Restriction fragment length polymorphism and multiple copies of DNA sequences homologous with probes for P-fimbriae and hemolysin genes among uropathogenic Escherichia coli

Hemolysin and P-fimbriae are two virulence traits frequently found together in uropathogenic Escherichia coli. Previous studies have discovered evidence both for linkage between the genes for these traits and for their duplication in the chromosomes of a limited number of strains. To test whether these observations are characteristic of uropathogenic Escherichia coli, the method of DNA hybridization to DNA restriction fragments separated by electrophoresis and transferred to nylon was used to determine copy number of genes for P-fimbriae (pap) among 51 E. coli strains isolated from symptomatic urinary tract infections. Twenty percent of the strains had more than one copy of pap homologous sequences. Fifteen strains, each representing a unique clone, were examined for the presence of sequences homologous with cloned hemolysin genes (hly). Samples of DNA from 14 of the 15 strains hybridized with hly probes. In eight strains the number of copies of pap equalled the number of copies of hly, including one strain with two apparent copies of each. Five strains appeared to have one more copy of pap than of hly, and one strain had an extra copy of hly.     

Ultrastructure of sperm development and mature sperm morphology in three species of commensal bivalves (Mollusca: Galeommatoidea)

Ultrastructural features of the ovotestes, spermatogenesis, and the mature sperm are described for three galeommatid bivalves, Divariscintilla yoyo, Divariscintilla troglodytes, and Scintilla sp., from stomatopod burrows in eastern Florida. All three species yielded similar results except with respect to mature sperm dimensions. The ovotestis contains three types of somatic cells within the testicular portion: flattened myoepithelial cells defining the outer acinal wall; underlying pleomorphic follicle cells containing abundant glycogen deposits; and scattered, amoeboid cells containing lysosomal-like inclusions which are closely associated with developing sperm. Early spermatogenesis is typical of that reported from other bivalves. In contrast, the late stages of spermiogenesis involve the migration and gradual rotation of the acrosomal vesicle, resulting in a mature acrosome tilted about 70° from the long axis of the cell. The mature sperm possesses an elongated, slightly curved nucleus; a subterminal, concave acrosome with a nipple-like central projection; five spherical mitochondria and two centnoles in the middlepiece; and a long flagellum. The rotational asymmetry and the presence of perimitochondrial glycogen deposits in these sperm are unusual in the Bivalvia and may be associated with fertilization specializations and larval brooding common among galeommatoideans.     

Chroman-like cyclic prenylflavonoids promote neuronal differentiation and neurite outgrowth and are neuroprotective

Flavonoids target a variety of pathophysiological mechanisms and are therefore increasingly considered as compounds encompassed with therapeutic potentials in diseases such as cancer, diabetes, arteriosclerosis, and neurodegenerative diseases and mood disorders. Hops (Humulus lupulus L.) is rich in flavonoids such as the flavanone 8-prenylnaringenin, which is the most potent phytoestrogen identified so far, and the prenylchalcone xanthohumol, which has potent tumor-preventive, anti-inflammatory and antiviral activities. In the present study, we questioned whether hops-derived prenylflavonoids and synthetic derivatives thereof act on neuronal precursor cells and neuronal cell lines to induce neuronal differentiation, neurite outgrowth and neuroprotection. Therefore, mouse embryonic forebrain-derived neural precursors and Neuro2a neuroblastoma-derived cells were stimulated with the prenylflavonoids of interest, and their potential to activate the promoter of the neuronal fate-specific doublecortin gene and to stimulate neuronal differentiation and neurite outgrowth was analyzed. In this screening, we identified highly “neuroactive” compounds, which we termed “enhancement of neuronal differentiation factors” (ENDFs). The most potent molecule, ENDF1, was demonstrated to promote neuronal differentiation of neural stem cells and neurite outgrowth of cultured dorsal root ganglion neurons and protected neuronal PC12 cells from cobalt chloride-induced as well as cholinergic neurons of the nucleus basalis of Meynert from deafferentation-induced cell death. The results indicate that hops-derived prenylflavonoids such as ENDFs might be powerful molecules to promote neurogenesis, neuroregeneration and neuroprotection in cases of chronic neurodegenerative diseases, acute brain and spinal cord lesion and age-associated cognitive impairments.     

Conserved sequence motifs, alignment, and secondary structure for the third domain of animal 12S rRNA

Secondary structure models are an important step for aligning sequences, understanding probabilities of nucleotide substitutions, and evaluating the reliability of phylogenetic reconstructions. A set of conserved sequence motifs is derived from comparative sequence analysis of 184 invertebrate and vertebrate taxa (including many taxa from the same genera, families, and orders) with reference to a secondary structure model for domain III of animal mitochondrial small subunit (12S) ribosomal RNA. A template is presented to assist with secondary structure drawing. Our model is similar to previous models but is more specific to mitochondrial DNA, fitting both invertebrate and vertebrate groups, including taxa with markedly different nucleotide compositions. The second half of the domain III sequence can be difficult to align precisely, even when secondary structure information is considered. This is especially true for comparisons of anciently diverged taxa, but well-conserved motifs assist in determining biologically meaningful alignments. Patterns of conservation and variability in both paired and unpaired regions make differential phylogenetic weighting in terms of "stems" and "loops" unsatisfactory. We emphasize looking carefully at the sequence data before and during analyses, and advocate the use of conserved motifs and other secondary structure information for assessing sequencing fidelity.      

Spermatogenic ultrastructure in the anomalodesmatan bivalve Myochama anomioides (Mollusca: Myochamidae) – does the nucleus help position the ‘temporary’ acrosome?

Spermatogenic ultrastructure in the marine bivalve mollusc Myochama anomioides (Myochamidae) is described and contrasted with other bivalves, especially other euheterodonts. Small (0.1 μm diameter), primary proacrosomal vesicles produced in spermatocytes give rise to much larger (0.4 μm diameter) secondary proacrosomal vesicles in early spermatids, which in turn form the dished‐shaped, definitive acrosomal vesicle (diameter 1.0 μm) of later spermatids. The acrosomal vesicle acquires a deposit of subacrosomal material and comes to lie close to or in contact with the plasma membrane. The acrosomal complex (acrosomal vesicle + subacrosomal material) initially positions itself at the apex of the condensing, fibrous nucleus (the so‐called temporary acrosome position), but subsequently begins to move posteriorly. The condensing nucleus becomes markedly folded so that its apex is posteriorly orientated towards the migrating acrosomal complex and the midpiece (mitochondria and centrioles). The close spatial relationship of nuclear apex to acrosomal complex during this folding strongly suggests that acrosomal migration in M. anomioides is assisted, at least in part, by movement of the late spermatid nucleus. Similar nuclear folding has previously been demonstrated in an early stage of fertilization in another anomalodesmatan (Laternula limicola) raising the possibility that one event might be a reversal of the other.     

Neonatal Restriction of Tactile Inputs Leads to Long-Lasting Impairments of Cross-Modal Processing

Optimal behavior relies on the combination of inputs from multiple senses through complex interactions within neocortical networks. The ontogeny of this multisensory interplay is still unknown. Here, we identify critical factors that control the development of visual-tactile processing by combining in vivo electrophysiology with anatomical/functional assessment of cortico-cortical communication and behavioral investigation of pigmented rats. We demonstrate that the transient reduction of unimodal (tactile) inputs during a short period of neonatal development prior to the first cross-modal experience affects feed-forward subcortico-cortical interactions by attenuating the cross-modal enhancement of evoked responses in the adult primary somatosensory cortex. Moreover, the neonatal manipulation alters cortico-cortical interactions by decreasing the cross-modal synchrony and directionality in line with the sparsification of direct projections between primary somatosensory and visual cortices. At the behavioral level, these functional and structural deficits resulted in lower cross-modal matching abilities. Thus, neonatal unimodal experience during defined developmental stages is necessary for setting up the neuronal networks of multisensory processing.     

Single-molecule anisotropy imaging

A novel method, single-molecule anisotropy imaging, has been employed to simultaneously study lateral and rotational diffusion of fluorescence-labeled lipids on supported phospholipid membranes. In a fluid membrane composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, in which the rotational diffusion time is on the order of the excited-state lifetime of the fluorophore rhodamine, a rotational diffusion constant, D(rot) = 7 x 10(7) rad(2)/s, was determined. The lateral diffusion constant, measured by direct analysis of single-molecule trajectories, was D(lat) = 3.5 x 10(-8) cm(2)/s. As predicted from the free-volume model for diffusion, the results exhibit a significantly enhanced mobility on the nanosecond time scale. For membranes of DPPC lipids in the L(beta) gel phase, the slow rotational mobility permitted the direct observation of the rotation of individual molecules characterized by D(rot) = 1.2 rad(2)/s. The latter data were evaluated by a mean square angular displacement analysis. The technique developed here should prove itself profitable for imaging of conformational motions of individual proteins on the time scale of milliseconds to seconds.     

Assessment of the Microbiological Potential for the Natural Attenuation of Petroleum Hydrocarbons in a Shallow Aquifer System

Abstract A multidisciplinary field study investigating the fate and transport of petroleum hydrocarbons commonly associated with jet-fuel contamination is currently underway at Columbus Air Force Base (AFB), Mississippi. Sixty sediment cores from 12 boreholes were recovered from the study aquifer. The goal of this initial sampling was to characterize the potential microbial activity using 14C-labeled substrates, as well as the presence, abundance, and distribution of specific hydrocarbon degrading genotypes using DNA:DNA hybridization. Enumeration of total microbial abundance using a 16S rDNA universal oligonucleotide probe was compared to traditional enumeration methods. Total culturable populations determined by spread plate analysis ranged from a low of 10(4) to more than 10(6) organisms per gram sediment. Microbial abundance estimated by DNA hybridization studies with 16S rDNA genes ranged from 10(7) to 10(8) organisms per gram sediment. Molecular analysis of aquifer samples using DNA probes targeting genes encoding the degradative enzymes alkane hydroxylase (alkB), catechol 2,3-dioxygenase (nahH), naphthalene dioxygenase (nahA), toluene dioxygenase (todC1C2), toluene monooxygenase (tomA), and xylene monooxygenase (xylA), as well as two probes measuring methanogenic microorganisms, codh (carbon monoxide dehydrogenase) and mcr (methyl coenzyme reductase), revealed that each target gene sequence was present in nearly all 60 samples. The presence of organisms demonstrating the phenotype to degrade BTEX and naphthalene was further supported using mineralization assays with 14C-labeled benzene, toluene, naphthalene, and phenanthrene. Minimal activity occurred during the first 24 hours. After a period of 5-7 days, greater than 40% of the target compounds were mineralized in aquifer sediments.