Identification of the membrane component of the anion pump encoded by the arsenical resistance operon of R-factor R773

The arsenical resistance (ars) operon of the conjugative R-factor R773 encodes an ATP-driven anion extrusion pump, producing bacterial resistance to arsenicals. There are three structural genes, of which the product of the middle gene, arsB, has not previously been identified. From nucleotide sequence data, the ArsB protein is predicted to be a 45577 Dalton hydrophobic protein. A mini-Mu transposition procedure was used to construct an arsB-lacZ gene fusion, producing a hybrid ArsB-beta-galactosidase protein which was localized in the inner membrane. The operon was cloned into a T7 RNA polymerase expression vector. In addition to the previously identified ArsA and ArsC proteins, the cells synthesized an inner membrane protein with an apparent mass of 36 kD identified as the ArsB protein.     

A comparison of two plating techniques to estimate plasmid stability of a prolonged chemostat culture

Summary A comparison of two plating techniques to estimate the segregational stability ofEscherichia coli RR1 harboring plasmid pBR322 in a chemostat was studied. No significant differences were observed between the spread and replica plating techniques in the beginning of the experiments. However, a noticeable discrepancy between these two methods appeared after approximately 100 hours. This inconsistency can be shown to be statistically significant.     

Morphological and molecular characterization of fertile tetraploid somatic hybrids produced by protoplast electrofusion and PEG-induced fusion between Lycopersicon esculentum Mill. and Lycopersicon peruvianum Mill.

Summary Mesophyl protoplasts of two genotypes of cultivated tomato (Lycopersicon esculentum Mill.) and one of its wild relative species (Lycopersicon peruvianum Mill.) were fused by using electrofusion and polyethyleneglycol-induced fusion. Forty-three fertile tetraploid somatic hybrid plants, each deriving from separate calli, were recovered from both fusion procedures. Electrofusion appeared more efficient than chemical fusion for the production of somatic hybrids. These plants appeared morphologically similar, whatever the fusion procedure and tomato genotype. They had intermediate leaf, inflorescence, and flower morphology. After self-pollination, the hybrids set fruit of intermediate size and color. The hybrid nature of these plants was confirmed by isoelectric focusing of the Rubisco small subunits used as nuclear markers. L. esculentum and L. peruvianum were distinguished by means of two chloroplast markers: CF1-ATPase subunit as analyzed by isoelectro-focusing and ct DNA restriction patterns. All hybrids displayed both ct markers of only one parent with no biased transmission. Mitochondrial (mt) DNAs were prepared from flower buds by using miniaturized CsCl gradients. Preliminary analysis indicated that mt genomes from the hybrids all differed from those of both parents. mt DNA Sall restriction enzyme analysis revealed that all but two hybrids contained one novel fragment of 13.5 kb. Gene mapping experiments showed that the mt apocytochrome b and ATPase subunit 9 homologies in the somatic hybrid mt DNA resembled L. esculentum and L. peruvianum, respectively; the mt nad5 probe distinguished at least four distinct patterns in the hybrids. These results indicated that mt DNA rearrangements involving intergenomic recombinations occurred through protoplast fusion. A greater mt DNA polymorphism was induced with chemical fusion than with electrofusion.     

Fine needle aspiration of peripheral neuroepithelioma of soft tissues.

Peripheral neuroepithelioma of soft tissue is a malignant primitive neuroectodermal tumor that appears both in children and adults and usually has a poor outcome. Fine needle aspiration on two patients with tumors in the lower limbs showed small round cells with unipolar processes and a tendency to form Homer-Wright rosettes. The cells had a round to oval nucleus with fine chromatin, up to four small, conspicuous nucleoli and vacuolated, periodic acid-Schiff-positive cytoplasm. The diagnosis was supported by electron microscopic study of the aspirate, which showed features of neuroblastic differentiation (i.e., neurosecretory granules), and by histologic, immunohistochemical and cytogenetic study of the resected tumors.     

Detection of hepatitis B virus DNA in human serum samples: use of digoxigenin-labeled oligonucleotides as modified primers for the polymerase chain reaction.

Nonradioactive techniques have been used for the direct detection of hepatitis B virus DNA in human serum samples. A comparison of two different systems using digoxigenin-labeled DNA probes is presented. Furthermore, oligonucleotides containing one molecule of the hapten digoxigenin at the 5'-end were prepared and used as primers for the polymerase chain reaction. Amplified DNA can be directly analyzed with anti-digoxigenin Fab fragments labeled with alkaline phosphatase and chemiluminescent substrates.     

Effect of ethanol and fatty acids on malolactic activity ofLeuconostoc oenos

Medium-chain fatty acids (C₆ to C₁₂), produced by yeast metabolism during alcoholic fermentation, are known to be inhibitory to lactic acid bacteria. The purpose of this work was to clarify the effect of both ethanol and decanoic and dodecanoic acids on the growth and malolactic activity of aLeuconostoc oenos strain isolated from Portuguese red wine. Ethanol in concentrations up to 12% had no significant effect on malolactic activity but strongly inhibited cell growth. The fatty acids decanoic acid, in concentrations up to 12.5 mg l^–1, and, dodecanoic acid up to 2.5 mg l^–1 seemed to act as growth factors stimulating also malolactic activity; at higher concentrations they exerted an inhibitory effect. We found clear pH dependence between pH 3.0 and pH 6.0, between decanoic acid concentration and its effect on malolactic activity, indicating that the undissociated molecule is the active form. At pH 3.0 the results can be explained by considering that fatty acids enter the cell as protonated molecules and dissociate in the cytoplasm due to the higher internal pH, leading to increased intracellular hydrogenous concentration. This may be the basis of two different effects that contribute to the observed inhibition: decrease in the intracellular pH and dissipation of the transmembrane proton gradient, thus inhibiting intracellular enzymes and ApH-dependent transport systems.     

Locus Heterogeneity for Waardenburg Syndrome is Predictive of Clinical Subtypes

Waardenburg syndrome (WS) is a dominantly inherited and clinically variable syndrome of deafness, pigmentary changes, and distinctive facial features. Clinically, WS type I (WS1) is differentiated from WS type II (WS2) by the high frequency of dystopia canthorum in the family. In some families, WS is caused by mutations in the PAX3 gene on chromosome 2q. We have typed microsatellite markers within and flanking PAX3 in 41 WS1 kindreds and 26 WS2 kindreds in order to estimate the proportion of families with probable mutations in PAX3 and to study the relationship between phenotypic and genotypic heterogeneity. Evaluation of heterogeneity in location scores obtained by multilocus analysis indicated that WS is linked to PAX3 in 60% of all WS families and in 100% of WS1 families. None of the WS2 families were linked. In those families in which equivocal lod scores (between −2 and +1) were found, PAX3 mutations have been identified in 5 of the 15 WS1 families but in none of the 4 WS2 families. Although preliminary studies do not suggest any association between the phenotype and the molecular pathology in 20 families with known PAX3 mutations and in four patients with chromosomal abnormalities in the vicinity of PAX3, the presence of dystopia in multiple family members is a reliable indicator for identifying families likely to have a defect in PAX3.     

Expression of a maize proteinase inhibitor gene is induced in response to wounding and fungal infection: systemic wound-response of a monocot gene

The isolation and characterization of cDNA and genomic clones encoding a proteinase inhibitor protein (MPI) in maize is reported. Accumulation of the MPI mRNA is induced in response to fungal infection in germinating maize embryos. The expression pattern of the MPI gene, in healthy and fungal infected maize tissues, was examined and compared with the expression pattern of a gene that codes for a pathogenesis-related protein (the PRms protein) from maize. These two genes are induced by fungal infection, however different signals trigger their activation. Accumulation of the proteinase inhibitor mRNA is more a consequence of the wound produced by the penetration and colonization of the host tissues by the pathogen, than the result of a direct molecular recognition of the pathogen by the plant, as is the case for the induction of the PRms gene. Wounding, or treatment with abscisic acid or methyl jasmonate, stimulate MPI mRNA accumulation, but not PRms mRNA accumulation. Local and systemic induction of the MPI gene expression in response to wounding occurs in maize plants. To the authors' knowledge, this is the first example of a gene from a monocotyledonous species that clearly shows a systemic wound response. The possible functional implications for the existence of different signal transduction pathways that simultaneously activate a battery of defense mechanisms against potential pathogens are discussed.     

The metabolism of sugar and malic acid by Leuconostoc oenos: effect of malic acid, pH and aeration conditions

The co-metabolism of sugars by Leuconostoc oenos was studied under different environmental conditions. Under aerobic conditions, growth and sugar metabolism were poorer than under CO₂ or N₂ atmosphere and acetic acid accumulated to a larger extent. Glycerol was found in the aerobic cultures while erythritol was detected under N₂ or CO₂. When medium conditions make growth difficult (low pH, aerobic conditions, low nutrients), sugars were only slightly metabolized and growth was very slow while malic acid was rapidly and completely degraded, leading to an increase in the y _ATP. Aeration effects on the malic acid degradation rate depended on the nutrients and carbon source in the medium. Malic acid clearly stimulated bacterial growth, allowing an increase in the molar growth yields and ATP production. The results suggest that under adverse conditions cells are not able to grow and malic degradation supplies additional energy production.