Transfer of the broad-host-range IncQ plasmid RSF1010 and other plasmid vectors to the Gram-positive methylotroph Brevibacterium methylicum by electrotransformation

Gram-positive facultative methylotrophic coryneform bacterium Brevibacterium methylicum was efficiently transformed with various plasmids using electroporation of intact cells. In addition to the plasmid vectors pEC71 and pZ6-1 constructed on the basis of cryptic plasmids from coryneform bacteria, broad-host-range plasmids pLS5 (derivative of plasmid pMV158 from Streptococcus agalactiae) and RSF1010 belonging to the incompatibility group IncQ from Gram-negative bacteria were found to be present as autonomous structurally unchanged DNA molecules in B. methylicum transformants. With the exception of pZ6-1, all these plasmids were stably maintained in B. methylicum cells grown under non-selective conditions. When plasmid DNAs isolated from B. methylicum were used, the highest efficiency of transformation (10⁵ transformants/g DNA) was achieved. Correspondence to: J. Nevera     

Carbon Incorporation and Anion Dynamics as Synergistic Drivers for Ultrafast Diffusion in Superionic LiCB11H12 and NaCB11H12

The disordered phases of LiCB₁₁H₁₂ and NaCB₁₁H₁₂ possess superb superionic conductivities that make them suitable as solid electrolytes. In these materials, cation diffusion correlates with high orientational mobilities of the CB₁₁H₁₂^? anions; however, the precise relationship has yet to be demonstrated. In this work, ab initio molecular dynamics and quasielastic neutron scattering are combined to probe anion reorientations and their mechanistic connection to cation mobility over a range of timescales and temperatures. It is found that anions do not rotate freely, but rather transition rapidly between orientations defined by the cation sublattice symmetry. The symmetry‐breaking carbon atom in CB₁₁H₁₂^? also plays a critical role by perturbing the energy landscape along the instantaneous orientation of the anion dipole, which couples fluctuations in the cation probability density directly to the anion motion. Anion reorientation rates exceed 3 × 10¹⁰ s^?1, suggesting the underlying energy landscape fluctuates dynamically on diffusion‐relevant timescales. Furthermore, carbon is found to modify the orientational preferences of the anions and aid rotational mobility, creating additional symmetry incompatibilities that inhibit ordering. The results suggest that synergy between the anion reorientational dynamics and the carbon‐modified cation–anion interaction accounts for the higher ionic conductivity in CB₁₁H₁₂^? salts compared with B₁₂H₁₂^2?.     

Responses of cell populations of three chlorococcal algae to the action of streptomycin

Time doses of a single concentration of streptomycin and its concentration doses acting for the same time period in a liquid medium had different effects on three strains of chlorococcal algae. This concerned both the physiological responses and permanent changes in the characteristics of cell colonies growing from treated cells. Significant differences were recorded in: the number of autospores produced during the first division of the treated cells on the surface of a solid medium, the length of the lag phase, the growth rate of the diameter of cell colonies, and the survival of the treated cells. The permanent changes in the characteristics of the growing colonies were very different in the individual algal strains in quality and frequency. Physiological and the mutation effects were compared and discussed.     

Isolation of Streptomyces rimosus Mutants with Reduced Actinophage Susceptibility

The infection of Streptomyces rimosus by the virulent actinophage RP1 was partially characterized. RP1 infection of the host cells results in a dramatic decrease in viable cell count, followed by reduced antibiotic production. Phage-resistant mutants were isolated after mutagenic treatment and RP1 selective pressure. Characterization of the isolated mutants has revealed that RP1 infection had no influence on their growth and antibiotic production. However, multiplication of the phage particles in the lawns of resistant mutants was detected. Since these strains differ from the wild type in RP1 relative efficiency of plating, plaque morphology, and the time necessary for plaque appearance, they are considered to be semiresistant mutants. The propagation of RP1 on semiresistant strains is characterized by lower adsorption of phage particles and longer latent and rise periods. As a consequence, the multiplication of the phage is slower than that of their host, which consequently reduces the ratio of phage to its host, thus diluting out the phage.     

A cherry map from the inter-specific cross Prunus avium ‘Napoleon’ × P. nipponica based on microsatellite, gene-specific and isoenzyme markers

One hundred and sixty microsatellite (simple sequence repeat (SSR)) and six gene-specific markers revealing 174 loci were scored in 94 seedlings from the inter-specific cross of Prunus avium ‘Napoleon’ × Prunus nipponica accession F1292. The co-segregation data from these markers were used to construct a linkage map for cherry which spanned 680 cM over eight linkage groups with an average marker spacing of 3.9 cM per marker and just six gaps longer than 15 cM. Markers previously mapped in Prunus dulcis ‘Texas’ × Prunus persica ‘Earlygold’ allowed the cherry map to be anchored to the peach × almond map and showed the high level of synteny between the species. Eighty-four loci segregated in P. avium ‘Napoleon’ versus 159 in P. nipponica. The segregations of 32 isoenzyme loci in a subset of 47 seedlings from the progeny were scored, using polyacrylamide gel electrophoresis and/or isoelectric focusing separation followed by activity staining, and the co-segregation data were analysed along with those for 39 isoenzymes reported previously and for the 174 sequence-tagged site loci plus an additional two SSR loci. The second map incorporates 233 loci and spans 736 cM over eight linkage groups with an average marker spacing of 3.2 cM per marker and just two gaps greater than 15 cM. The microsatellite map will provide a useful tool for cherry breeding and marker-assisted selection and for synteny studies within Prunus; the gene-specific markers and isoenzymes will be useful for comparisons with maps of other rosaceous fruit crops. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

The CFTR M470V gene variant as a potential modifier of COPD severity: study of Serbian population

Chronic obstructive pulmonary disease (COPD) is a complex disease influenced by genetic and environmental factors. Cystic fibrosis transmembrane conductance regulator (CFTR) protein is an important component of the lung tissue homeostasis, involved in the regulation of the rate of mucociliary clearance. As it is known that certain CFTR variants have consequences on the function of CFTR protein, the aim of this study was to examine the possible role of F508del, M470V, Tn locus, and R75Q variants in COPD development and modulation. Total number of 86 COPD patients and 102 control subjects were included in the study. Possible association between COPD susceptibility, severity, and onset of the disease and allele or genotype of four analyzed CFTR variants was examined. No associations were detected between COPD development, onset of the disease and tested CFTR alleles and genotypes. However, VV470 genotype was associated with mild/moderate COPD stages in comparison to severe/very severe ones (OR = 0.29, 95%CI = 0.11-0.80, p = 0.016). Our study showed that patients with VV470 genotype had a 3.4-fold decreased risk for the appearance of severe/very severe COPD symptoms, and the obtained results indicate that this genotype may have a protective role. These results also suggest the importance of studying CFTR gene as a modifier of this disease.     

Monitoring the orientation of myosin bridges on two-dimensional maps of birefringence in a single muscle fiber

Two-dimensional maps of birefringence in sarcomers of a single fiber of rabbit m.psoas were obtained by an automated interference microscope developed at our laboratory. The changes in birefringence of muscle fibers reflect the movement of myosin cross-bridges. The orientation of cross-bridges was modified by varying the pH (pH 7.0, 6.0, 8.0) and ionic strength (mu = 0.115, 0.085, 0.235) of the bathing rigor solution. The maximum value of total birefringence in the rigor state was observed at neutral pH. Total birefringence markedly decreased (by 40%) as pH was changed from 7.0 to both 8.0 and 6.0. No significant changes in light phase shifts were found at a 1.5 reduction of ionic strength in the rigor solution. The calculated birefringence values were 45% higher in rigor solutions of a high (mu = 0.235) ionic strength. The results observed are discussed in terms of changes in the orientation of cross-bridges due to the movement of the alpha-helical subfragment-2 away from the filament shaft (pH 8) or coming closer to it (mu = 0.235). The available data do not allow one to explain the results obtained at pH 6.0.     

Transformation of a new Gram-positive methylotroph,Brevibacterium methylicum,by plasmid DNA

Summary Brevibacterium methylicum is a newly isolated Gram-positive facultatively methylotrophic bacterium that uses the NAD⁺-dependent methanol dehydrogenase for methanol oxidation and assimilates its carbon via the ribulose monophosphate cycle. Protoplasts prepared by lysozyme treatment of B. methylicum cells grown in the presence of glycine were transformed by plasmid shuttle vectors pCEM500 (16.5 kb; Sm^r/Sp^r, Km^r/Gm^r) and pEC71 (7.1 kb; Km^r/Nm^r) constructed on the basis of B. lactofermentum plasmid pAM330 and replicating in Escherichia coli and in amino-acid-producing coryneform bacteria. The resistance markers were found to be expressed in B. methylicum and autonomous plasmid DNAs of various size were isolated from the transformants. The presence of the pAM330 replicon in these plasmids was demonstrated by DNA-DNA hybridization experiments. Offprint requests to: J. Nevera     

Influence of the war in Croatia on the frequency of gynecological cancer in the University Hospital Osijek in the period from 1985 to 2002

The aim of this study is to estimate the influence of war in Croatia on the frequency of gynecological cancer (cancer of corpus and cervix uteri and ovary) in the Clinical Hospital Osijek, particularly the relation between the pre-war and post-war period. We analyzed 1455 patients with corpus uteri and cervix uteri cancer and ovarian cancer treated in the Clinical Hospital Osijek in the period 1985-2002 (group I). Patients from Osjecko-Baranjska County were analyzed separately--1273 women, (group II) and in the group III there were 182 patients from other counties. The analyzed period was divided into: pre-war 1985-1990, war 1991-1993 and post-war period 1997-2002. In all three groups the number of patients treated for gynecological cancer was significantly larger in the post-war period (group I, N = 611, group II, N = 498, group III, N = 113) than in the pre-war period (group I, N = 457, group II, N = 433, group III, N = 24). The analysis of cancer frequency in relation to the site shows that a total number of patients treated for cervical cancer was larger in the post-war (N = 229) than in the pre-war period (N = 214), but the difference wasn't significant. However, the number of patients from Osjecko-baranjska County treated for cervical cancer was larger in the pre-war (N = 207) than in the post-war period (N = 178) but still, the difference wasn't significant. The number of patients treated for corpus uteri cancer and ovarian cancer was significantly larger for the I group in the post-war (N = 225 and N = 157 respectively) than in the pre-war period (N = 136, and N = 107 respectively). In the group II the number of patients treated for corpus uteri cancer and ovarian cancer was larger in the post-war (N = 196 and N = 124 respectively) than in the pre-war period (N = 130 and N = 96 respectively) but the difference was significant only for corpus uteri cancer. Significantly more women were treated for gynecological cancer in the post-war than in the pre-war period. However, the war had probably an indirect influence on the increased number of patients treated for gynecological cancer mainly because many more women arrived from other counties.     

Gas chromatography-mass spectrometry of oleanane- and ursane-type triterpenes—application to Chenopodium quinoa triterpenes

Nine trimethylsilylated pentacyclic triterpenes were separated by GLC on an OV-101 column employing temperature programming. Characteristic retro-Diels-Alder fragmentation was observed in their mass spectra. The fragmentation patterns allowed individual characterization except for certain isomers which, nevertheless, were resolved by GLC, thus permitting their identification. Oleanolic acid and hederagenin were confirmed to be major triterpenes of Chenopodium quinoa seed saponins.