The dual functions of biphenyl-degrading ability of Pseudomonas sp. KKS102: energy acquisition and substrate detoxification

The bph operon of Pseudomonas sp. KKS102 is constituted of 11 bph genes which encode enzymes for biphenyl assimilation. Growth of a mutant in which a large part of the bph operon was deleted was inhibited by biphenyl in a concentration-dependent manner. We constructed a series of bph operon deletion mutants and tested for their biphenyl sensitivity. Growth inhibition by biphenyl was more prominent with the mutants defective in bphA1, bphB, bphC, and bphD, which were clustered in the bph operon and working in the early stage of the biphenyl degradation. The mutant defective in bphE, which was working at the late stage and forming a different cluster from the early stage genes, was not much inhibited by biphenyl. These indicate that biphenyl is detoxified by enzymes which function in the early stage of biphenyl assimilation and thus detoxification of substrates as well as energy acquisition could have played an important role in the evolution of the KKS102 bph operon.     

The stability and complexity of antibody responses to the major surface antigen of Plasmodium falciparum are associated with age in a malaria endemic area

Individuals that are exposed to malaria eventually develop immunity to the disease with one possible mechanism being the gradual acquisition of antibodies to the range of parasite variant surface antigens in their local area. Major antibody targets include the large and highly polymorphic Plasmodium falciparum Erythrocyte Membrane Protein 1 (PfEMP1) family of proteins. Here, we use a protein microarray containing 123 recombinant PfEMP1-DBLα domains (VAR) from Papua New Guinea to seroprofile 38 nonimmune children (<4 years) and 29 hyperimmune adults (≥15 years) from the same local area. The overall magnitude, prevalence and breadth of antibody response to VAR was limited at <2 years and 2-2.9 years, peaked at 3-4 years and decreased for adults compared with the oldest children. An increasing proportion of individuals recognized large numbers of VAR proteins (>20) with age, consistent with the breadth of response stabilizing with age. In addition, the antibody response was limited in uninfected children compared with infected children but was similar in adults irrespective of infection status. Analysis of the variant-specific response confirmed that the antibody signature expands with age and infection. This also revealed that the antibody signatures of the youngest children overlapped substantially, suggesting that they are exposed to the same subset of PfEMP1 variants. VAR proteins were either seroprevalent from early in life, (<3 years), from later in childhood (≥3 years) or rarely recognized. Group 2 VAR proteins (Cys2/MFK-REY+) were serodominant in infants (<1-year-old) and all other sequence subgroups became more seroprevalent with age. The results confirm that the anti-PfEMP1-DBLα antibody responses increase in magnitude and prevalence with age and further demonstrate that they increase in stability and complexity. The protein microarray approach provides a unique platform to rapidly profile variant-specific antibodies to malaria and suggests novel insights into the acquisition of immunity to malaria.     

Three-dimensional lithographically defined organotypic tissue arrays for quantitative analysis of morphogenesis and neoplastic progression

Here, we describe a simple micromolding method to construct three-dimensional arrays of organotypic epithelial tissue structures that approximate in vivo histology. An elastomeric stamp containing an array of posts of defined geometry and spacing is used to mold microscale cavities into the surface of type I collagen gels. Epithelial cells are seeded into the cavities and covered with a second layer of collagen. The cells reorganize into hollow tissues corresponding to the geometry of the cavities. Patterned tissue arrays can be produced in 3-4 h and will undergo morphogenesis over the following 1-3 d. The protocol can easily be adapted to study a variety of tissues and aspects of normal and neoplastic development.     

Individual ontogenetic trajectories and ontogenetic channels of three forms of large African barbs (<Emphasis Type="Italic">Barbus intermedius</Emphasis> complex). Analysis of experimental data

Changes of external morphological characters in three forms (morphotypes) of African barbs from Lake Tana (Ethiopia) belonging to Barbus intermedius complex were traced using electronic tags. It was shown that within age interval from two to four years individual ontogenetic allometric curves often cannot be described with a single power function equation. Individual ontogenetic trajectories in the principal components space within this interval strongly differ in shape meandering within ontogenetic channels outlined on the basis of cross-sectional data. Morphological differences between siblings are found that do not allow to classify some of them with the morphotype of their parents.     

Characterization of the intermediate filament proteins of Murine Mammary Gland epithelial cells : Response to collagen substratum

The insoluble cytoskeletal material remaining after detergent lysis of 'Normal' Murine Mammary Gland (NMuMG) cells, growing on plastic or collagen gel substrata, was analyzed by two-dimensional gel electrophoresis. The identity of the cytoskeletal elements was determined by their solubility properties, electrophoretic separation pattern, and immunoreactivity using monoclonal antibodies against intermediate filament proteins (AIF), keratins (AE1 and AE3) and actin. The electrophoretic pattern of the cytoskeletal elements from the NMuMG cell strain was found to be very similar to that of primary mouse mammary epithelial cells. Both NMuMG and primary mammary epithelial cells when grown on collagen exhibited an increased expression of a 49 kD protein with a pI of 5.6, that appeared to be a cytokeratin. Many of the cytoskeletal proteins remained tightly attached to the collagen gel substratum after cell lysis. These results demonstrate that the NMuMG cell strain has retained a stable expression of cytokeratins that remains responsive to the presence of extracellular matrix material.     

Spawning of large <Emphasis Type="Italic">Barbus</Emphasis> (<Emphasis Type="Italic">Barbus intermedins</Emphasis> Complex) in a small river of the Lake Tana basin (Ethiopia) and relationships of some putative species

Direct observations on three forms (morph types) of spawning barbs, considered by some authors as different species, were carried out in September–October 2005 at the Gumara River and its small tributary, the Ducalit, in the Lake Tana basin. The spawning barbs were sampled using cast nets. Barbs of different morphotypes composed common spawning aggregations and were caught together. It is likely that troutlike females mate extensively with intermedius males and, probably, with bigmouth small-eye males. Experiments in artificial fertilization and rearing showed that, during the embryonic period, there was no increase in mortality of progeny from crosses of barbs of different morphotypes, in comparison with homonomic crosses, thus suggesting an absence of postzygotic reproductive isolation between the morphotypes. At the same time, mortality of progeny from crossing barbs with Varicorhinus beso Rüppell, 1836 was very high during the earliest stages of development, suggesting strong, even if incomplete, reproductive isolation ensured by postzygotic mechanisms. Published in Voprosy Ikhtiologii, 2007, Vol. 47, No. 5, pp. 676–683. The article was translated by the authors.     

Characterization of the centromere and peri-centromere retrotransposons in Brassica rapa and their distribution in related Brassica species

We report the identification and characterization of the major repeats in the centromeric and peri-centromeric heterochromatin of Brassica rapa. The analysis involved the characterization of 88 629 bacterial artificial chromosomes (BAC) end sequences and the complete sequences of two BAC clones. We identified centromere-specific retrotransposons of Brassica (CRB) and various peri-centromere-specific retrotransposons (PCRBr). Three copies of the CRB were identified in one BAC clone as nested insertions within a tandem array of 24 copies of a 176 bp centromeric repeat, CentBr. A complex mosaic structure consisting of nine PCRBr elements and large blocks of 238 bp degenerate tandem repeats (TR238) were found in or near a derivative of 5S-25S rDNA sequences. The chromosomal positions of selected repeats were determined using in situ hybridization. These revealed that CRB is a major component of all centromeres in three diploid Brassica species and their allotetraploid relatives. However, CentBr was not detected in the most distantly related of the diploid species analyzed, B. nigra. PCRBr and TR238 were found to be major components in the peri-centromeric heterochromatin blocks of four chromosomes of B. rapa. These repetitive elements were not identified in B. oleracea or B. nigra, indicating that they are A-genome-specific. GenBank accession numbers: KBrH001P13 (AC 166739); KBrH015B20 (AC 166740); end sequences of KBrH BAC library (CW 978640 - CW 988843); end sequences of KBrS BAC library (DU 826965 - DU 835595); end sequences of KBrB BAC library (DX 010661 - DX 083363).     

Mode of infection of Hokkaido virus (Genus Hantavirus) among grey red-backed voles, Myodes rufocanus, in Hokkaido, Japan

Hokkaido virus (HOKV) is a member of the genus Hantavirus, in the family Bunyaviridae. To investigate HOKV infection in the host Myodes rufocanus, the grey red-backed vole, 199 animals were captured at Tobetsu (October 2004 and July 2005) and Nakagawa (October 2004) in Hokkaido, Japan, for detection of antibody, antigen, and viral RNA. In the surveys in Tobetsu (2004) and Nakagawa (2004), seropositive animals were detected at a frequency of 6.0% (5/84) and 10.4% (5/48), respectively. No seropositive animals were detected in Tobetsu in 2005. Seroprevalence in males in Tobetsu and Nakagawa in 2004 was 25% (1/4) and 45.5% (5/11), respectively, which was higher than in females, at 5.0% (4/80) and 0% (0/37), respectively (P<0.01). These results suggest that male animals play an important role in the maintenance of HOKV in M. rufocanus. Two females were seronegative but viral RNA-positive, indicating that these animals had acute infections before antibody was produced. Another five infected animals in Nakagawa were all male and had high levels of antibodies and viral RNA, suggesting that they had persistent infections. Viral RNA copies in organs of infected animals in Nakagawa were quantified by real-time polymerase chain reaction. Two acutely infected animals had > or = 10 times the number of RNA copies in their lungs compared to those of persistently infected animals. In most cases, lungs or spleen had the highest RNA copy number, regardless of infection status.     

Mutations in HPCA Cause Autosomal-Recessive Primary Isolated Dystonia

Reports of primary isolated dystonia inherited in an autosomal-recessive (AR) manner, often lumped together as “DYT2 dystonia,” have appeared in the scientific literature for several decades, but no genetic cause has been identified to date. Using a combination of homozygosity mapping and whole-exome sequencing in a consanguineous kindred affected by AR isolated dystonia, we identified homozygous mutations in HPCA, a gene encoding a neuronal calcium sensor protein found almost exclusively in the brain and at particularly high levels in the striatum, as the cause of disease in this family. Subsequently, compound-heterozygous mutations in HPCA were also identified in a second independent kindred affected by AR isolated dystonia. Functional studies suggest that hippocalcin might play a role in regulating voltage-dependent calcium channels. The identification of mutations in HPCA as a cause of AR primary isolated dystonia paves the way for further studies to assess whether “DYT2 dystonia” is a genetically homogeneous condition or not.