Use of green fluorescent protein to tag and investigate gene expression in marine bacteria

Two broad-host-range vectors previously constructed for use in soil bacteria (A. G. Matthysse, S. Stretton, C. Dandie, N. C. McClure, and A. E. Goodman, FEMS Microbiol. Lett. 145:87-94, 1996) were assessed by epifluorescence microscopy for use in tagging three marine bacterial species. Expression of gfp could be visualized in Vibrio sp. strain S141 cells at uniform levels of intensity from either the lac or the npt-2 promoter, whereas expression of gfp could be visualized in Psychrobacter sp. strain SW5H cells at various levels of intensity only from the npt-2 promoter. Green fluorescent protein (GFP) fluorescence was not detected in the third species, Pseudoalteromonas sp. strain S91, when the gfp gene was expressed from either promoter. A new mini-Tn10-kan-gfp transposon was constructed to investigate further the possibilities of fluorescence tagging of marine bacteria. Insertion of mini-Tn10-kan-gfp generated random stable mutants at high frequencies with all three marine species. With this transposon, strongly and weakly expressed S91 promoters were isolated. Visualization of GFP by epifluorescence microscopy was markedly reduced when S91 (mini-Tn10-kan-gfp) cells were grown in rich medium compared to that when cells were grown in minimal medium. Mini-Tn10-kan-gfp was used to create an S91 chitinase-negative, GFP-positive mutant. Expression of the chi-gfp fusion was induced in cells exposed to N'-acetylglucosamine or attached to chitin particles. By laser scanning confocal microscopy, biofilms consisting of microcolonies of chi-negative, GFP+ S91 cells were found to be localized several microns from a natural chitin substratum. Tagging bacterial strains with GFP enables visualization of, as well as monitoring of gene expression in, living single cells in situ and in real time.     

Dual effect of erbB-2 depletion on the regulation of DNA repair and cell cycle mechanisms in non-small cell lung cancer cells

Overexpression of the erbB-2 tyrosine kinase receptor, p185erbB-2, is a common alteration in non-small cell lung cancer (NSCLC) and has been associated with poor prognosis and a tumor drug resistance phenotype. In this study, we have examined the consequences of erbB-2 depletion on DNA repair, cell cycle, and apoptosis using a panel of NSCLC cell lines constitutively overexpressing erbB-2 receptor. Depletion of the erbB-2 was achieved using the tyrosine kinase inhibitor CP127,374 which promotes erbB-2 degradation. Treatment with CP127,374 concentrations which deplete erbB-2 and inhibit tyrosine phosphorylation resulted in downregulation of DNA repair mechanisms and cell accumulation at G1 phase of the cell cycle. GI arrest was observed in cells with mutated p53 as well as cells lacking p53 protein, suggesting a p53-independent mechanisms. NSCLC cells which overexpress erbB-2 were more resistant to cisplatin-induced cytotoxicity in comparison to cells expressing low levels of erbB-2. Treatment with CP127,374 alone did not result in any induction of apoptosis. A combination of CP127,374 and cisplatin, however, was more potent in cell growth inhibition and induction of apoptosis compared to treatment with cisplatin alone. Together, our results further support a pivotal role of erbB-2 signaling in the regulatory balance between DNA repair, cell cycle checkpoints and apoptosis; all these mechanisms are essential determinants for tumor cell destiny following chemotherapy stress.     

Axial patterning in the leech: developmental mechanisms and evolutionary implications

The subcutaneous distribution and number of Pacinian corpuscles were studied in ten fresh cadaver hands. They were found to cluster close to nerves and vessels at the metacarpophalangeal joints and the proximal phalanx. The total mean number in the hand was 300 (192-424). The percentage of the total was 44 to 60% in the fingers, 23 to 48% in the metacarpophalangeal area and 8 to 18% in the thenar and hypothenar regions. Corpuscles in palmar skin overlying the distal phalanx were smaller than receptors in the metacarpophalangeal area. The lowest density of corpuscles was along the nerves and vessels of the middle phalanx.     

Influence of nutritional iron deficiency development on some aspects of iron, copper and zinc metabolism

The permanent fibrocyte-like fish cell line RTG-2 from rainbow trout (Oncorhynchus mykiss) gonads was investigated by means of light and scanning electron microscopy with respect to alterations as a consequence of sublethal exposure to 0, 1, 10, 16, 25, and 50 mg/liter 3,5-dichlorophenol (3,5-DCP) for 24 h. Control RTG-2 cells were spindle-like in shape and almost three times longer than wide. In subconfluent cultures, they displayed widely spread, flat leading lamellae and formed small groups. Along the edges of the cell protrusions, numerous retraction fibrils could be identified. Except for shallow ridges and small folds, the surface of untreated cells was completely smooth. Following exposure to 3,5-DCP, distinct dose-dependent alterations in cell shape, the appearance of surface blebs and invaginations, as well as progressive retraction of cell extensions could be observed from the lowest test concentration. Morphological changes could be correlated to suppression of lactate dehydrogenase and reduced neutral red retention capacity.     

Midwall fractional shortening is an independent predictor of left ventricular diastolic dysfunction in asymptomatic patients with systemic hypertension

Conventional measures of left ventricular (LV) systolic performance suggest that diastolic dysfunction precedes the development of systolic dysfunction in hypertension. Midwall fractional shortening is a new measure of systolic function that identifies hypertensive patients who have evidence of target-organ damage, impaired contractile reserve, and increased mortality. We therefore sought to determine whether depressed midwall fiber shortening is associated with abnormal diastolic function. Echocardiograms were obtained in 102 otherwise healthy hypertensive patients without treatment with normal conventional measures of systolic function. Of these, 15 had depressed midwall shortening based on previously described normative relations. Patients with depressed midwall shortening had slightly higher blood pressure. Abnormal diastolic function, defined as late (A) LV inflow velocity greater than early (E) velocity, was observed in 33% of those with normal midwall shortening but in 60% of those with depressed shortening (p <0.05). Patients with A/E >1 had lower absolute midwall fiber shortening (15 +/- 3% vs 18 +/- 3%, p <0.0001) but similar endocardial shortening. Patients with abnormal midwall shortening had higher A/E and longer isovolumic relaxation times (both p <0.05). In multivariate analysis, midwall fractional shortening, age, and heart rate were independent predictors (p <0.01) of A/E in a model including blood pressure, LV mass, and endocardial shortening. We conclude that subnormal midwall shortening predicts LV diastolic abnormalities in this population of hypertensive patients with otherwise normal measures of LV systolic function. Contrary to our previous understanding, depressed LV systolic performance, when identified with this newer method, occurs coincidentally with impaired diastolic function.