Transgene inactivation inPetunia hybrida is influenced by the properties of the foreign gene

Petunia mutant RL01 was transformed with maizeA1 and gerberagdfr cDNAs, which both encode dihydroflavonol-4-reductase (DFR) activity. The sameAgrobacterium vector and the same version of the CaMV 35S promoter were used in both experiments. Transformation with the cDNAs resulted in production of pelargonidin pigments in the transformants. However, theA1 andgdfr transformants showed clearly different phenotypes. The flowers of the primaryA1 transformants were pale and showed variability in pigmentation during their growth, while the flowers of thegdfr transformants showed intense and highly stable coloration. The color difference in the primary transformants was reflected in the expression levels of the transgenes as well as in the levels of anthocyanin pigment. As previously reported by others, the instability in pigmentation in theA1 transformants was more often detected in clones with multiple copies of the transgene and was associated with methylation of the 35S promoter and of the transgene cDNA itself. In thegdfr transformants, the most intense pigmentation was observed in plants with multiple transgenes in their genome. Only rarely was partial methylation of the 35S promoter detected, while thegdfr cDNA always remained in an unmethylated state. We conclude that the properties of the transgene itself strongly influence the inactivation process. The dicotyledonousgdfr cDNA with a lower GC content and fewer possible methylation sites is more compatible the genomic organization of petunia and this prevents it being recognized as a foreign gene and hence silenced by methylation.     

Molecular Epidemiology and Transmission Dynamics of Recent and Long-Term HIV-1 Infections in Rural Western Kenya

Objective

To identify unique characteristics of recent versus established HIV infections and describe sexual transmission networks, we characterized circulating HIV-1 strains from two randomly selected populations of ART-naïve participants in rural western Kenya.

Methods

Recent HIV infections were identified by the HIV-1 subtype B, E and D, immunoglobulin G capture immunoassay (IgG BED-CEIA) and BioRad avidity assays. Genotypic and phylogenetic analyses were performed on the pol gene to identify transmitted drug resistance (TDR) mutations, characterize HIV subtypes and potential transmission clusters. Factors associated with recent infection and clustering were assessed by logistic regression.

Results

Of the 320 specimens, 40 (12.5%) were concordantly identified by the two assays as recent infections. Factors independently associated with being recently infected were age ≤19 years (P = 0.001) and history of sexually transmitted infections (STIs) in the past six months (P = 0.004). HIV subtype distribution differed in recently versus chronically infected participants, with subtype A observed among 53% recent vs. 68% chronic infections (p = 0.04) and subtype D among 26% recent vs. 12% chronic infections (p = 0.012). Overall, the prevalence of primary drug resistance was 1.16%. Of the 258 sequences, 11.2% were in monophyletic clusters of between 2–4 individuals. In multivariate analysis factors associated with clustering included having recent HIV infection P = 0.043 and being from Gem region P = 0.002.

Conclusions

Recent HIV-1 infection was more frequent among 13–19 year olds compared with older age groups, underscoring the ongoing risk and susceptibility of younger persons for acquiring HIV infection. Our findings also provide evidence of sexual networks. The association of recent infections with clustering suggests that early infections may be contributing significant proportions of onward transmission highlighting the need for early diagnosis and treatment as prevention for ongoing prevention. Larger studies are needed to better understand the structure of these networks and subsequently implement and evaluate targeted interventions.     

Fatty Acid Degradation in Escherichia coli: Requirement of Cyclic Adenosine Monophosphate and Cyclic Adenosine Monophosphate Receptor Protein for Enzyme Synthesis

The strong repression of inducible synthesis of the enzymes of fatty acid degradation by glucose can be partially relieved by the addition of cyclic adenosine 3',5' monophosphate (cyclic AMP) to the growth medium. This reversal of the glucose effect by cyclic AMP is not observed in a mutant (K29) that is unable to grow on fatty acids as sole carbon source and that was found to synthesize low levels of several enzymes specified by the fad regulon. In a revertant selected for the ability to grow on oleate these effects are concomitantly relieved. By both genetic (co-transduction of the mutation with the strA locus) and biochemical experiments (an extract of the mutant strain does not show the cyclic AMP-dependent stimulation of the deoxyribonucleic acid-directed in vitro synthesis of the enzymes of the gal operon), it is demonstrated that the mutant lacks functional cyclic AMP receptor protein (CR protein). It is concluded that, like many other inducible enzyme systems, expression of the enzymes of the fad system requires cyclic AMP and the CR protein.     

Mitochondrial accumulations in nerve fibres of human sympathetic ganglia

Summary The fine structure of mitochondrial accumulations in axonal swellings of human sympathetic ganglia is described. A typical swelling contained, in addition to regularly organized mitochondria, bundles of neurofilaments and vesicles as well as large dense-cored vesicles and myelin figures. Synaptic contacts between axonal swellings with mitochondrial accumulations and ganglion cells were not found.A three-dimensional model of the mitochondrial accumulation based on serial sectioning is presented. The possible degenerative and regenerative features of these accumulations are discussed. It is possible that mitochondrial accumulations are functionally active energy producers rather than results of degenerative processes.     

The isolation and characterization in vitro of normal epithelial cells,endothelial cells and fibroblasts from rat urinary bladder

Epithelial cells, microvascular endothelial cells, and fibroblasts have been isolated in culture from normal urinary bladders of Fischer rats. Normal epithelial cells were cultured most efficiently when transitional epithelial sheets were plated on to collagen-coated roller flasks. The epithelial sheets were obtained by two micro-dissection techniques. In the first method, the epithelium was peeled as a large coherent sheet from the submucosal connective tissue following subepithelial injection of a collagenase solution, and after incubation of the bladders in the same enzyme solution. Epithelial sheets with intact basal cell layers were essential for culture success. On collagenous matrices, epithelial differentiation was similar to that in vivo. The in vitro transitional epithelium was composed of three cell layers, namely superficial, intermediate, and basal cells. Basal cells were attached to newly synthesized basal lamina by means of hemidesmosomes. Superficial cells were sealed at their apical lateral membranes by a junctional complex, i.e. a terminal bar. Asymmetric luminal membrane plaques were not apparent. In the second method, the epithelium was separated from the underlying connective tissue after collagenase-trypsin digestion of everted urinary bladders. Although the digest consisted mainly of epithelial cells, these rarely survived the first passage when plated on conventional plastic growth surfaces. After the third culture week, epithelial cells usually died and slowly growing colonies of fibroblasts or large flattened epitheloid cells became apparent. Epitheloid cells were identified by their typical ultrastructure as endothelial cells, showing Weibel-Palade bodies and pinocytotic caveolae. These cells were reactive with antiserum against factor VIII. The free surface of monolayer cultures was non-thrombogenic when incubated in the presence of platelets. Fibroblasts were isolated from heavily contaminated epithelial cell cultures after differential trypsinization. These three cell types represent the normal control cells of an in vitro tumor model for the study of invasiveness. All three cell types are involved in the formation and functional maintenance of the epithelial-stromal junction. The study of cell-cell and cell-matrix interactions may provide important clues for the understanding of tumor invasiveness, a process that starts at the epithelial-stromal junction and proceeds with its destruction.