The role of environmental antigens in the spontaneous development of autoimmunity in MRL-lpr mice.

It has been proposed that the "normal" stimulation of the immune system that occurs from interactions with environmental stimuli, whether infectious or dietary, is necessary for the initiation and/or continuation of autoimmunity. We tested this hypothesis by deriving a group of MRL-lpr mice into a germfree (GF) environment. At 5 mo of age, no differences between GF and conventional MRL-lpr mice were noted in lymphoproliferation, flow cytometric analysis of lymph node cells (LN), or histologic analysis of the kidneys. Autoantibody levels were comparably elevated in both groups. A second experiment tested the role of residual environmental stimuli by contrasting GF mice fed either a low m.w., ultrafiltered Ag-free (GF-AF) diet or an autoclaved natural ingredient diet (GF-NI). At 4 mo of age, both groups showed extensive lymphoproliferation and aberrant T cell formation, although the GF-AF mice had approximately 50% smaller LNs compared with sex-matched GF-NI controls. Autoantibody formation was present in both groups. Histologic analysis of the kidneys revealed that GF-AF mice had much lower levels of nephritis, while immunofluorescence analysis demonstrated no difference in Ig deposits but did reveal a paucity of C3 deposition in the kidneys of GF-AF mice. These data do not support a role for infectious agents in the induction of lymphoproliferation and B cell autoimmunity in MRL-lpr mice. Furthermore, they suggest that autoantibodies do not originate from B cells that were initially committed to exogenous Ags. They do suggest a possible contributory role for dietary exposure in the extent of lymphoproliferation and development of nephritis in this strain.     

Genetic diversity of phlD gene from 2,4-diacetylphloroglucinol-producing Pseudomonas spp. strains from the maize rhizosphere

In biocontrol Pseudomonads, phlD is an essential gene involved in the biosynthesis of 2,4-diacetylphloroglucinol (DAPG). HaeIII restriction of amplified phlD gene, previously proposed as the most discriminant analysis, showed no polymorphism among 144 Pseudomonas strains isolated from maize roots. However, these strains fell into three statistically significant DAPG production level groups. phlD sequences of 13 strains belonging to the three DAPG groups revealed a KspI restriction site only in good DAPG-producing strains. This result was confirmed on the 144 strains, 82 of which were identified as good-DAPG producers by both biochemical and amplified phlD KspI restriction analysis. They are candidates as potential biocontrol agents.     

Evolution of the primate lentiviruses: evidence from vpx and vpr.

The genomes of the four primate lentiviral groups are complex and contain several regulatory or accessory genes. Two of these genes, vpr and vpx, are found in various combinations within the four groups and encode proteins whose functions have yet to be elucidated. Comparison of the encoded protein sequences suggests that the vpx gene within the HIV-2 group arose by the duplication of an ancestral vpr gene within this group. Evolutionary distance analysis showed that both genes were well conserved when compared with viral regulatory genes, and indicated that the duplication occurred at approximately the same time as the HIV-2 group and the other primate lentivirus groups diverged from a common ancestor. Furthermore, although the SIVagm vpx proteins are homologous to the HIV-2 group vpx proteins, there are insufficient grounds from sequence analysis for classifying them as vpx proteins. Because of their similarity to the vpr proteins of other groups, we suggest reclassifying the SIVagm vpx gene as a vpr gene. This creates a simpler and more uniform picture of the genomic organization of the primate lentiviruses and allows the genomic organization of their common precursor to be defined; it probably contained five accessory genes: tat, rev, vif, nef and vpr.     

Structural analysis of heparin by methylation and g.l.c.-m.s.: preliminary results

Heparin is a complex mixture of polysaccharides differing in biological activity and structure, and attempts to relate this activity to structure have suffered, owing to a lack of sufficiently sensitive and specific analytical methods. Application of methylation analysis to determination of the structure of heparin is described. Carboxyl-reduced heparin was converted into its pyridinium salt, this was dissolved in Me2SO, and free OH and NH groups were methylated with dimethylsulfinyl anion. Sulfate groups were removed by solvolysis, and after dialysis, the polymer was acetylated and depolymerized by acetolysis. The resulting monosaccharides were converted into alditol acetates, which were separated by capillary, gas-liquid chromatography, and identified by both electron impact and chemical ionization mass spectrometry. Seventeen different monosaccharides were identified in the hydrolyzate. All of the expected internal hexosaminyl and glycosyluronic residues were identified. Although several sugars were identified as nonreducing termini, only a hexosamine 6-sulfate was identified as a reducing-terminus sugar. The results indicate that methylation analysis of heparins and other complex, sulfated glycosaminoglycans is feasible.     

Multivariate analysis for the understanding of typical diabetic hemostasis criteria. 2. Prevalent thrombocytic markers]

Various thrombocyte functions and metabolic criteria were investigated in 309 test persons, among them 198 Patients affected with diabetes mellitus, 67 of type I and 131 of type II as well as 111 healthy control persons. From the variety of these factors an optimal amount was determined by means of the statistical method of multivariance analysis separating both types of diabetes and healthy control persons. In addition to haemoglobin A1 concentration, thrombocyte parameters have been found together with the degree of capillary fragility, adrenalin-induced aggregation, platelet aggregation test and clot retraction, which allow individuals to be assigned diagnostically to various groups of test persons. Thus, thrombocyte functions found in both types of diabetes and normal persons exhibit differences which may contribute to a diagnostic classification.     

Heterogeneity of genetic control of blood pressure in ethnically different populations.

We review the literature on statistical genetic analyses of blood pressure in samples from various ethnic backgrounds using different statistical methods and packages. We then provide the results of a complex segregation analysis performed on familial data on systolic and diastolic blood pressure in 2 ethnically different populations, Chuvashans and Turkmenians. Two types of major gene models were tested in the segregation analysis: Model type 1 tests for a Mendelian mode of transmission and estimates genotype-specific averages regardless of age and sex effect, and model type 2 estimates age and sex effects on each of 3 genotypes within the putative major genotype. In both total samples, by both types of segregation analysis, familial aggregation of both systolic and diastolic blood pressure was inconsistent with the Mendelian mode of inheritance. In the next step of analysis the pedigrees in both samples were sorted into 2 groups on the basis of 2 likelihoods as obtained under Mendelian and nontransmission models for each entire sample. This procedure resulted in the appearance of 2 subsamples (large and small) in each ethnic sample. The segregation analysis that was carried out then on the larger subsample provided consistent evidence to support the major gene effect on systolic and diastolic blood pressure in 2 ethnic groups. Interestingly, model type 2 showed that in both ethnically different large subsamples, for each sex the genotype predisposing to a larger mean value of systolic (or diastolic) blood pressure also displayed the highest rate of blood pressure increase with age. We discuss in detail possible sources of heterogeneity in familial transmission of blood pressure observed in our 2 samples, and we suggest a method to improve the analysis of heterogeneity for trait inheritance.     

Structure and sequence relationships in the lipocalins and related proteins.

The lipocalins and fatty acid-binding proteins (FABPs) are two recently identified protein families that both function by binding small hydrophobic molecules. We have sought to clarify relationships within and between these two groups through an analysis of both structure and sequence. Within a similar overall folding pattern, we find large parts of the lipocalin and FABP structures to be quantitatively equivalent. The three largest structurally conserved regions within the lipocalin common core correspond to characteristic sequence motifs that we have used to determine the constitution of this family using an iterative sequence analysis procedure. This afforded a new interpretation of the family, which highlighted the difficulties of determining a comprehensive and coherent classification of the lipocalins. The first of the three conserved sequence motifs is also common to the FABPs and corresponds to a conserved structural element characteristic of both families. Similarities of structure and sequence within the two families suggests that they form part of a larger "structural superfamily"; we have christened this overall group the calycins to reflect the cup-shaped structure of its members.     

Salmonella plasmids of the pre-antibiotic era.

In order to provide a profile of the plasmid gene pool in Salmonella prior to the clinical use of antibiotics, a molecular genetic analysis was made of plasmids in strains collected by E.D.G. Murray between 1917 and 1954. These pre-antibiotic era (PAE) salmonellae contain conjugative plasmids of the same incompatibility groups as contemporary enterobacterial plasmids. Upon analysis of total plasmid content, 42 plasmids, sized between 23 and 72 MDa, were found. We defined and investigated six groups of these PAE Salmonella plasmids in terms of three groups of genes; those involved in plasmid maintenance and incompatibility, DNA repair and virulence. Of the five groups, three were replicon-typed to groups IncI1, IncX and IncFII; one group exhibited no homology to contemporary Inc/Rep probes, and one group represented virulence plasmids containing a common plasmid-partitioning locus. The results indicated that most of the PAE groups were progenitors of contemporary R-plasmids, except for the virulence plasmids, which have generally not evolved as vectors of antibiotic resistance.     

Genetic Analysis of the Heterochromatin of Chromosome 3 in Drosophila Melanogaster. II. Vital Loci Identified through Ems Mutagenesis

Chromosome 3 of Drosophila melanogaster contains the last major blocks of heterochromatin in this species to be genetically analyzed. Deficiencies of heterochromatin generated through the detachment of compound-3 chromosomes revealed the presence of vital loci in the heterochromatin of chromosome 3, but an extensive complementation analysis with various combinations of lethal and nonlethal detachment products gave no evidence of tandemly repeated vital genes in this region. These findings indicate that the heterochromatin of chromosome 3 is genetically similar to that of chromosome 2. A more thorough genetic analysis of the heterochromatic regions has been carried out using the chemical mutagen ethyl methanesulfonate (EMS). Seventy-five EMS-induced lethals allelic to loci uncovered by detachment-product deficiencies were recovered and tested for complementation. In total, 12 complementation groups were identified, ten in the heterochromatin to the left of the centromere and two to the right. All but two complementation groups in the left heterochromatic block could be identified as separate loci through deficiency mapping. The interallelic complementation observed between some EMS-induced lethals, as well as the recovery of a temperature-sensitive allele for each of the two loci, provided further evidence that single-copy, transcribed vital genes reside in the heterochromatin of chromosome 3. Cytological analysis of three detachment-product deficiencies provided evidence that at least some of the genes uncovered in this study are located in the most distal segments of the heterochromatin in both arms. This study provides a detailed genetic analysis of chromosome 3 heterochromatin and offers further information on the genetic nature and heterogeneity of Drosophila heterochromatin.     

On the complex formation of acridine dyes with DNA. VII. Dependence of the binding on the dye structure

The binding constants for the complex formation of more than twenty ring nitrogen-and amino-substituted acridine derivatives with calf thymas DNA were measured by a fluorescence method. DNA quenches the fluorescence of the aminoacridine dyes so long as both amino hydrogens are not substituted. These dyes show an enhancement of their fluorescence intensity in the presence of DNA. Typical representatives of both are proflavine and acridine orange derivatives, respectively. A discussion of steric and electronic influences of various substituents attached to the ring nitrogen and amino groups on the binding led to the concept of different conformations for intercalated acridines without amino groups and the aminoacridines. The electrostatic binding site of the former seems to be the positively charged ring nitrogen, while the binding sites in the aminoacridines are so located that the amino groups are directed towards the negatively charged DNA phosphates.     

Quantitative-morphometric and functional-anatomical studies on the locomotor apparatus of athletes. IV. Discussion, resume and literature

The locomotor system of 155 athletes from 3 athletic disciplines was examined, and the data of 20 anthropometrical measurements and the results for the corresponding disciplines were recorded subjected to a multivariate morphometrical analysis with the following aim: 1. To evaluate the morphological and functional-anatomical criteria of the locomotor system which quantified the variation differences in the body composition of the athletic groups, and which indicated a quantifiable functional-anatomical relationship between the variation in the body composition and the result in the corresponding disciplines. 2. To show the bivariate relationship between the morphological parameter and the results in the corresponding disciplines, and furthermore to evaluate the amount of result-increasing or result-decreasing components in a morphological variable. 3. To analyse the morphological variation of the 20 body composition variables and the corresponding results in the disciplines by means of factor analysis. This is achieved by the use of multivariate methods to analyse the variation in the athlete's body composition and the result-influencing-components. The following results were obtained: 1. There are partly pronounced morphological differences between the 3 athletic groups, quantified by discriminant functions. The differences in the locomotor system were reduced to 2 independent axes of variation, with one axis being identified as the general size variation between the 3 groups, and the other as the difference in the variation of the degree of strength of the musculature of the trunk and the limbs by considering the length and breadth measurements. For both canonical axes, the shotputters have above-average high canonical numbers compared to the other 2 groups. The sprinters and long jumpers show conformity in the general size variation of the locomotor system, but they differ in the second axis. Whilst in both groups the lower limbs are similarly constituted in form, shape and size, the canonical values of the trunk and the upper limbs of the sprinters are higher. In this respect, they tend more towards the shot-putter group. The morphological differences obtained for the athlete's locomotor system are functional-anatomically related to the corresponding athletic disciplines. 2. In almost every case, the regression functions for the shot-putters show an approximately linear relationship between the morphological variables and the result of the shot-putt. The quadratic and cubic regression functions only deviate slightly from the linearity, i.e. the result will be altered depending on the height and size of the body composition (within the possible range of biological variation). On the other hand, the sprinting result, either increased or decreased, is determined by a limited morphological variation of the parameter of the locomotor system...     

Analysis of a pyruvic acid acetal-containing polysaccharide by the reductive-cleavage method.

The applicability of the reductive-cleavage method to the analysis of polysaccharides bearing pyruvic acid acetals has been demonstrated. Direct reductive cleavage of fully methylated gum xanthan yielded the expected products, including 1,5-anhydro-4,6-O-[(S)-1-methoxycarbonylethylidene]-2,3-di-O-methy l-D- mannitol. The latter product was not observed when reductive cleavage was performed subsequent to reduction of ester groups in the fully methylated polysaccharide and mild hydrolysis to remove pyruvic acid acetal substituents. Instead, the latter experiment yielded 1,5-anhydro-2,3-di-O-methyl-D-mannitol, establishing the presence in the polysaccharide of terminal (nonreducing) D-mannopyranosyl groups bearing 4,6-O-(1-carboxyethylidene) substituents. The products of reductive cleavage were characterized, where appropriate, by comparison of the gas chromatographic retention times and chemical ionization- and electron ionization-mass spectra of their acetates to those of authentic standards. Alternatively, the products of reductive cleavage could be characterized without resort to comparison with authentic standards by analysis of the 1H-n.m.r. spectra of their benzoates, which were obtained in pure form by high-performance liquid chromatography. By either method of product characterization, this two-step procedure of analysis reveals the presence of pyruvic-acetal residues in polysaccharides and establishes both the identity of the sugar residue to which they are attached and their positions of attachment.     

RNA-protein interactions in the Escherichia coli ribosome.

Over the last two decades essentially three different approaches have been used to study the topography of RNA-protein interactions in the ribosome. These are: (a) the analysis of binding sites for individual ribosomal proteins or groups of proteins on the RNA; (b) the determination of protein footprint sites on the RNA by the application of higher order structure analytical techniques; and (c) the localisation of RNA-protein cross-link sites on the RNA. This article compares and contrasts the types of data that the three different approaches provide, and gives a brief and highly simplified summary of the results that have been obtained for both the 16S and 23S ribosomal RNA from E coli.     

Estimation of the deamidation rate of asparagine side chains.

Statistical analysis of data from the literature concerning the deamidation reaction of asparagine side-chains in short peptides reveals that the logarithm of rate constants can be solved into a constant plus contributions from the residues closest to asparagine. A table of amino acid contributions has been derived, from which deamidation rate constants can be estimated with good approximation. Assuming the contribution of glycine to be zero, the mean of the absolute values of the contributions for the residues following asparagine is approximately seven times that for the preceding residues. In both positions residues with no bulk side chains or with functional side groups contribute markedly to the increase in the rate constant.     

Thiamine transport in thiamine-deficient rats. Role of the unstirred water layer.

As part of a systematic study of alcoholism and thiamine absorption, the effect of diet-induced thiamine deficiency and the role of the unstirred water layer on the thiamine transport were investigated. Using 3H-labeled dextran as a marker of adherent mucosal volume, jejunal uptake of 14C-labeled thiamine hydrochloride was measured, in vitro, in thiamine-deficient rats and pair-fed controls. Uptake of low thiamine concentrations (0.2 and 0.5 muM) was greater in the thiamine-deficient rats than in the controls. In contrast, uptake rates for high thiamine concentrations (20 and 50 muM) were similar in both groups. While Jmax was unaltered, Km was decreased in thiamine deficiency, suggesting a decrease in unstirred water layer thickness. Accordingly, the thickness of the water layer was measured in both groups of animals and correlated with Jmax and Km under unstirred and stirred conditions. Without stirring, there was no difference in Jmax between the two groups. In contrast, both Km and the water layer were reduced in the thiamine-deficient rats. With stirring, Jmax was not affected, but both Km and the water layer thickness were reduced to similar values in both groups. Reversal of thiamine deficiency resulted in the return of thiamine uptake and the unstirred water layer thickness to control values. These data support the concept of a dual system of thiamine transport and emphasize the role of the unstirred water layer as an important determinant of transport kinetics not only under physiologic situations but also in diet-induced rat thiamine deficiency, a model for a clinical patholigical state. The decrease in the unstirred water layer thickness in thiamine deficiency may be also viewed as a possible adaptive mechanism to facilitate absorption of meager supplies of thiamine.     

Urban plant ecology patterns and processes: a case study of the flora of the City of Plymouth, Devon, U. K.

Aim Using a large database that has been created over the past 5 years with the RECORDER package, presence/absence data for 829 species of vascular plants in the 103 1-km² squares that cover the city of Plymouth (pop. 243,373) have been analysed by two-way indicator species analysis (TWINSPAN ) and canonical correspondence analysis (CCA) to establish the major species assemblages and to examine their spatial distribution across the city in relation to variation in land use. Location The City of Plymouth. Methods Nine groupings of squares emerged and their distribution was mapped across the city. Interpretation of those groups and their variation in relation to the land use ordination axes showed that TWINSPAN groups lying along the first axis of variation correlated floristic variation with the process of urban development and the historical evolution of urban structure. The second axis appeared to be related to particular remnant semi-natural habitats within the city that could be regarded as ‘hot-spots’ for survival of many plant species. Species were categorized into four types on the basis of their recency and mode of arrival in the city and by using the historical flora of Plymouth produced by T. R. Archer-Briggs in 1880. Variations in the sources of species in relation to the TWINSPAN classification groups were then examined. A second ordination was also computed; this time with geological, altitudinal and distance variables. TWINSPAN groups were then superimposed on the ordinations using polygons to assist with biogeographical interpretation. Results and conclusions The results of both the multivariate analyses and distribution of species by source are then discussed in the context of previous research into urban plant ecology, particularly in Central Europe. The problems of inferring process from pattern in this meso-scale study are reviewed and suggestions are made for further research into urban ecology and biogeography in the city.     

Analysis of expressed sequence tags of the water flea Daphnia magna.

To study gene expression in the water flea Daphnia magna we constructed a cDNA library and characterized the expressed sequence tags (ESTs) of 7210 clones. The EST sequences clustered into 2958 nonredundant groups. BLAST analyses of both protein and DNA databases showed that 1218 (41%) of the unique sequences shared significant similarities to known nucleotide or amino acid sequences, whereas the remaining 1740 (59%) showed no significant similarities to other genes. Clustering analysis revealed particularly high expression of genes related to ATP synthesis, structural proteins, and proteases. The cDNA clones and EST sequence information should be useful for future functional analysis of daphnid biology and investigation of the links between ecology and genomics.     

Molecular cloning of a gut-specific chitinase cDNA from the beetle Phaedon cochleariae.

A cDNA encoding a chitinase of Pheadon cochleariae was isolated from a larval gut library. The cDNA encodes a preenzyme with a putative 20 amino-acid signal peptide and a 385 amino-acid mature enzyme of calculated mass of 42.7 kDa. Amino-acid alignment shows 24-33% identity to other insect and crustacea chitinases. The sequence lacks C-terminus domains but active site residues are conserved. Northern analysis localizes the mRNA to guts of feeding larvae. Southern blot analysis, with a complete cDNA probe, suggests that the P. cochleariae genome may contain several chitinase genes. Activity gels show that two groups of chitinases are expressed in the insect. One group comprises chitinases of 30-40 kDa that are active at pH 5.0 and detected in guts of feeding larvae and adults, as well as in pre-pupae and pupae. The other group comprises chitinases of 40-70 kDa that are more active at pH 7.0 and are mainly expressed in pre-pupae and pupae. The biological significance of both groups of chitinases is discussed.     

Studies on the mechanism of the changes in serum and liver gamma-glutamyl transpeptidase activity. I. Experimental extrahepatic cholestasis in rabbits.

Serum, liver and renal gamma-glutamyl transpeptidase (GGT) activities were studied in four groups of rabbits: controls, rabbits with obstructive extrahepatic cholestasis, rabbits with obstructive anuria, and animals with combined obstructive extrahepatic cholestasis and obstructive anuria. Serum GGT was essentially increased in rabbits with obstructive extrahepatic cholestasis, showing peak values in the combined cholestasis + obstructive anuria group, and practically normal values in animals with anuria. Liver GGT was increased in both cholestasis groups, but the increase was less prominent than the increase in serum GGT and there was no correlation between them. In both anuric groups renal GGT was reduced, probably as a result of inhibited enzyme synthesis secondary to the altered conditions for adequate renal function. The results obtained are suggestive of a probable renal involvement in the formation of the serum GGT activity level.     

Coxsackie A 13 virus in the foci of rheumatism.

Faeces and nasopharyngeal washing were examined in 12 patients and 27 contacts of these patients in family foci (primary foci) of rheumatism and in 37 patients and 32 contacts of these patients in secondary foci of rheumatism (in hospitals). Results of stimultaneous examinations of 127 children of the control groups served as the control. An analysis of the results of the investigation showed the Coxsackie A 13 virus was found considerably more frequently in both the patients (83.3%) and their contacts (148.1%) in the primary foci of rheumatism than outside these foci (11.9--15.3%). The difference is statistically significant. Wide distribution of Coxsackie A 13 virus was also observed in clinical departments in which children were hospitalized at the acute stage of rheumatism. We failed to establish marked differences in the rate of detection of other viruses in the foci of rheumatism in comparison with the control groups.