The mink gene for the λ light immunoglobulin chain: Characterization of cDNA and chromosomal localization

A cDNA library from mink spleen was constructed by use of the phage gt11. The library was screened using polyvalent serum raised against the mink immunoglobulin chain. As a result, several clones expressing mink immunoglobulin light chains were identified. Sequencing of one of the clones with an 803 bp insert was performed. The insert comprised nearly the entire coding region for the mature light immunoglobulin gene with the exception of the leader polypeptide and several amino acids of the RF1 region of the V segment. Compared with the rabbit, mouse and human light immunoglobulin genes, the homology of the cloned sequence was found to be highest relative to the rabbit gene. With the cloned mink cDNA containing the C-region only as a probe, the DNAs from mink-Chinese hamster hybrid clones were studied. The results of segregation analysis of this mink cDNA sequence and mink chromosomes in the mink-Chinese hamster clone panel allowed us to assign the gene for the light immunoglobulin constant polypeptide (IGLC) to mink Chromosome (Chr) 4.     

Are bloodstream leukocytes Trojan Horses for the metastasis of Staphylococcus aureus?

Staphylococcus aureus bacteraemia remains very difficult to treat, and a large proportion of cases result in potentially lethal metastatic infection. Unpredictable and persistent bacteraemia in the face of highly active, usually bactericidal antibiotics is the strongest predictor of death or disseminated disease. Although S. aureus has conventionally been considered an extracellular pathogen, much evidence demonstrates that it can survive intracellularly. In this Opinion article, we propose that phagocytes, and specifically neutrophils, represent a privileged site for S. aureus in the bloodstream, offering protection from most antibiotics and providing a mechanism by which the bacterium can travel to and infect distant sites. Furthermore, we suggest how this can be experimentally confirmed and how it may prompt a change in the current paradigm of S. aureus bacteraemia and identify better treatment options for improved clinical outcomes.     

New 18S·26S ribosomal RNA gene loci: chromosomal landmarks for the evolution of polyploid wheats

Three new 18S·26S rRNA gene loci were identified in common wheat by sequential N-banding and in situ hybridization (ISH) analysis. Locus Nor-A7 is located at the terminal area of the long arm of 5A in both diploid and polyploid wheats. Locus Nor-B6 is located in N-band 1BL2.5 of the long arm of chromosome 1B in Triticum turgidum and Triticum aestivum. ISH sites, similar to Nor-B6, were also detected on the long arms of chromosomes 1G in Triticum timopheevii and 1S in Aegilops speltoides, but their locations on the chromosomes were different from that of Nor-B6, indicating possible chromosome rearrangements in 1GL and 1BL during evolution. The third new locus, Nor-D8, was only found on the short arm of chromosome 3D in the common wheat Wichita. The loss of rRNA gene locus Nor-A3 and gain of repetitive DNA sequence pSc119 on the terminal part of 5AS suggest a structural modification of 5AS. Comparative studies of the location of the 18S·26S rRNA gene loci in polyploid wheats and putative A and B (G) genome progenitor species support the idea that: (1) Triticum monococcum subsp. urartu is the donor of both the A and A^t genome of polyploid wheats. (2) Ae. speltoides is closer to the B and G genome of polyploid wheats than Aegilops longissima and is the most probable progenitor of these two genomes.     

Mycelium of fungi isolated from mouldy foods inhibits Staphylococcus aureus including MRSA – A rationale for the re-introduction of mycotherapy?

Fungal mycelium capable of producing antibacterial agents was isolated from samples of apple, beetroot, lemon and orange; the mycelium of all isolates produced penicillin, while the apple and beetroot samples also produced the antibacterial mycotoxin patulin. The known penicillin-producing fungi were shown to produce penicillin, but not patulin. The mycelial discs of all of fruit and vegetable isolates, as well as the two known penicillin producing fungi, inhibited Staphylococcus aureus, and mycelium of all isolates inhibited MRSA, in contrast, only one of the two known penicillin-producers did so. The results are discussed in relation to the possibility of using the mycelium of Penicillium species in mycotherapy.     

Human aldehyde dehydrogenase: chromosomal assignment of the gene for the isozyme that metabolizes γ-aminobutyraldehyde

A cDNA clone of the E3 isozyme of human liver aldehyde dehydrogenase consisting of a 1320-base pair (bp) coding region and a 180-bp non-coding region at the 3 end was used for chromosomal localization of the E3 gene. Using a panel of human/hamster somatic cell hybrids we have localized, the gene coding for the E3 isozyme to human chromosome 1.     

Characterization of NADPH–cytochrome P450 reductase gene from the cotton bollworm, Helicoverpa armigera

A complete cDNA encoding the NADPH–cytochrome P450 reductase (haCPR) and its genomic sequence from the cotton bollworm Helicoverpa armigera were cloned and sequenced. The open reading frame of haCPR codes for a protein of 687 amino acid residues with a calculated molecular mass of 77.4 kDa. The haCPR gene spans over 11 kb and its coding region is interrupted by 11 introns. haCPR is ubiquitously expressed in various tissues and at various stages of development. Escherichia coli produced haCPR enzyme exhibited catalytic activity for NADPH-dependent reduction of cytochrome c, following Michaelis–Menten kinetics. The functionality of CPR was further demonstrated by its capacity to support cytochrome P450 (e.g. haCYP9A14 and chicken CYP1A5) mediated O-dealkylation activity of alkoxyresorufins. The flavoprotein-specific inhibitor (diphenyleneiodonium chloride, DPI) showed a potent inhibition to haCPR activity (IC₅₀ = 1.69 μM). Inhibitory effect of secondary metabolites in the host plants (tannic acid, quercetin and gossypol) on CPR activity (with an IC₅₀ value ranged from 15 to 90 μM) was also observed.     

β-Thalassemia major resulting from a compound heterozygosity for the β-globin gene mutation: further evidence for multiple origin and migration of the thalassemia gene

Summary We describe in a Japanese family -thalassemia resulting from a compound heterozygosity for a -globin gene mutation. One mutation is a C-to-T transition at IVS-2 nucleotide position 654 on the background of Mediterranean haplotype IX. Another mutation is a G-to-A transition at IVS-2 nucleotide position 1, associated with a novel haplotype XL The occurrence of these mutations on various chromosomal backgrounds provides strong evidence for an interplay of gene migration, interallelic gene conversion, and multiple origins of the same mutation.     

Characterization of Staphylococcus aureus from Humans and a Comparison with ?solates of Animal Origin,in North Dakota,United States

Different clones of methicillin-susceptible (MSSA) and methicillin-resistant (MRSA) Staphylococcus aureus have been found in humans as well as in animals and retail meat. However, more information about the genetic characteristics and similarities between strains is needed. The aim of this study was to identify and characterize Staphylococcus aureus from humans, and to compare their characteristics with isolates of animal origin. A total of 550 nasal swabs were taken from healthy humans, and S. aureus was isolated and identified. Positive S. aureus isolates were subjected to molecular typing and susceptibility testing. In addition, 108 MRSA isolates recovered from clinical patients in the state of North Dakota and 133 S. aureus isolates from animals and meat previously analyzed were included. The nasal carriage of S. aureus in healthy people was 7.6% and, in general, clones were genetically diverse. None of the S. aureus strains obtained from healthy people were mecA- or PVL-positive. A total of 105 (97.2%) MRSA isolates from clinical cases harbored the mecA gene and 11 (10.2%) isolated from blood stream infections harbored the PVL gene. The most common resistance profile among S. aureus from healthy people was penicillin, and from clinical cases were erythromycin-penicillin-ciprofloxacin. The rate of multidrug resistance (MDR) was 70% in humans. Most of the S. aureus harboring mecA and PVL genes were identified as ST5 and ST8, and exhibited MDR. However, S. aureus isolates of animal origin used for comparison exhibited a lower rate of MDR. The most common resistance profiles in isolates of animal origin were penicillin-tetracycline and penicillin-tetracycline-erythromycin, in animals and raw meat, respectively. The ST5 was also found in animals and meat, with ST9 and ST398 being the major clones. The genetic similarity between clones from humans and meat suggests the risk of spread of S. aureus in the food chain.     

The evolution and maintenance of virulence in Staphylococcus aureus: a role for host-to-host transmission?

Despite progress in our understanding of infectious disease biology and prevention, the conditions that select for the establishment and maintenance of microbial virulence remain enigmatic. To address this aspect of pathogen biology, we focus on two members of the Staphylococcus genus - Staphylococcus aureus and Staphylococcus epidermidis - and consider why S. aureus has evolved to become more virulent than S. epidermidis. Several hypotheses to explain this phenomenon are discussed and a mathematical model is used to argue that a complex transmission pathway is the key factor in explaining the evolution and maintenance of virulence in S. aureus. In the case of S. epidermidis, where skin contact affords easier transmission between hosts, high levels of virulence do not offer an advantage to this pathogen.     

Dissection of events in the resistance to β-lactam antibiotics mediated by the protein BlaR1 from Staphylococcus aureus

A heterologous expression system was used to evaluate activation of BlaR1, a sensor/signal transducer protein of Staphylococcus aureus with a central role in resistance to β-lactam antibiotics. In the absence of other S. aureus proteins that might respond to antibiotics and participate in signal transduction events, we documented that BlaR1 fragmentation is autolytic, that it occurs in the absence of antibiotics, and that BlaR1 directly degrades BlaI, the gene repressor of the system. Furthermore, we disclosed that this proteolytic activity is metal ion-dependent and that it is not modulated directly by acylation of the sensor domain by β-lactam antibiotics.     

Human Vκ immunoglobulin gene number: Implications for the origin of antibody diversity

To assess the relative contributions of germline versus somatically mutated genes in the human immune system, we have examined the size of the kappa light-chain variable region (V_κ) gene pool. Two cloned kappa subgroup 1 (V_κ1) gene probes detected the same family of 15 to 20 crosshybridizing restriction fragments in human DNA, whereas flanking region probes detected fewer hybridizing fragments. Most of the hybridizing bands represent single-copy genes, as judged by a “gene titration” experiment. Furthermore, the number of hybridization bands is a good estimate of the haploid gene number, since we observed little polymorphism of restriction sites in the V_κ locus of eight unrelated people. A cloned V_κ3 probe hybridized to essentially the same 15–20 genes in human DNA as the V_κ1 probes. These results strongly suggest that a discrete family of 15–20 genes constitutes a large proportion of the V genes from three of the four V_κ subgroups. The small number of V_κ genes in the human genome supports the idea that somatic mutation plays a major role in the origin of antibody diversity in man.     

Molecular characterization of nitrite reductase gene (aniA) and gene product in Neisseria meningitidis isolates: is aniA essential for meningococcal survival?

The present study evaluates sequence conservation in the gene coding for nitrite reductase (aniA) and AniA expression from a panel of Neisseria meningitidis isolates. Sequence analysis of the coding region in 19 disease-associated and 4 carrier strains notwithstanding a high degree of sequence similarity showed a number of nucleotide changes, some of which possibly resulted in premature translation termination or function loss. In particular, in one disease-associated strain a 9-residues insertion was found to be located close to the type I Cu-site and a catalytic histidine at position 280 was mutated into a leucine. In two strains from carriers, a sequence corresponding to a portion of a transposase gene within the aniA was also found. The AniA protein was always expressed, except for these two carriers strains and for other two strains in which the presence of the premature stop codons was recognized. The biochemical properties of the cloned soluble domain of the enzyme (sAniA) from N. meningitidis reference MC58 strain and from a clinical invasive isolate were studied. In particular, biochemical analysis of sAniA from MC58 demonstrated a clear dependence of its catalytic activity upon acidification, while the clinical isolate-derived sAniA was not functional. Thus, the results obtained suggest that the presence of a conserved and functional aniA gene is not essential for meningococcal survival.     

Neuroblastoma in a dwarfed newborn. Possible clue to the chromosomal localization of the gene for achondroplasia?

The authors report a premature achondroplastic child with connatal neuroblastoma. Though this association could be coincidental, we suggest that a microdeletion inducing a contiguous gene syndrome involving the locus of neuroblastoma suppressor gene could be an alternative hypothesis. The gives a working hypothesis for the localization of the gene for achondroplasia.     

Characterization of the plasma-membrane-bound nitrate reductase in Chlorella saccharophila (Krüger) Nadson

The plasma membranes of Chlorella saccharophila (Krüger) Nadson cells contained a membrane-bound nitrate reductase. This form of nitrate reductase was purified and characterized. Several differences from the soluble form of nitrate reductase were apparent, the most important being: (i) the greater hydrophobicity, as proven using Triton X-114 phase separation, hydrophobic interaction chromatography and stimulation by phosphilipids; (ii) the differences in the native molecular mass compared with Chlorella sorokiniana (Krüger) Nadson; and (iii) the different polypeptide pattern obtained by two-dimensional polyacrylamide gel electrophoresis. Only the plasma-membrane-bound nitrate reductase could be found in both inside-out and right-side-out plasma-membrane vesicles.Abbreviations HIC hydrophobic interaction chromatography - IEF isoelectric focusing - MV methyl viologen - NR nitrate reductase - PM plasma membrane - PMNR plasma-membrane-bound nitrate reductase - SDS-PAGE sodium dodecyl sulfatepolyacrylamide gel electrophoresis This work is part of the Ph.D. Thesis of Christine Stöohr, University of Göttingen. This work was supported by the Deutsche Forschungsgemeinschaft.     

Crystal Structures of Penicillin-Binding Protein 3 (PBP3) from Methicillin-Resistant Staphylococcus aureus in the Apo and Cefotaxime‐Bound Forms

Staphylococcus aureus is a widespread Gram‐positive opportunistic pathogen, and a methicillin‐resistant form (MRSA) is particularly difficult to treat clinically. We have solved two crystal structures of penicillin‐binding protein (PBP) 3 (PBP3) from MRSA, the apo form and a complex with the β-lactam antibiotic cefotaxime, and used electrospray mass spectrometry to measure its sensitivity to a variety of penicillin derivatives. PBP3 is a class B PBP, possessing an N-terminal non-penicillin‐binding domain, sometimes called a dimerization domain, and a C-terminal transpeptidase domain. The model shows a different orientation of its two domains compared to earlier models of other class B PBPs and a novel, larger N-domain. Consistent with the nomenclature of “dimerization domain”, the N-terminal region forms an apparently tight interaction with a neighboring molecule related by a 2-fold symmetry axis in the crystal structure. This dimer form is predicted to be highly stable in solution by the PISA server, but mass spectrometry and analytical ultracentrifugation provide unequivocal evidence that the protein is a monomer in solution.     

Comparative genomic analysis of the proteasome β5t subunit gene: implications for the origin and evolution of thymoproteasomes

The thymoproteasome is a recently discovered, specialized form of 20S proteasomes expressed exclusively in the thymic cortex. Although the precise molecular mechanism by which the thymoproteasome exerts its function remains to be elucidated, accumulating evidence indicates that it plays a crucial role in positive selection of T cells. In the present study, we analyzed the evolution of the β5t subunit, a β-type catalytic subunit uniquely present in thymoproteasomes. The gene coding for the β5t subunit, designated PSMB11, was identified in the cartilaginous fish, the most divergent group of jawed vertebrates compared to the other jawed vertebrates, but not in jawless vertebrates or invertebrates. Interestingly, teleost fish have two copies of apparently functional PSMB11 genes, designated PSMB11a and PSMB11b, that encode β5t subunits with distinct amino acids in the S1 pocket. BLAST searches of genome databases suggest that birds such as chickens, turkey, and zebra finch lost the PSMB11 gene, and have neither thymoproteasomes nor immunoproteasomes. In mammals, reptiles, amphibians, and teleost fishes, the PSMB11 gene (the PSMB11a gene in teleost fish) is located next to the PSMB5 gene coding for the β5 subunit of the standard 20S proteasome, indicating that the PSMB11 gene arose by tandem duplication from the evolutionarily more ancient PSMB5 gene. The general absence of introns in PSMB11 and an unusual exon–intron structure of jawed vertebrate PSMB5 suggest that PSMB5 lost introns and duplicated in tandem in a common ancestor of jawed vertebrates, with PSMB5 subsequently gaining two introns and PSMB11 remaining intronless.