Effects of hyposmolar solutions on membrane currents of hippocampal interneurons and mossy cells in vitro

Whole cell voltage-clamp recordings in rat hippocampal slices were used to investigate the effect of changes in extracellular osmolarity on voltage-activated potassium currents. Currents were evoked from oriens/alveus (O/A) interneurons, hilar interneurons, and mossy cells. Hyposmolar external solutions produced a significant potentiation of K+ current recorded from O/A and hilar interneurons, but not from mossy cells. Hyposmolar solutions also dramatically potentiated the spontaneous excitatory postsynaptic currents recorded from mossy cells. These results suggest that hippocampal excitability can be modulated by the complex actions exerted by changes in extracellular osmolarity.     

Chromosomal duplication accompanies allelic loss in non-small cell lung carcinoma

Hemizygous deletion in the short (p) arm of chromosome 3 is a common finding in non-small cell lung carcinoma (NSCLC) and is postulated to be a crucial early change in lung tumorigenesis. Yet one of the most frequent nuclear abnormalities in both NSCLC and premalignant bronchial epithelium is increase in chromosomal copy number. Deletion and duplication have not been assessed in the same tumor set by both molecular and cytogenetic methods to determine whether allelic loss correlates with chromosomal duplication in the same tumor cell populations. It is also not established what biological mechanisms might lead to allelic deletion and chromosomal duplication. We have investigated changes in the copy number of chromosome 3 in touch preparations of 38 NSCLCs (19 adenocarcinomas and 19 squamous cell carcinomas) using dual-target, dual-color fluorescence in situ hybridization (FISH) assays. Chromosome 3 centromere probe was matched with a 3p14.2 probe [intron 4 of the fragile histidine triad (FHIT) gene] and a 3p21.31 probe (HSemaIV gene). We then correlated FISH results with results of molecular analyses for allelic losses at loci in the regions to which the FISH probes mapped in 20 of these cases. Although various combinations of FISH abnormalities were sometimes detected within the same specimens, individual cases could be classified according to the predominant FISH pattern, usually with one abnormality present in >60% of tumor cells. Chromosomal duplication, indicated by the presence of more than two centromeric signals, was the most frequent abnormality observed by FISH and was accompanied by loss of specific sequences on 3p in approximately one-half of the specimens in which it was observed. The most frequent abnormality observed by molecular analysis was loss of heterozygosity (LOH) in both of the chromosomal regions tested and was demonstrated in 83% of cases with chromosomal duplication. We conclude that LOH may occur in the presence of chromosomal duplication, suggesting that the duplicated chromosome is homozygous. Our findings imply that LOH occurs before chromosomal duplication during lung carcinogenesis.     

Effects of phosphorylation of troponin I and C protein on isometric tension and velocity of unloaded shortening in skinned single cardiac myocytes from rats

Effects on isometric tension generation and maximum velocity of unloaded shortening after exposure to cAMP-dependent protein kinase (PKA) were investigated in rat enzymatically isolated, tritonized ventricular myocytes. Exposure of myocytes to PKA in the presence of [32P]ATP resulted in phosphorylation of troponin I and C protein. Ca2+ sensitivity of isometric tension was assessed as pCa50, ie, the [Ca2+] at which tension was 50% of maximum, and was lower after PKA treatment (pCa50 5.58) than before PKA treatment (pCa50 5.74). This suggests beta-adrenergic stimulation of the heart and subsequent increases in PKA activity and phosphorylation of troponin I and C protein lead to a significant decrease in tension-generating ability at a given submaximum [Ca2+]. Unloaded shortening velocity was determined by measuring the time required to take up various amounts of slack imposed at one end of the cardiac myocyte preparation. Unloaded shortening velocity during maximum activation was 2.88 +/- 0.11 muscle lengths per second (mean +/- SEM) before PKA exposure and 2.86 +/- 0.13 muscle lengths per second after PKA exposure. Unloaded shortening velocity during 40% of maximum activation was 1.91 +/- 0.25 muscle lengths per second before PKA exposure and 2.17 +/- 0.15 muscle lengths per second after PKA exposure. The absence of an effect of PKA on unloaded shortening velocity in skinned ventricular myocytes suggests that beta-adrenergic stimulation of myocardium either does not affect myofilament velocity of shortening or alters velocity of shortening by a non-PKA-dependent process.     

Quantitation of transcription and clonal selection of single living cells with beta-lactamase as reporter

Gene expression was visualized in single living mammalian cells with beta-lactamase as a reporter that hydrolyzes a substrate loaded intracellularly as a membrane-permeant ester. Each enzyme molecule changed the fluorescence of many substrate molecules from green to blue by disrupting resonance energy transfer. This wavelength shift was detectable by eye or color film in individual cells containing less than 100 beta-lactamase molecules. The robust change in emission ratio reveals quantitative heterogeneity in real-time gene expression, enables clonal selection by flow cytometry, and forms a basis for high-throughput screening of pharmaceutical candidate drugs in living mammalian cells.     

Matrilineal inheritance of complex I dysfunction in a multigenerational Parkinson's disease family

Recent data suggesting complex I dysfunction in Parkinson's disease (PD) arises from mitochondrial DNA (mtDNA) mutation does not conclusively answer whether the responsible genetic lesion is inherited (primary) or somatic (secondary). To address this question, we identified a family in which multiple members over three generations are affected with PD through exclusively maternal lines. Cytoplasmic hybrids (cybrids) were created for 15 family members over two generations by transferring each individual's mtDNA to mtDNA-depleted human neuroblastoma cells. Eight of the 15 cybrid lines contained mtDNA obtained from maternally descended family members and seven contained mtDNA from paternally descended family members. After 6 weeks of culture, cybrid cell lines were assayed for complex I activity and oxidative stress, and mitochondrial morphology was analyzed by electron microscopy. Compared with the cybrid lines containing mtDNA from paternal descendants, cybrid lines containing mtDNA from maternal descendants had lower complex I activity, increased reactive oxygen species production, increased radical scavenging enzyme activities, and more abnormal mitochondrial morphologic features. These findings were present in cybrid lines containing mtDNA from maternal descendants with PD as well as in currently asymptomatic young maternal descendants, and support a precedent for inherited mtDNA mutation in some persons with PD.     

Regulated nucleo/cytoplasmic exchange of HOG1 MAPK requires the importin beta homologs NMD5 and XPO1

MAP kinase signaling modules serve to transduce extracellular signals to the nucleus of eukaryotic cells, but little is known about how signals cross the nuclear envelope. Exposure of yeast cells to increases in extracellular osmolarity activates the HOG1 MAP kinase cascade, which is composed of three tiers of protein kinases, namely the SSK2, SSK22 and STE11 MAPKKKs, the PBS2 MAPKK, and the HOG1 MAPK. Using green fluorescent protein (GFP) fusions of these kinases, we found that HOG1, PBS2 and STE11 localize to the cytoplasm of unstressed cells. Following osmotic stress, HOG1, but neither PBS2 nor STE11, translocates into the nucleus. HOG1 translocation occurs very rapidly, is transient, and correlates with the phosphorylation and activation of the MAP kinase by its MAPKK. HOG1 phosphorylation is necessary and sufficient for nuclear translocation, because a catalytically inactive kinase when phosphorylated is translocated to the nucleus as efficiently as the wild-type. Nuclear import of the MAPK under stress conditions requires the activity of the small GTP binding protein Ran-GSP1, but not the NLS-binding importin alpha/beta heterodimer. Rather, HOG1 import requires the activity of a gene, NMD5, that encodes a novel importin beta homolog. Similarly, export of dephosphorylated HOG1 from the nucleus requires the activity of the NES receptor XPO1/CRM1. Our findings define the requirements for the regulated nuclear transport of a stress-activated MAP kinase.     

Using focus groups to identify psychosocial issues of urban black individuals with diabetes

BACKGROUND: Human serum represents an important barrier to the entry of most mucosal organisms into tissues and to the systemic circulation. If at all present, Helicobacter pylori within gastric tissue is rare, and bacteremia for this organism has been described only once. METHODS: To assess the susceptibility of H. pylori to the bactericidal activity present in normal human serum (NHS), we examined 13 H. pylori isolates. To assess the contributions of the classical and alternative complement pathways to killing, we added either C2-deficient or factor B-deficient serum, respectively, to heat-inactivated NHS. Also we assessed the ability of the strains to bind 125I-C3. RESULTS: After incubation for 60 minutes at 37 degrees C, all 13 H. pylori strains were killed by NHS; heating to 56 degrees C for 30 minutes ablated killing, indicating complement dependence for this phenomenon. In the absence of an antibody source, there was no killing when either an alternative or classical complement pathway source was used. Adding B-deficient serum to heat-inactivated normal human serum did not restore killing, but adding C2-deficient serum permitted partial killing. All of the 13 strains bound 125I-C3. Although the kinetics varied from strain to strain, C3 bound was significantly correlated (r = 0.61, p = 0.03) with serum susceptibility. CONCLUSIONS: H. pylori are susceptible to complement, alternative pathway activation appears critical, and C3 binding is a major locus of variability.