Age-dependent Expression of S100β in the Brain of Mice

S100β is a soluble calcium binding protein released by glial cells. It has been reported as a neurotrophic factor that promotes neurite maturation and outgrowth during development. This protein also plays a role in axonal stability and in long term potentiation in the adult brain. The ability of S100β to modulate neuronal morphology raises the important question whether there is an age-related difference in the expression of S100β in the cerebral and cerebellar cortices of AKR strain mice and is this change is region specific. Our RT–PCR and Western blotting experiments show that the expression of S100β gene in the cerebral and cerebellar cortices starts from 0 day, peaks at about 45 days. However, in 70-week old mice its expression is significantly up-regulated as compared to that of 20-week old mice. S100β follows the same age-related pattern in both cerebral and cerebellar cortices. These results suggest that S100β is important for brain development and establishment of proper brain functions. Up-regulation of S100β in old age may have some role in development of age-related pathological systems in the brain.     

Protein S3 in the human 80S ribosome adjoins mRNA 3′ of the A-site codon

The protein environment of mRNA 3′ of the A-site codon (the decoding site) in the human 80S ribosome was studied using a set of oligoribonucleotide derivatives bearing a UUU triplet at the 5′-end and a perfluoroarylazide group at one of the nucleotide residues 3′ of this triplet. Analogues of mRNA were phased into the ribosome using binding at the tRNA^Phe P-site, which recognizes the UUU codon. Mild UV irradiation of ribosome complexes with tRNA^Phe and mRNA analogues resulted in the predominant crosslinking of the analogues with the 40S subunit components, mainly with proteins and, to a lesser extent, with rRNA. Among the 40S subunit ribosomal proteins, the S3 protein was the main target for modification in all cases. In addition, minor crosslinking with the S2 protein was observed. The crosslinking with the S3 and S2 proteins occurred both in ternary complexes and in the absence of tRNA. Within ternary complexes, crosslinking with S15 protein was also found, its efficiency considerably falling when the modified nucleotide was moved from positions +5 to +12 relative to the first codon nucleotide in the P-site. In some cases, crosslinking with the S30 protein was observed; it was most efficient for the derivative containing a photoreactive group at the +7 adenosine residue. The results indicate that the S3 protein in the human ribosome plays a key role in the formation of the mRNA binding site 3′ of the codon in the decoding site.     

Monitoring of the dissemination of Salmonella in the chicken Frankfurt-sausage production line of a sausage factory in the state of São Paulo, Brazil

Poultry meat and its derivatives are among the foodstuffs considered by environmental health authorities to present the highest risks to the public. A total of 185 samples were collected in five monthly batches, from different processing stages in a sausage plant that uses mechanically-deboned chicken meat (MDCM), and tested for the presence of Salmonella. Enrichment was carried out in both Kauffman's tetrathionate broth and Rappaport-Vassiliadis broth and isolation on Salmonella-Shigella agar and brilliant-green agar. Live Salmonella bacteria were isolated from six samples of the raw meat and from the emulsion, in batches three, four, and five, but not from any sample in batches one or two. The six isolated strains were all classified as Salmonella Albany, which has not previously been reported in MDCM. Of the two enrichment broths, Rappaport-Vassiliadis gave the better results. The pattern of contamination suggests a probable common source, given that a new supplier was used in the third, fourth, and fifth months. It was also shown that the industrial cooking was effective in preventing Salmonella surviving in the final product.     

A metagenomic study of the gut microbiome in PTB’S disease

BackgroundThere is an abundant link between the gut microbiota and human health and it plays a critical role in the clinic. It is recognized that microbial dysregulation contributes to the pathogenesis of tuberculosis (TB) but the underlying mechanisms remain unclear. In this study, we investigated the association of gut microbiome composition with TB as well as its possible roles in the development of this disease.MethodsFecal samples were collected from 10 TB patients and 20 healthy control samples. DNA extracted from fecal samples was subjected to 16S rDNA gene sequencing analysis on the Illumina MiSeq platform.ResultsCompared with healthy control samples, the gut microbiome of patients with TB was characterized by the decreased Alpha diversity. Perhaps, the decrease of microbial diversity which results in microbial dysregulation is the reason for clinical patients with more symptoms. The PTB group showed the most unique microbiota by higher abundance of Bifidobacteriaceae, Bifidobacteriales, Coriobacteriaceae, Coriobacteriales, Actinobacteria, Caulobacteraceae, Phyllobacteriaceae, Rhizobiales, Burkholderiaceae, Burkholderiaceae. Inflammatory status in PTB patients may be associated with the increased abundance of Clostridia and decreased abundance of Prevotella. We found that the abundance of Solobacterium and Actinobacteria was higher in the patients. There were 4 significant differences (p < 0.05) in the two groups which belonged to four metabolic categories, including endocytosis, phosphotransferase system (PTS), toluene degradation, and amoebiasis.ConclusionWe applied the approach of metagenomic sequencing to characterize the features of gut microbiota in PTB patients. The present study provided a detailed analysis of the characterization of the gut microbiota in patients based on the clinic. According to the metagenome analysis, our results indicated that the gut microbiota in PTB patients was significantly different from healthy control samples as characterized by the bacteria and metabolic pathway. The richness of the gut microbiota in patients was revealed. It was hypothesized that the above-mentioned changes of the gut microbiota could exert an impact on the development of PTB through the downstream regulation of the immune status of the host by way of the gut–lung axis.     

Identification of the 7S globulin with β-conglycinin in soybean seeds

The 7S globulin, a major ultracentrifugal component with the 11S globulin, was identical with β-conglycinin one of four antigenic components in the reserve proteins of soybean seeds (Glycine max). Double gel immunodiffusion and immunoelectrophoresis in agar gel were used for their identification. In addition, some characteristic properties on ultracentrifugation and in carbohydrate content agreed well between the proteins. Their MWs were ca 180000.     

Glutamine Substitution at Alanine1649 in the S4–S5 Cytoplasmic Loop of Domain 4 Removes the Voltage Sensitivity of Fast Inactivation in the Human Heart Sodium Channel

Normal activation–inactivation coupling in sodium channels insures that inactivation is slow at small but rapid at large depolarizations. M1651Q/M1652Q substitutions in the cytoplasmic loop connecting the fourth and fifth transmembrane segments of Domain 4 (S4–S5/D4) of the human heart sodium channel subtype 1 (hH1) affect the kinetics and voltage dependence of inactivation (Tang, L., R.G. Kallen, and R. Horn. 1996. J. Gen. Physiol. 108:89–104.). We now show that glutamine substitutions NH₂-terminal to the methionines (L¹⁶⁴⁶, L¹⁶⁴⁷, F¹⁶⁴⁸, A¹⁶⁴⁹, L¹⁶⁵⁰) also influence the kinetics and voltage dependence of inactivation compared with the wild-type channel. In contrast, mutations at the COOH-terminal end of the S4–S5/D4 segment (L¹⁶⁵⁴, P¹⁶⁵⁵, A¹⁶⁵⁶) are without significant effect. Strikingly, the A1649Q mutation renders the current decay time constants virtually voltage independent and decreases the voltage dependences of steady state inactivation and the time constants for the recovery from inactivation. Single-channel measurements show that at negative voltages latency times to first opening are shorter and less voltage dependent in A1649Q than in wild-type channels; peak open probabilities are significantly smaller and the mean open times are shorter. This indicates that the rate constants for inactivation and, probably, activation are increased at negative voltages by the A1649Q mutation reminiscent of Y1494Q/ Y1495Q mutations in the cytoplasmic loop between the third and fourth domains (O''Leary, M.E., L.Q. Chen, R.G. Kallen, and R. Horn. 1995. J. Gen. Physiol. 106:641–658.). Other substitutions, A1649S and A1649V, decrease but fail to eliminate the voltage dependence of time constants for inactivation, suggesting that the decreased hydrophobicity of glutamine at either residues A¹⁶⁴⁹ or Y¹⁴⁹⁴Y¹⁴⁹⁵ may disrupt a linkage between S4–S5/D4 and the interdomain 3–4 loop interfering with normal activation–inactivation coupling.     

Survey of bovine mycotic mastitis in dairy herds in the State of São Paulo,Brazil

The purpose of this study was to isolate fungi from the quarter milk of cow udders from several dairy herds and to identify the different genera and species involved in mastitis. A total of 2078 milk samples from normal, clinical and subclinical mastitis quarters from 22 dairy herds of 16 districts in the State of São Paulo, Brazil was utilized in this survey. Two hundred and fifty one (12.07%) fungi were isolated from the samples. Two hundred and eight of these (82.86%) were yeasts and 30 (11.95%) were moulds. The fungi were isolated in pure culture (24.77%) or in cultures mixed with bacteria (72.22%). The yeasts isolated were:Cryptococcus spp. (71 strains),Rhodotorula spp. (40),Candida spp. (68),Trichosporon cutaneum (21),Aureobasidium pullulans (7), andPichia ohmeri (1). Moulds classified in following genera were also isolated:Aspergillus (3),Penicillium (3),Alternaria (3),Phoma (3),Epicoccum (2), andGeotrichum (16).     

Rickettsia infection in five areas of the state of São Paulo, Brazil

This study investigated rickettsial infection in animals, humans, ticks, and fleas collected in five areas of the state of S?o Paulo. Eight flea species (Adoratopsylla antiquorum antiquorum, Ctenocephalides felis felis, Polygenis atopus, Polygenis rimatus, Polygenis roberti roberti, Polygenis tripus, Rhopalopsyllus lugubris, and Rhopalopsyllus lutzi lutzi), and five tick species (Amblyomma aureolatum, Amblyomma cajennense, Amblyomma dubitatum, Ixodes loricatus, and Rhipicephalus sanguineus) were collected from dogs, cats, and opossums. Rickettsia felis was the only rickettsia found infecting fleas, whereas Rickettsia bellii was the only agent infecting ticks, but no animal or human blood was shown to contain rickettsial DNA. Testing animal and human sera by indirect immunofluorescence assay against four rickettsia antigens (R. rickettsii, R. parkeri, R. felis, and R. bellii), some opossum, dog, horse, and human sera reacted to R. rickettsii with titers at least four-fold higher than to the other three rickettsial antigens. These sera were considered to have a predominant antibody response to R. rickettsii. Using the same criteria, opossum, dog, and horse sera showed predominant antibody response to R. parkeri or a very closely related genotype. Our serological results suggest that both R. rickettsii and R. parkeri infected animals and/or humans in the studied areas.     

Binding of the S7 Protein with Fragment 926–986/1219–1393 of the 16S rRNA As a Key Step in the Assembly of the Small Subunit of Prokaryotic Ribosomes

Both structural and thermodynamic studies are necessary to understand the ribosome assembly. An initial step was made in studying the interaction between a 16S rRNA fragment and S7, a key protein in assembling the prokaryotic ribosome small subunit. The apparent dissociation constant was obtained for complexes of recombinant Escherichia coliandThermus thermophilusS7 with a fragment of the 3" domain of the E. coli16S rRNA. Both proteins showed high rRNA-binding activity, which was not observed earlier. Since RNA and proteins are conformationally labile, their folding must be considered to correctly describe the RNA–protein interactions.     

Use of the STR loci D18S53, D18S59, and D18S488 in the diagnosis of Edwards’ syndrome

The aim of this study was to investigate the feasibility of using short tandem repeats (STRs) to diagnose Edwards’ syndrome (ES). Quantitative fluorescence polymerase chain reaction (QF-PCR) was performed to amplify STR loci on chromosome 18, specifically D18S53, D18S59, and D18S488. The amplified products were subjected to a fluorescence signal analysis and their application to ES diagnosis was examined. Among the 807 cases that showed normal results in the karyotype analysis, 793 showed one or two fluorescence bands with a fluorescence intensity ratio of 1:1, and 14 cases showed 3 bands, which were false-positive results. ES was diagnosed in 9 samples. The sensitivities of D18S53, D18S59, and D18S488 for the diagnosis of ES were 77.78, 44.44, and 55.56 % and the specificities were 96.16, 96.03, and 96.28 %, respectively. The combined sensitivity of the three loci for diagnosing DS was 100 % (9/9), with a specificity of 98.27 % (793/807). QF-PCR amplification of STR loci had high sensitivity, strong specificity, and was simple and rapid. Thus, it might have wide clinical applications, and could be an ideal tool for large-scale genetic and prenatal diagnosis of ES.     

Healing in the Sámi North

There is a special emphasis today on integrating traditional healing within health services. However, most areas in which there is a system of traditional healing have undergone colonization and a number of pressures suppressing tradition for hundreds of years. The question arises as to how one can understand today’s tradition in light of earlier traditions. This article is based on material collected in Sámi areas of Finnmark and Nord-Troms Norway; it compares local healing traditions with what is known of earlier shamanic traditions in the area. The study is based on 27 interviews among healers and their patients. The findings suggest that although local healing traditions among the Sámi in northern Norway have undergone major transformations during the last several hundred years, they may be considered an extension of a long-standing tradition with deep roots in the region. Of special interest are also the new forms tradition may take in today’s changing global society.     

Perturbation of the vesicle cycle in reticulospinal synapses of lamprey after presynaptic microinjections of GTPγS

The synaptic vesicle cycle sustains neurotransmission and keeps exo- and endocytosis in synapses in dynamic equilibrium. GTP-binding proteins function as key regulators of this cycle. The large GTPase dynamin is implicated in the fission of clathrin-coated vesicles from presynaptic membrane during endocytosis. The present study addresses the effect of the nonhydrolysable GTP analog GTPγS on assembly of the dynamin fission complex in situ. Intraaxonal microinjections of GTPγS induced the following distinct ultrastructural changes in the synapses: the number of synaptic vesicles in a cluster decreased while the number of the docked vesicles at the active zone increased; at the same time, the clathrin-coated intermediates also increased in number, indicating the inhibition of synaptic vesicle recycling. Unusual clathrin-coated intermediates were found. At low concentrations of GTPγS, they were presented by long tubules wreathed with a dynamin helix (spiral) and topped with a clathrin-coated vesicle. At high concentrations of GTPγS the tubular structures were much shorter and branched, with each branch topped with a clathrin-coated vesicle. The spiral pitch and the tubule diameter were significantly reduced as the concentration of GTPγS built up (23.1 ± 0.4 and 26.6 ± 0.4 nm, respectively, at low and 19.0 ± 0.5 and 23.3 ± 0.4 nm at high concentration of GTPγS, p < 0.001). We suggest that these ultrastructural changes reflect different steps in dynamin-mediated fission of clathrin-coated vesicles and propose a model for this process. The model implies that at first, GTP hydrolysis leads to a fast elongation of the helix due to a straightening of its dynamin dimmers. This entails an increase both in a pitch and a diameter of the dynamin helix. The shift in diameter disrupts local hydrophobic interactions between the inner and the outer lipid layers of the membrane at the sites of dynamin binding. Concurrent stretching of the helix and the clathrin-coated vesicle’s neck disintegrates the neck membrane and results finally in a release of the clathrincoated vesicle.     

The Template Region 5′ of the E-Site Codon Is Close to S26 in the Human 80S Ribosome

The environment of the template sequence 5 of the E-site codon on the 80S ribosome was studied with nonaribonucleotide or dodecaribonucleotide derivatives containing Phe codon UUU at the 3 end and a perfluoroarylazido group at the first or third nucleotide. A photoreactive group was linked to C5 of U or N7 of G. The analogs were positioned on the ribosome with the use of tRNA^Phe, which is cognate to the UUU codon and directs it to the P site, bringing a modified nucleotide in position –4 to –9 relative to the first nucleotide of the P-site codon. Upon irradiation of ribosome complexes with tRNA^Phe and the mRNA analogs with mild UV light, the analogs crosslinked predominantly to the 40S subunit, modifying the proteins. The major target of modification was S26 in all cases. In addition, S3 was modified to a low extent when the reactive nucleotide was in position –4 and S14 was in position –6. In the absence of tRNA, all mRNA analogs modified S3.     

Identification of the protein cross-linked to 3′-terminus of 5 S RNA in rat liver ribosomal 60 S subunits

To cross-link the 3′-terminus of 5 S RNA to its neighbouring proteins, ribosomal 60 S subunits of rat liver were oxidized with sodium periodate and reduced with sodium borohydride. 5 S RNP was then isolated by EDTA treatment followed by sucrose density-gradient centrifugation and subjected to SDS-polyacrylamide gel electrophoresis. The protein with a slower mobility than the L5 protein, which was thought to be cross-linked 5 S RNP, was labeled with ¹²⁵I, treated with RNAase, and analyzed by two-dimensional polyacrylamide gel electrophoresis, followed by radioautography. A radioactive spot located anodically from L5 protein was observed, suggesting that it is the L5 protein-oligonucleotide complex. When analyzed by SDS slab polyacrylamide gel electrophoresis followed by radioautography, the peptide pattern of the α-chymotrypsin digest of this ¹²⁵I-labeled protein-oligonucleotide complex was similar to that of the digest of ¹²⁵I-labeled L5 protein. The results indicate that L5 protein binds to the 3′-terminal region of 5 S RNA in rat liver 60 S subunits.     

Complete cloning of the chloroplast genome of safflower in λ EMBL3 and mapping of 23S and 16S rRNA genes

Summary A recombinant DNA library was constructed from partial BamHI or MboI digests of safflower (Carthamus tinctorius L.) chloroplast DNA, in the BamHI site of EMBL3. Seventeen recombinants, selected by chromosome walking, were found to contain overlapping fragments of the entire chloroplast genome. These clones were mapped using single and double digests of BamHI, EcoRI and HindIII. cDNAs synthesized from isolated 16S and 23S chloroplast rRNAs were used to map the ribosomal RNA genes relative to physical maps of the above restriction enzymes. The mapped positions of the rRNA genes for the safflower chloroplast DNA are in good agreement with previously published data for tobacco, spinach and several other higher plants.     

Analysis of the Gut Microbiota by High-Throughput Sequencing of the V5–V6 Regions of the 16S rRNA Gene in Donkey

Considerable evidence suggests that the gut microbiota is complex in many mammals and gut bacteria communities are essential for maintaining gut homeostasis. To date the research on the gut microbiota of donkey is surprisingly scarce. Therefore, we performed high-throughput sequencing of the 16S rRNA genes V5–V6 hypervariable regions from gut fecal material to characterize the gut microbiota of healthy donkeys and compare the difference of gut microbiota between male and female donkeys. Sixty healthy donkeys (30 males and 30 females) were enrolled in the study, a total of 915,691 validated reads were obtained, and the bacteria found belonged to 21 phyla and 183 genera. At the phylum level, the bacterial community composition was similar for the male and female donkeys and predominated by Firmicutes (64 % males and 64 % females) and Bacteroidetes (23 % males and 21 % females), followed by Verrucomicrobia, Euryarchaeota, Spirochaetes, and Proteobacteria. At the genus level, Akkermansia was the most abundant genus (23 % males and 17 % females), followed by Sporobacter, Methanobrevibacter, and Treponema, detected in higher distribution proportion in males than in females. On the contrary, Acinetobacter and Lysinibacillus were lower in males than in females. In addition, six phyla and 15 genera were significantly different between the male and female donkeys for species abundance. These findings provide previously unknown information about the gut microbiota of donkeys and also provide a foundation for future investigations of gut bacterial factors that may influence the development and progression of gastrointestinal disease in donkey and other animals.