Transgenic and herbicide resistant pearl millet (Pennisetum glaucum L.) R.Br. via microprojectile bombardment of scutellar tissue

Four different pearl millet breeding lines were transformed and led to the regeneration of fertile transgenic plants. Scutellar tissue was bombarded with two plasmids containing the bar selectable marker and the -glucuronidase reporter gene (gus or uidA) under control of the constitutive CaMV 35S promoter or the maize Ubiquitin1 promoter (the CaMV 35S is not a maize promoter). For the delivery of the DNA-coated microprojectiles, either the particle gun PDS 1000/He or the particle inflow gun was used. The calli and regenerants were selected for their resistance to the herbicide Basta (glufosinate ammonium) mediated by the bar gene. Putative transformants were screened for enzyme activity by painting selected leaves or spraying whole plants with an aqueous solution of the herbicide Basta and by the histochemical GUS assay using cut leaf segments. PCR and Southern blot analysis of genomic DNA indicated the presence of introduced foreign genes in the genomic DNA of the transformants. Five regenerated plants represent independent transformation events and have been grown to maturity and set seed. The integration of the bar selectable and the gus reporter gene was confirmed by genomic Southern blot analysis in all five plants. All five plants had multiple integrations of both marker genes. To date, the T₁ progeny of three out of four lines generated by the PDS particle gun shows co-segregating marker genes, indicating an integration of the bar and the gus gene at the same locus in the genome.     

A novel transmembrane MSP-containing protein that plays a role in right ventricle development

We have identified and characterized a gene, Mospd3 on mouse chromosome 5 using gene trapping in ES cells. MOSPD3 is part of a family of proteins, including MOSPD1, which is defined by the presence of a major sperm protein (MSP) domain and two transmembrane domains. Interestingly Mospd3 is mammalian specific and highly conserved between mouse and man. Insertion of the gene trap vector at the Mospd3 locus is mutagenic and breeding to homozygosity results in a characteristic right ventricle defect and neonatal lethality in 50% of mice. The phenotypic defect is dependent on the genetic background, indicating the presence of genetic modifier loci. We speculate that the further characterization of Mospd3 will shed light on the complex genetic interactions involved in cardiac development and disease.     

Preparation of internally labelled rat pituitary somatotropin (growth hormone).

Rat somatotropin (growth hormone) was labelled biosynthetically by incubating anterior pituitary lobes with radioactive amino acids for 24 h in a simple buffered salts medium containing glucose. The labelled hormone was isolated by preparative polyacrylamide-gel electrophoresis or by chromatography on Sephadex G-100 and then DEAE-cellulose. The labelled material was pure by several criteria and cross-reacted immunologically with unlabelled rat somatotropin. When a mixture of 14C-labelled amino acids was used for labelling the protein, label could be introduced into these same amino acids of somatotropin, though relative specific radioactivities varied considerably. Somatotropin labelled by the procedures described in the present paper was suitable for structural studies and could be used for a variety of other biochemical experiments.     

Virus Aggregation as the Cause of the Non-neutralizable Persistent Fraction

The non-neutralizable or persistent fraction of virus populations has been found to be caused by aggregated virus. Detailed investigation was performed with the prototype strain of echovirus type 4 (Pesascek), as this virus is notorious for its large non-neutralizable fraction. When Pesascek virus was clarified by low-speed centrifugation, homologous antiserum hardly neutralized the virus. However, when the virus was filtered through membranes having a porosity only twice the diameter of the virus, monodispersed virus was obtained which was efficiently neutralized. Serum titers were up to 1,000 times higher if the neutralization test was carried out with monodispersed virus. Virus in non-neutralizable aggregates was found to constitute 30% of the infective units of unfiltered Pesascek virus but only 0.1% of the antigenically related DuToit strain. This explains why DuToit strain has been a more satisfactory indicator strain for detecting type 4 antibodies, regardless of the echo 4 strain used for inducing the antibodies. Clarified suspensions and ultrafiltrates of viruses belonging to the picorna-, reo-, myxo-, adeno-, herpes-, and poxvirus groups were studied. Clarified suspensions yielded persistent fractions of 0.005% for poliovirus, of 0.1% for reovirus, of 0.6% for influenza virus, of <0.001% for adenovirus, of 0.06% for herpesvirus, and of 10 to 30% for vaccinia virus. In all cases the persistent fractions were removed by membrane filters which had a pore diameter no larger than twice that of the virus under test, and the high concentration of virus in each ultrafiltrate was completely neutralized by antiserum.     

The type III protein translocation system of enteropathogenic Escherichia coli involves EspA-EspB protein interactions

Enteropathogenic Escherichia coli (EPEC), like many bacterial pathogens, use a type III secretion system to deliver effector proteins across the bacterial cell wall. In EPEC, four proteins, EspA, EspB, EspD and Tir are known to be exported by a type III secretion system and to be essential for 'attaching and effacing' (A/E) lesion formation, the hallmark of EPEC pathogenicity. EspA was recently shown to be a structural protein and a major component of a large, transiently expressed, filamentous surface organelle which forms a direct link between the bacterium and the host cell. In contrast, EspB is translocated into the host cell where it is localized to both membrane and cytosolic cell fractions. EspA and EspB are required for translocation of Tir to the host cell membrane suggesting that they may both be components of the translocation apparatus. In this study, we show that EspB co-immunoprecipitates with the EspA filaments and that, during EPEC infection of HEp-2 cells, EspB localizes closely with EspA. Using a number of binding assays, we also show that EspB can bind and be copurified with EspA. Nevertheless, binding of EspA filaments to the host cell membranes occurred even in the absence of EspB. These results suggest that following initial attachment of the EspA filaments to the target cells, EspB is delivered into the host cell membrane and that the interaction between EspA and EspB may be important for protein translocation.     

Rhizodeposition shapes rhizosphere microbial community structure in organic soil

The aims of the study were to determine group specificity in microbial utilization of root-exudate compounds and whole rhizodeposition; quantify the proportions of carbon acquired by microbial groups from soil organic matter and rhizodeposition, respectively; and assess the importance of root-derived C as a driver of soil microbial community structure. Additions of 13C-labelled root-exudate compounds to organic soil and steady-state labelling of Lolium perenne, coupled to compound-specific isotope ratio mass spectrometry, were used to quantify group-specific microbial utilization of rhizodeposition. Microbial utilization of glucose and fumaric acid was widespread through the microbial community, but glycine was utilized by a narrower range of populations, as indicated by the enrichment of phospholipid fatty acid (PLFA) analysis fractions. In L. perenne rhizospheres, high rates of rhizodeposit utilization by microbial groups showed good correspondence with increased abundance of these groups in the rhizosphere. Although rhizodeposition was not the quantitatively dominant C source for microbes in L. perenne rhizospheres, relative utilization of this C source was an important driver of microbial group abundance in organic soil.     

Neighbourhood Factors and Depression among Adolescents in Four Caribbean Countries

Background

Past research suggests that perceived neighbourhood conditions may influence adolescents'' emotional health. Relatively little research has been conducted examining the association of perceived neighbourhood conditions with depressive symptoms among Caribbean adolescents. This project examines the association of perceived neighbourhood conditions with levels of depressive symptoms among adolescents in Jamaica, the Bahamas, St. Kitts and Nevis, and St. Vincent.

Methods

Adolescents attending grade ten of the academic year 2006/2007 in Jamaica, the Bahamas, St. Vincent, and St. Kitts and Nevis were administered the Neighbourhood Characteristics Questionnaire along with the BDI-II. Social cohesion, attachment to the neighbourhood, neighbourhood quality, neighbourhood crime, and neighbourhood disorder scales were created by summing the relevant subscales of the Neighbourhood Characteristics Questionnaire. Multiple regression analyses were used to examine the relationships of perceived neighbourhood conditions to depressive symptoms.

Results

A wide cross-section of tenth grade students in each nation was sampled (n = 1955; 278 from Jamaica, 217 from the Bahamas, 737 St. Kitts and Nevis, 716 from St. Vincent; 52.1% females, 45.6% males and 2.3% no gender reported; 12 to 19 years, mean = 15.3 yrs, sd = .95 yr). Nearly half (52.1%) of all adolescents reported mild to severe symptoms of depression with 29.1% reporting moderate to severe symptoms of depression. Overall, Jamaican adolescents perceived their neighbourhoods in a more positive manner than those in the Bahamas, St. Vincent and St. Kitts and Nevis. Results of a series of hierarchical multiple regression analyses suggested that a different pattern of neighbourhood factors for each island were associated with depressive symptoms. However, neighbourhood factors were more highly associated with depressive symptoms for Jamaican students than for students in the other three islands.

Conclusions

Neighbourhood factors appear to be partially associated with adolescents'' self-reports of depressive symptoms. However, other factors may mitigate this relationship.     

SSCP and segregation analysis of the human type X collagen gene (COL10A1) in heritable forms of chondrodysplasia.

Type X collagen is a homotrimeric, short chain, nonfibrillar collagen that is expressed exclusively by hypertrophic chondrocytes at the sites of endochondral ossification. The distribution and pattern of expression of the type X collagen gene (COL10A1) suggests that mutations altering the structure and synthesis of the protein may be responsible for causing heritable forms of chondrodysplasia. We investigated whether mutations within the human COL10A1 gene were responsible for causing the disorders achondroplasia, hypochondroplasia, pseudoachondroplasia, and thanatophoric dysplasia, by analyzing the coding regions of the gene by using PCR and the single-stranded conformational polymorphism technique. By this approach, seven sequence changes were identified within and flanking the coding regions of the gene of the affected persons. We demonstrated that six of these sequence changes were not responsible for causing these forms of chondrodysplasia but were polymorphic in nature. The sequence changes were used to demonstrate discordant segregation between the COL10A1 locus and achondroplasia and pseudoachondroplasia, in nuclear families. This lack of segregation suggests that mutations within or near the COL10A1 locus are not responsible for these disorders. The seventh sequence change resulted in a valine-to-methionine substitution in the carboxyl-terminal domain of the molecule and was identified in only two hypochondroplasic individuals from a single family. Segregation analysis in this family was inconclusive, and the significance of this substitution remains uncertain.