent on the factor Xa concentration. Sodium dodecyl sulfate gradient polyacrylamide gel electrophoresis was used to study complex formation between alpha 2M and factor Xa. Under nonreducing conditions, four factor Xa-alpha 2M complexes were observed. Reduction of these complexes showed the formation of two new bands. One complex (Mr 225,000) consisted of the heavy chain of the factor Xa molecule covalently bound to a subunit of alpha 2M, while the second complex (Mr 400,000) consisted of the heavy chain of factor Xa molecule and two subunits of alpha 2M. Factor Xa was able to form a bridge between two subunits of alpha 2M, either within one molecule of alpha 2M or by linking two molecules of alpha 2M. Complexes involving more than two molecules of alpha 2M were not formed.     

Contribution of each membrane binding domain of the CTP:phosphocholine cytidylyltransferase-alpha dimer to its activation, membrane binding, and membrane cross-bridging

CTP:phosphocholine cytidylyltransferase (CCT), a rate-limiting enzyme in phosphatidylcholine synthesis, is regulated by reversible membrane interactions mediated by an amphipathic helical domain (M) that binds selectively to anionic lipids. CCT is a dimer; thus the functional unit has two M domains. To probe the functional contribution of each domain M we prepared a CCT heterodimer composed of one full-length subunit paired with a CCT subunit truncated before domain M that was also catalytically dead. We compared this heterodimer to the full-length homodimer with respect to activation by anionic vesicles, vesicle binding affinities, and promotion of vesicle aggregation. Surprisingly for all three functions the dimer with just one domain M behaved similarly to the dimer with two M domains. Full activation of the wild-type subunit was not impaired by loss of one domain M in its partner. Membrane binding affinities were the same for dimers with one versus two M domains, suggesting that the two M domains of the dimer do not engage a single bilayer simultaneously. Vesicle cross-bridging was also unhindered by loss of one domain M, suggesting that another motif couples with domain M for cross-bridging anionic membranes. Mutagenesis revealed that the positively charged nuclear localization signal sequence constitutes that second motif for membrane cross-bridging. We propose that the two M domains of the CCT dimer engage a single bilayer via an alternating binding mechanism. The tethering function involves the cooperation of domain M and the nuclear localization signal sequence, each engaging separate membranes. Membrane binding of a single M domain is sufficient to fully activate the enzymatic activity of the CCT dimer while sustaining the low affinity, reversible membrane interaction required for regulation of CCT activity.     

The membrane (M1) protein of influenza virus occurs in two forms and is a phosphoprotein.

The membrane (M1) protein of influenza virus was found to be heterogenous and to occur in two forms in the virus particle. The two forms of M1 were found in virus which was produced both early and late after infection and in infected cells. The two forms could be separated on polyacrylamide gels under specific conditions. The two components of M1 contained similar tryptic peptides. However, a small proteolytic difference between the two proteins could not be ruled out. Both M1 proteins were present in phosphorylated form in the virus particle. The phosphorylated M1 components were not readily distinguished from phosphorylated nonstructural protein (NS1) when cytoplasm of infected cells was analyzed on polyacrylamide gels. The two phosphorylated M1 components could, however, be detected in infected cells by immunoprecipitation. One M1 component contained only phosphoserine whereas the second contained phosphoserine and a small amount of phosphothreonine as well. In addition to the phosphorylated nucleoprotein and M1, a third phosphorylated protein was routinely detected in virus particles. It was a surface component of the virus, since it could be removed from whole virus with chymotrypsin and contained phosphate at serine residues. The identity of this component was not known.     

Identification of commercial hake and grenadier species by proteomic analysis of the parvalbumin fraction

Analysis of parvalbumin fractions through proteomic methodologies allowed the differential classification of ten commercial, closely related species of the family Merlucciidae. Muscle extracts from nine hake species of the genus Merluccius including two subspecies of Merluccius australis (australis and polylepsis) and one grenadier species Macruronus novaezelandiae with two populations (novaezelandiae and magellanicus) were evaluated by 2-DE and MALDI-TOF MS. 2-DE demonstrated that the species tested displayed a low intra-specific degree of polymorphism and the isoform patterns were noticeably species-specific. MALDI-TOF mass fingerprints showed clear differences in the pattern of peptides produced by tryptic digestion between the Merluccius and the Macruronus, making the genus differentiation possible. In addition, a selective peptide mass present in the spectra from certain hakes allowed its classification in two groups: Euro-African and American hakes. Besides, some specific masses allowed a clearly individual identification for M. bilinearis, M. australis polylepsis, M. australis australis, M. productus, M. paradoxus and M. polli, while the rest of the hake species can be grouped in two clusters, comprising M. hubbsi and M. gayi in one and M. merluccius and M. capensis in the other.     

试用聚类方法探讨中国雪雀的分类地位

应用聚类方法对雪雀属、种及部分亚种的分类地位进行初步探索,选取一定的数量性状,取平均值组成原始数据矩阵。原始数据矩阵再进一步标准化,然后应用距离系数和相关系数进行聚类分析。比较了数量性状的聚类结果与传统的形态特征分类之间的异同,并探讨了雪雀属种级分类单元的亲缘关系和分类地位。     

Direct photoaffinity labeling of the catalytic site of mouse ribonucleotide reductase by CDP

Ribonucleotide reductase reduces all four ribonucleoside diphosphates to the deoxyribonucleotides required for DNA synthesis. The enzyme is composed of two nonidentical subunits, M1 and M2. The 89-kilodalton M1 subunit contains at least two allosteric sites which, by binding nucleotide effectors, regulate the catalytic activity and substrate specificity of the enzyme. We now show that in addition, protein M1 contains a substrate-binding (catalytic) site which is specifically photolabeled after UV irradiation in the presence of the natural substrate, [32P]CDP. The photolabeling of protein M1 by [32P]CDP required the presence of the second subunit, protein M2, and ATP, the positive allosteric effector for CDP reduction. The negative effectors, dATP, dGTP, and dTTP, inhibited the photolabeling of wild type protein M1. Deoxy-ATP did not inhibit the labeling of a mutant protein M1 that is resistant to feedback inhibition by dATP. In addition, hydroxyurea and 4-methyl-5-aminoisoquinoline thiosemicarbazone, two inhibitors of ribonucleotide reductase which affect protein M2, also inhibited the [32P]CDP labeling of protein M1. These data provide new insights into the role and interaction of the two ribonucleotide reductase subunits, proteins M1 and M2, and the mechanism of action of the allosteric effectors.     

A new species of Mimulus endemic to copper mines in California

MACNAIR, M. R., 1989. A new species of Mimulus endemic to copper mines in California . The distribution and morphology of Mimulus cupriphilus , a new species restricted to two small copper mines in Calaveras County, California, is described. A comparison of flowers collected in the wild of M. guttatus and the new taxon shows that they differ considerably. These two species and three other species from the M. guttatus complex, M. nasutus, M. laciniatus and M. nudatus were grown in a glasshouse and compared. The five species were all clearly distinct. The distribution of M. cupriphilus is consistent with it having evolved comparatively recently on one or both of the two copper mines on which it is found.     

Breeding season, courtship behaviour, and territoriality of White and Japanese Wagtails Motacilla alba and M. grandis

The breeding ecology of White and Japanese Wagtails, Motacilla alba and M. grandis , was studied along a river in central Japan. The home ranges of the two species greatly overlapped along the river, but no interbreeding occurred. M. grandis spent more time on the river than M. alba and defended territories there. M. alba used the river as part of the entire home range, and did not defend the river areas as territories. Singing activity and breeding started earlier in M. grandis than in M. alba. The early breeding of M. grandis was related to the lack of moulting in spring, less necessity for pair formation due to the existence of pairs in the winter, and the greater dependency on larval than on adult insects. Songs were very different between the two species. The bowing display that preceded the pre-copulation display was found only in M. alba. During the pre-copulation display, male M. grandis lifted both wings above the horizontal while male M. alba drooped both wings. The pre-copulation display of M. grandis was similar to that of Large Pied Wagtails M. maderaspatensis in India, suggesting a closer relationship of the two species than to M. alba.     

山罗花和天柱山罗花四个居群的数值分析

对山罗花和天柱山罗花的4个居群的15项形态特征进行了数值分析。结果表明:形态性状在4个居群间均存在一定程度的变异,居群间变异系数的平均值从小到大排列为:天柱山居群(天柱山罗花)、天柱山居群(山罗花)、黄山居群(山罗花)、鹞落坪居群(山罗花);天柱山罗花与山罗花种间的形态差异已达到极显著水平,山罗花的3个居群间部分性状也具有极显著的形态差异;以形态特征为基础的Q-聚类分析可以把天柱山罗花和山罗花聚为两类,但山罗花(天柱山居群)和其他2个居群在较远处聚在一起,R-聚类分析发现了强正相关关系、弱正相关关系、弱负相关关系的性状。     

M BAND PROTEIN : Two Components Isolated from Chicken Breast Muscle

M band protein can be specifically extracted from fresh chicken breast muscle myofibrils suspended in 5 mM Tris-HCl pH 8.0. During discontinuous polyacrylamide gel electrophoresis the isolated protein separates into three bands which can be identified as two separate components (A, B) and a complex of the two. When partially purified fractions of the separated components are combined, an increase in the intensity of the band containing the complex can be shown. The polypeptide chain weights of the two components are 100,000 (A) and 40,000 (B) daltons as estimated by sodium dodecyl sulfate- (SDS-) polyacrylamide gel electrophoresis. Antibody prepared against total M band protein stains only the M band of the myofibril and is completely absorbed by M band protein. M band protein also absorbs the M band staining specifically from antibody which stains both I and M bands. Immunodiffusion data indicate that anti-M band is a mixture of two specific antibodies, one against each component.     

Characterization of the gene and protein of the common alpha 1-antitrypsin normal M2 allele.

The normal M2 variant of alpha 1-antitrypsin (alpha 1AT) was cloned from a genomic DNA library of an individual homozygous for this allele. Sequencing of all coding exons of the M2 gene revealed it was identical to the common M1(Val213) gene except for two bases (M1(Val213) CGT Arg101, M2 CAT His101; M1(Val213) GAA Glu376 M2 GAC Asp376). Analysis of the sequence of the M1(Val213) and M2 genes around residue 101 revealed the M1 Arg101----M2 His101 caused a loss of the cutting site for the restriction endonuclease RsaI. Using this enzyme, as well as 19-mer oligonucleotides probes centered at residues 101 and 376, evaluation of genomic DNA from 22 M1 alleles and 14 M2 alleles revealed that residue 101 was Arg in all M1 alleles and His in all M2 alleles, while residue 376 was Glu in all M1 alleles and Asp in all M2 alleles. Despite the differences in sequence at two amino acids, the M1(Val213) and M2 proteins function similarly as assessed by quantification of the association rate constant of each for their natural substrate neutrophil elastase. In the context that there are two mutations separating the M1(Val213) and M2 alleles, it is likely that there is another alpha 1AT variant that was an intermediate in the evolution of these genes.     

Finite element analysis of implant-supported prosthesis with pontic and cantilever in the posterior maxilla

The aim of this study was to evaluate the influence of pontic and cantilever designs (mesial and distal) on 3-unit implant-retained prosthesis at maxillary posterior region verifying stress and strain distributions on bone tissue (cortical and trabecular bones) and stress distribution in abutments, implants and fixation screws, under axial and oblique loadings, by 3D finite element analysis. Each model was composed of a bone block presenting right first premolar to the first molar, with three or two external hexagon implants (4.0 × 10 mm), supporting a 3-unit splinted dental fixed dental prosthesis with the variations: M1 – three implants supporting splinted crowns; M2 – two implants supporting prosthesis with central pontic; M3 – two implants supporting prosthesis with mesial cantilever; M4 – two implants supporting prosthesis with distal cantilever. The applied forces were 400 N axial and 200 N oblique. The von Mises criteria was used to evaluate abutments, implants and fixation screws and maximum principal stress and microstrain criteria were used to evaluate the bone tissue. The decrease of the number of implants caused an unfavorable biomechanical behavior for all structures (M2, M3, M4). For two implant-supported prostheses, the use of the central pontic (M2) showed stress and strain distributions more favorable in the analyzed structures. The use of cantilever showed unfavorable biomechanical behavior (M3 and M4), mainly for distal cantilever (M4). The use of three implants presented lower values of stress and strain on the analyzed structures. Among two implant-supported prostheses, prostheses with cantilever showed unfavorable biomechanical behavior in the analyzed structures, especially for distal cantilever.     

Habitat separation of sympatric Microcebus spp. in the dry spiny forest of south-eastern Madagascar

We investigated whether or not habitat structure contributes to the separation of two sister species of lemurs and their hybrids. For this, we studied Microcebus murinus and M. griseorufus along a continuous vegetation gradient where populations of the two species occur in sympatry or in allopatry. In allopatry, the two species are generalists without any sign of microhabitat selectivity. In sympatry, both species differed significantly and discriminated against certain habitat structures: M. murinus was found in microhabitats with larger trees than average while M. griseorufus utilized microhabitats with smaller trees. Hybrids between the two species did not show any significant discrimination for or against microhabitat structure and did not differ in their habitat utilization from either parent species. Both species can go into torpor and hibernation. M. griseorufus is seen more frequently during the cool dry season than M. murinus. We assume that M. murinus goes into extended torpor or hibernation more frequently than M. griseorufus. We interpret the different occurrence of large-sized trees in microhabitats of M. murinus as a prerequisite for M. murinus to be able to spend extended periods of time in tree holes that are isolated and allow hibernation at reduced temperature levels.     

A Study of the Taxonomy of the Mycobacterium nonchromogenicum Complex and Report of Six Cases of Lung Infection Due to Mycobacterium nonchromogenicum

In numerical classification, four species of the Mycobacterium nonchromogenicum complex, Mycobacterium nonchromogenicum, M. terrae, M. novum, and M. triviale, formed one cluster. These four species appeared to be reduced to one species, Mycobacterium nonchromogenicum. Furthermore, relationships between the species were numerically analyzed by using the hypothetical median organism pattern. The results showed that the M. nonchromogenicum complex can be divided into two subgroups: M. nonchromogenicum and the other three. These two subgroups were differentiated from each other by scores based on two or more positive reactions in the following three characteristics: resistance to bleomycin (5 μg/ml); heat-stable acid phosphatase activity; nicotinamidase or pyrazinamidase activity or both activities. M. nonchromogenicum gave two or three positive reactions among these three, and M. terrae, M. novum, and M. triviale gave two or three negative reactions. Three cases of lung infection due to M. nonchromogenicum, as well as three other cases of probable lung infection due to M. nonchromogenicum, were observed in this study. Only one organism isolated from one doubtful case was M. terrae. Up to now, M. nonchromogenicum was considered a nonpathogen. It was shown, however, that this organism causes lung infection in humans.     

Patterns of nucleotide variation and reproductive isolation between a Mimulus allotetraploid and its progenitor species

Here we report our characterization of a widespread, highly selfing Mimulus allotetraploid formed by interspecific hybridization between M. nasutus and M. guttatus. Nucleotide variation at two nuclear loci (mCYCA and mAP3) within and among tetraploid populations resolves two haplotype clusters for each locus: one shares near identity with sequences from M. nasutus and the other group shares substantial variation with M. guttatus. With respect to the two loci studied, each allotetraploid individual is a 'fixed heterozygote' carrying sequences from both clusters. Moreover, mCYCA variation is consistent with at least two evolutionary origins for the Mimulus allotetraploid. We show that the allotetraploid is strongly reproductively isolated from M. nasutus and M. guttatus; interploidy crosses produce almost no viable seeds. By extension, we infer strong triploid block and argue that Mimulus allotetraploid formation might proceed in one step via the union of unreduced gametes in an M. nasutus-M. guttatus F(1) hybrid. We also discuss the potential roles of mating system and flowering asynchrony in allotetraploid establishment.     

Recurrent replacement of mtDNA and cryptic hybridization between two sibling bat species Myotis myotis and Myotis blythii

The two sibling bat species Myotis myotis and Myotis blythii occur in sympatry over wide areas of Southern and Central Europe. Morphological, ecological and previous genetic evidence supported the view that the two species constitute two well-differentiated groups, but recent phylogenetic analyses have shown that the two species share some mtDNA haplotypes when they occur in sympatry. In order to see whether some genetic exchange has occurred between the two species, we sequenced a highly variable segment of the mitochondrial control region in both species living in sympatry and in allopatry. We also analysed the nuclear diversity of 160 individuals of both species found in two mixed nursery colonies located north and south of the Alps. MtDNA analysis confirmed that European M. blythii share multiple, identical or very similar haplotypes with M. myotis. Since allopatric Asian M. blythii presents mtDNA sequences that are very divergent from those of the two species found in Europe, we postulate that the mitochondrial genome of the European M. blythii has been replaced by that of M. myotis. The analysis of nuclear diversity shows a strikingly different pattern, as both species are well differentiated within mixed nursery colonies (F(ST) = 0.18). However, a Bayesian analysis of admixture reveals that the hybrids can be frequently observed, as about 25% of sampled M. blythii show introgressed genes of M. myotis origin. In contrast, less than 4% of the M. myotis analysed were classified as non-parental genotypes, revealing an asymmetry in the pattern of hybridization between the two species. These results show that the two species can interbreed and that the hybridization is still ongoing in the areas of sympatry. The persistence of well-differentiated nuclear gene pools, in spite of an apparent replacement of mitochondrial genome in European M. blythii by that of M. myotis, is best explained by a series of introgression events having occurred repeatedly during the recent colonization of Europe by M. blythii from Asia. The sharp contrast obtained from the analysis of mitochondrial and nuclear markers further points to the need to cautiously interpret results based on a single class of genetic markers.