A group translocator for sucrose assimilation in tonoplast vesicles of sugarcane cells

Existence of a group translocator for sucrose transfer into vacuoles of sugarcane (Saccharum sp.) cells has been further confirmed by the use of tonoplast vesicles isolated from intact vacuoles. The group translocator depends on external UDP-Glucose (Glc) and, via a series of enzymic reactions within the tonoplast, sucrose phosphate and sucrose are deposited inside the vesicles. Fructose-6-phosphate was not required for UDP-Glc uptake, nor was it taken up. None of the other sugar phosphates tested were taken up nor were the nucleotide sugars, UDP-Galactose and ADP-Glc. The uptake of UDP-Glc was concentration-dependent with a K_m of 1.2 millimolar and a V_max of 83.3 nanomoles per minute per milligram protein. The optimum pH for UDP-Glc uptake was 7.0. Uptake of UDP-Glc was inhibited by para-chloromercuribenzene-sulfonic acid, UDP, and GDP; carbonyl cyanide m-chlorophenylhydrazone inhibited to a lesser extent.     

Evidence for the involvement of a UDP-glucose-dependent group translocator in sucrose uptake into vacuoles of storage roots of red beet

Vacuoles isolated from the storage roots of red beet (Beta vulgaris L.) accumulate sucrose via two different mechanisms. One mechanism transports sucrose directly, and its rate is increased by the addition of MgATP. The other mechanism utilizes uridine diphosphate glucose (UDP-glucose) to synthesize and simultaneously transport sucrose phosphate and sucrose into the vacuole. This group translocation mechanism has also been found in sugarcane vacuoles. As in sugarcane, the beet group translocator does not require fructose 6-phosphate, nor is the latter substance transported into the vacuole. The uptake of UDP[¹⁴C]glucose in inhibited by high concentrations of osmoticum.Abbreviations EDTA ethylenediaminetetraacetic acid - Hepes 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid - UDP uridine 5-diphosphate     

Analysis of Sarcoptes scabiei finds no evidence of infection with Wolbachia

The endosymbiont Wolbachia has been detected in a range of filarial nematodes and parasitic mites and is known to affect host reproductive compatibility and potentially evolutionary processes. PCR of Wolbachia surface protein (wsp), ftsZ and 16SrRNA genes from individual Sarcoptes scabiei mites obtained from a series of individual hosts, and database searches of an S. scabiei var. hominis EST library failed to detect Wolbachia genes. Therefore, Wolbachia appears not to be involved in the genetic subdivision observed between varieties of host-associated S. scabiei or, involved in the inflammatory disease pathogenesis of scabies unlike its activity in filarial infection.     

Huntington disease: no evidence for locus heterogeneity

A total of 63 families with Huntington disease (HD) were examined for linkage between HD and G8 (D4S10). The families included 57 Caucasian, four Black American, and two Japanese. The combined maximum lod score was 87.69 at theta = 0.04 (99% confidence interval 0.018-0.071). The maximum frequency of recombination was 0.03 in males and 0.05 in females. Fifty-seven families gave positive lod scores; five small families gave mildly negative lod scores. The maximum likelihood estimate of alpha, the proportion of linked loci, was 1.0 with a lower 99% confidence interval of 0.88. These data suggest that there is only one HD locus, although a second rare locus cannot be ruled out.     

Structure of the Haemophilus influenzae HMW1B translocator protein: evidence for a twin pore

Secretion of the Haemophilus influenzae HMW1 adhesin occurs via the two-partner secretion pathway and requires the HMW1B outer membrane translocator. HMW1B has been subjected to extensive biochemical studies to date. However, direct examination of the structure of HMW1B has been lacking, leaving fundamental questions about the oligomeric state, the membrane-embedded beta-barrel domain, the approximate size of the beta-barrel pore, and the mechanism of translocator activity. In the current study, examination of purified HMW1B by size exclusion chromatography and negative staining electron microscopy revealed that the predominant species was a dimer. In the presence of lipid, purified HMW1B formed two-dimensional crystalline sheets. Examination of these crystals by cryo-electron microscopy allowed determination of a projection structure of HMW1B to 10 A resolution. The native HMW1B structure is a dimer of beta-barrels, with each beta-barrel measuring 40 A by 50 A in the two orthogonal directions and appearing largely occluded, leaving only a narrow pore. These observations suggest that HMW1B undergoes a large conformational change during translocation of the 125-kDa HMW1 adhesin.     

Visualizing clinical evidence: citation networks for the incubation periods of respiratory viral infections

Simply by repetition, medical facts can become enshrined as truth even when there is little empirical evidence supporting them. We present an intuitive and clear visual design for tracking the citation history of a particular scientific fact over time. We apply this method to data from a previously published literature review on the incubation period of nine respiratory viral infections. The resulting citation networks reveal that the conventional wisdom about the incubation period for these diseases was based on a small fraction of available data and in one case, on no retrievable empirical evidence. Overall, 50% of all incubation period statements did not provide a source for their estimate and 65% of original sources for incubation period data were not incorporated into subsequent publications. More standardized and widely available methods for visualizing these histories of medical evidence are needed to ensure that conventional wisdom cannot stray too far from empirically supported knowledge.     

The identification of trihydroxyeicosatrienoic acids as products from the incubation of arachidonic acid with washed blood platelets

Arachidonic acid is converted by washed platelets from man, horse and dog into a mixture of 8, 9, 12-trihydroxyeicosa-5, 10, 14-trienoic acid and 8, 11, 12-trihydroxyeicosa-5, 9, 14-trienoic acid (termed 8, 9, 12-THETA and 8, 11, 12-THETA respectively and THETA collectively). Gas chromatographic — mass spectrometric evidence of structure is discussed.     

Enzymatic reaction of chlorotrifluoroethene with glutathione: 19F NMR evidence for stereochemical control of the reaction

Chlorotrifluoroethene, a potent nephrotoxin, is a substrate for the glutathione S-transferases present in the cytosolic and microsomal fractions of rat liver. The glutathione conjugate formed by both subcellular fractions has been identified as S-(2-chloro-1,1,2-trifluoroethyl)glutathione by 1H and 19F NMR and by secondary ion mass spectrometry. The conjugate formed by the cytosolic fraction is an equimolar mixture of two diastereomers, whereas the conjugate formed by the microsomal fraction is predominantly one diastereomer, as judged by the 19F NMR spectra. No evidence for the formation of S-(trihalovinyl)glutathione derivatives by an addition/elimination reaction was found. High-performance liquid chromatography was employed to measure the rates of glutathione conjugate formation in vitro. The rates of S-(2-chloro-1,1,2-trifluoroethyl)glutathione formation were 75-107 nmol min-1 (mg of protein)-1 and 151-200 nmol min-1 (mg of protein)-1 catalyzed by the cytosolic and microsomal fractions, respectively (measured at pH 7.4, 37 degrees C, with 5 mM glutathione). These results suggest that glutathione conjugation occurs at high rates in vivo to produce the highly nephrotoxic S-(2-chloro-1,1,2-trifluoroethyl)glutathione.     

First crystallographic evidence for the formation of beta-D-ribopyranosylamine from the reaction of ammonia with of D-ribose

Beta-D-Ribopyranosylamine was synthesized and characterized using analytical, spectral and single-crystal X-ray diffraction methods. The molecule exists in the chair form with the 4C(1) conformation. The beta anomeric form of C-1 is supported by the dihedral angles. The molecule exhibits both intra- and intermolecular hydrogen-bond interactions of the type O-H...O, Nz-H...O and C-H...O, and these are interconnected to each other to form chains.     

Direct evidence for nitric oxide formation from glyceryl trinitrate during incubation with intact bovine pulmonary artery.

It has been proposed that the mechanism of the vasodilator action of glyceryl trinitrate (GTN) involves biotransformation to nitric oxide. A sensitive chemiluminescence method for nitric oxide determination was used to test this hypothesis. In four experiments, bovine pulmonary artery (BPA) was incubated with GTN (0.1 mM) in Krebs' solution (2 mL) containing 30 mM KCl, and in anaerobic conditions using 95% Ar - 5% CO2, in a sealed micro-Fernbach flask (6.2-mL volume). After incubation for 2, 5, 10, or 20 min at 37 degrees C, 400-microL aliquots of headspace gas were removed and injected into a redox chemiluminescence detector. Nitric oxide formation was first measurable at 5 min (76 +/- 53 pmol/g wet wt. BPA), and increased with incubation time (174 +/- 46 pmol/g wet wt. BPA after 10 min and 310 +/- 67 pmol/g wet wt. BPA after 20 min). This is the first direct chemical measurement of nitric oxide formation during interaction of GTN with vascular smooth muscle. These data support the concept that GTN is a nitrovasodilator prodrug acting via the formation of nitric oxide.     

A de novo recombination in the ABO blood group gene and evidence for the occurrence of recombination products

We have encountered a paternity case where exclusion of the putative father was only observed in the ABO blood group (mother, B; child, A1; putative father, O), among the many polymorphic markers tested, including DNA fingerprints and microsatellite markers. Cloning a part of the ABO gene, PCR-amplified from the trio’s genomes, followed by sequencing the cloned fragments, showed that one allele of the child had a hybrid nature, comprising exon 6 of the B allele and exon 7 of the O1 allele. Based on the evidence that exon 7 is crucial for the sugar-nucleotide specificity of A1 and B transferases and that the O1 allele is only specified by the 261G deletion in exon 6 of the consensus sequence of the A1 allele, we concluded that the hybrid allele encodes a transferase with A1 specificity, resulting, presumably, from de novo recombination between the B and O1 alleles of the mother during meiosis. Screening of random populations demonstrated the occurrence of four other hybrid alleles. Sequencing of intron VI from the five hybrid alleles showed that the junctions of the hybrid alleles were located within intron VI, the intron VI-exon 7 boundaries, or exon 7. Recombinational events seem to be partly involved in the genesis of sequence diversities of the ABO gene. Received: 25 October 1996     

Immunoelectron microscopic evidence for different compartments in the secretory vacuoles of the rat seminal vesicles

Summary Immunoelectron microscopy of the rat seminal vesicle was performed using specific antibodies to secretory proteins. Proteins were precipitated from rat seminal vesicle secretion and were separated by SDS—polyacrylamide gel electrophoresis. Among the great number of bands the two most prominent bands were selected and designated SVS II and IV. Their apparent molecular weights were 48 kDa and 16.5 kDa respectively. The bands were excised from the gels and used for antibody production in rabbits. The respective antisera were used for immunohistochemical studies both at the light and electron microscopic levels in the rat seminal vesicle and the different prostatic lobes in infantile, adult and castrated animals. A positive immunoreaction was observed in seminal vesicle and lateral prostatic epithelium of the intact adult rat, while it was lacking in prepubertal and castrated animals. The subcellular distribution of both proteins was clearly different: SVS II was exclusively confined to the electron dense core of the secretory vacuoles, while SVS IV was detected only in the clear halo surrounding the central granule. It is suggested that the spatial arrangement of both proteins in the seminal vesicle secretion vacuole reflects a particular functional significance of each of these proteins. These proteins may serve as a tool in the study of regulation of androgendependent protein synthesis.     

Insights into the gut bacterial communities of spider from wild with no evidence of phylosymbiosis

In the present study, an effort has been made to elucidate the gut bacterial diversity of twelve species of the family Araneidae under three subfamilies collected from 5 states of India along with their predicted metabolic role in functional metabolism. Further, we also compared the host species phylogeny based on partial cytochrome c oxidase subunit I (COI) sequences with the gut bacteria composition dendrogram to decipher the phylosymbiotic relationships. Analysis revealed the presence of 22 bacterial phyla, 145 families, and 364 genera in the gut, with Proteobacteria, Firmicutes, Actinobacteria, and Deinococcus-Thermus as the highest abundant phyla. Moreover, phylum Bacteriodetes was dominated only in Cyclosa mulmeinensis and Chlamydiae in Neoscona bengalensis. At the genus level, Bacillus, Acinetobacter, Cutibacterium, Pseudomonas, and Staphylococcus were the most dominant genera. Furthermore, the genus Prevotella was observed only in Cyclosa mulmeinensis, and endosymbiont Wolbachia only in Eriovixia laglaizei. The differential abundance analysis (DeSeq2) revealed the 19 significant ASVs represented by the genera like Acinetobacter, Vagoccoccus, Prevotella, Staphylococcus, Curvibacter, Corynebacterium, Paracoccus, Streptococcus, Microbacterium, and Pseudocitrobacter. The inter- and intra-subfamilies comparison based on diversity indices (alpha and beta diversity) revealed that the subfamily Araneinae have high richness and diversity than Argiopinae and Gasteracanthinae. The phylosymbiotic analysis revealed that there is no congruence between the gut bacteria composition dendrogram with their host phylogeny.     

Phylogeography of lemmings (Lemmus): no evidence for postglacial colonization of Arctic from the Beringian refugium

Beringia is considered as an important glacial refugium that served as the main source for colonization of formerly glaciated Arctic regions. To obtain high resolution views of Arctic refugial history, we examined mitochondrial cytochrome b phylogeography in the northern genus of rodents, Lemmus (true lemmings), sampled across its circumpolar distribution. Strong phylogeographical structure suggests vicariant separation over several glacial-interglacial periods and does not provide evidence supporting the importance of Beringia for extensive colonization of formerly glaciated regions. Rather than a source of postglacial colonization, Beringia represents an area of intraspecific endemism previously undetected by biogeographical analysis. Existing phylogeographical structure suggests that vicariant separation by glacial barriers was an important factor generating genetic divergence and, thus, increasing genetic diversity in lemmings on continental and circumpolar scales. However, there is little evidence for the direct effect of the last glaciation on the level of genetic variation and allele genealogy in lemmings on a regional geographical scale. This finding implies that the population genetic models of postglacial colonization suggested for temperate taxa might have limited applicability for Arctic species.     

Allelopathy of Baltic Sea cyanobacteria: no evidence for the role of nodularin

Extracts of Aphanizomenon flos-aquae and Nodularia spumigena,the two most common cyanobacteria forming recurrent blooms inthe Baltic Sea, decrease the abundance of some phytoplanktonspecies via the release of allelopathic substances. We investigatedhow cell-free filtrates of the two cyanobacteria, as well aspurified hepatotoxin nodularin, produced by N. spumigena affectedcell numbers, chlorophyll a content and ¹⁴CO₂ uptake of thecryptophyte Rhodomonas sp. Both cyanobacterial filtrates significantlyretarded the growth of Rhodomonas sp., A. flos-aquae filtrateup to 46%, whereas purified nodularin showed no significanteffect on any of the growth parameters of the cryptophyte. Theseresults suggest that the allelopathic effect of N. spumigenais most probably due to metabolite(s) other than nodularin,possibly acting via the damage of the target cells.     

Analysis of ras oncogene products by two-dimensional gel electrophoresis: evidence for protein families with distinctive molecular forms

Protein products of the ras family of oncogenes were immunoprecipitated by an anti-p21 monoclonal antibody, separated by two-dimensional gel electrophoresis and subsequently detected by western immunoblot analysis using the same anti-p21 monoclonal antibody as a probe. Using this method, a 21 kDa oncogene protein (p21) was detected and characterized in cell lines containing Harvey (Ha), Kirsten (Ki), neuroblastoma (N), or cellular (proto) ras genes. The ras gene products from all cell types occurred with multiple forms differing in size, charge or in both parameters. Transforming ras oncogene proteins occurred in easily identifiable groups that were different from each other in molecular weight and charge, were distinctive for each ras gene type and were different from cellular ras equivalents. Similar, but not identical, family groups occurred in different cell types containing the same oncogene. The reproducible occurrence of unpredicted p21 forms suggests that previously unreported post-translational processing steps may be associated with the synthesis of certain oncogene products. This immunoprecipitation/two-dimensional gel/western blot technique is a simple method for the identification and characterization of p21 gene products.     

Analysis of cedar pollen time series: no evidence of low-dimensional chaotic behavior

Much of the current interest in pollen time series analysis is motivated by the possibility that pollen series arise from low-dimensional chaotic systems. If this is the case, short-range prediction using nonlinear modeling is justified and would produce high-quality forecasts that could be useful in providing pollen alerts to allergy sufferers. To date, contradictory reports about the characterization of the dynamics of pollen series can be found in the literature. Pollen series have been alternatively described as featuring and not featuring deterministic chaotic behavior. We showed that the choice of test for detection of deterministic chaos in pollen series is difficult because pollen series exhibit power spectra. This is a characteristic that is also produced by colored noise series, which mimic deterministic chaos in most tests. We proposed to apply the Ikeguchi–Aihara test to properly detect the presence of deterministic chaos in pollen series. We examined the dynamics of cedar (Cryptomeria japonica) hourly pollen series by means of the Ikeguchi–Aihara test and concluded that these pollen series cannot be described as low-dimensional deterministic chaos. Therefore, the application of low-dimensional chaotic deterministic models to the prediction of short-range pollen concentration will not result in high-accuracy pollen forecasts even though these models may provide useful forecasts for certain applications. We believe that our conclusion can be generalized to pollen series from other wind-pollinated plant species, as wind speed, the forcing parameter of the pollen emission and transport, is best described as a nondeterministic series that originates in the high dimensionality of the atmosphere.