Isozyme studies on the association of the herpes simplex virus type 1 thymidine kinase gene with human chromosomes in somatic cell hybrids.

To investigate chromosomal site(s) of integration of the herpes simplex virus type 1 (HSV-1) thymidine kinase (TK) gene in biochemically transformed [HeLa(BU25)/KOS 8-1] cells, these human cells which had been transformed by ultraviolet light-irradiated HSV-1 were fused with TK-negative mouse LM(TK-) cells, human-mouse somatic cell hybrid clones (LH81 clones 1-20) were isolated by HATG-ouabain selection and their chromosomes and isozymes were analyzed. Electrophoretic and serological analyses showed that all 20 clones expressed type-specific HSV-1 TK. Isozyme analyses on 29 gene-enzyme systems representing 22 human chromosomes revealed that all of the HSV-1 TK-positive clones expressed human aminoacylase-1 (ACY-1) and esterase D (ESD), which have been mapped to human chromosomes 3 and 13, respectively. Other human isozymes were detected in only one to four clones or in none of the clones. Chromosome analyses showed that: (1) the hybrid clones retained only a few human chromosomes; (2) a marker chromosome, designated M7, consisting of a chromosome 17 translocated to the short arm of chromosome 3, occurred in 36 out of the 41 metaphases examined of LH81-4 clones 1 to 4 and in 31 out of the 33 metaphases examined of LH81-12 clone 10; (3) a modified M7 chromosome, (M7/m), in which the distal 2/3 of the long arm of M7 was translocated to a small acrocentric mouse chromosome, was the only human chromosome found in metaphases of LH81-13 clone 17; and (4) an intact human chromosome 13 was not present in LH81-12 clone 10 or LH81-13 clone 17 cells. Counterselection with BrdUrd resulted in the isolation of subclones lacking HSV-1 TK, human ACY-1 and ESD, and the human marker M7 chromosomes. The experiments indicate that the HSV-1 TK gene is probably associated in HeLa (BU25)/KOS 8-1 cells with marker chromosome M7, but the possibility is not excluded that the segment of human chromosome 13 which codes for ESD is involved.     

Adolescent alcohol and drug abuse and health

Purpose: To examine the relationship of adolescent alcohol and drug use over a 5-year period to cumulative health problems in late adolescence and young adulthood.

Methods: We prospectively examined self-reported health problems in a sample of adolescents, some of whom received treatment for substance use disorders and had consistently poor substance use outcomes (n = 38), some of whom received treatment for substance use disorders and had positive substance use outcomes (n = 30), and a low alcohol and drug use community comparison group (n = 48). Data regarding health-related problems of these adolescents (mean, 15.9 years; 83% Caucasian; 56.5% female) were collected at 2, 4, and 6 years following initial assessments.

Results: Alcohol and/or drug involvement severe enough to warrant treatment during adolescence was associated with more cumulative health problems and severe health problems for girls and more cumulative health problems for boys. Protracted and continuous abuse of alcohol and drugs was associated with more cumulative and severe health problems for girls and more severe health problems for boys.

Conclusions: These results suggest that significant health problems and concerns are related to both brief and protracted alcohol and drug abuse during adolescence. Health problems will likely become even more evident as early-onset, chronic substance abusers continue to age.     

Antifungal and cancer cell growth inhibitory activities of 1-(3',4',5'-trimethoxyphenyl)-2-nitro-ethylene

The antifungal and cancer cell growth inhibitory activities of 1-(3',4',5'-trimethoxyphenyl)-2-nitro-ethylene (TMPN) were examined. TMPN was fungicidal for the majority of 132 reference strains and clinical isolates tested, including those resistant to fluconazole, ketoconazole, amphotericin B or flucytosine. Minimum fungicidal concentration/minimum inhibitory concentration (MFC/MIC) ratios were < or = 2 for 96% of Cryptococcus neoformans clinical isolates and 71% of Candida albicans clinical isolates. TMPN was fungicidal for a variety of other basidiomycetes, endomycetes and hyphomycetes, and its activity was unaffected by alterations in media pH. The frequency of occurrence of fungal spontaneous mutations to resistance was <10(-6). Kill-curve analyses confirmed the fungicidal action of TMPN, and demonstrated that killing was concentration- and time-dependent. At sub-MIC exposure to TMPN, C. albicans did not exhibit yeast/hyphae switching. TMPN was slightly cytotoxic for murine and human cancer cell lines (GI50=1-4 microg ml(-1)), and weakly inhibited mammalian tubulin polymerization (IC50=0.60 microg ml(-1)).     

Referral sources,diagnoses, and service types of youth in public outpatient mental health care: A focus on ethnic minorities

Racial/ethnic variations in clinical and service delivery characteristics among youth in public outpatient mental health services were examined using data from San Diego County mental health service programs for fiscal year 1996 to 1997 (N=3,962). Differences in referral sources, primary diagnoses, and service types were investigated for three racial/ethnic groups (African Americans, Asian/Pacific Islander Americans, and Latinos) compared to non-Hispanic whites. Controlling for age, gender, functional impairment, and prior service use, significant differences by race/ethnicity were found for all three variables studied. Possible explanations for these variations and future directions for research are discussed.     

Abundant phosphorus expected for possible life in Enceladus’s ocean

Saturn’s moon Enceladus has a potentially habitable subsurface water ocean that contains canonical building blocks of life (organic and inorganic carbon, ammonia, possibly hydrogen sulfide) and chemical energy (disequilibria for methanogenesis). However, its habitability could be strongly affected by the unknown availability of phosphorus (P). Here, we perform thermodynamic and kinetic modeling that simulates P geochemistry based on recent insights into the geochemistry of the ocean–seafloor system on Enceladus. We find that aqueous P should predominantly exist as orthophosphate (e.g., HPO₄²⁻), and total dissolved inorganic P could reach 10⁻⁷ to 10⁻² mol/kg H₂O, generally increasing with lower pH and higher dissolved CO₂, but also depending upon dissolved ammonia and silica. Levels are much higher than <10⁻¹⁰ mol/kg H₂O from previous estimates and close to or higher than ∼10⁻⁶ mol/kg H₂O in modern Earth seawater. The high P concentration is primarily ascribed to a high (bi)carbonate concentration, which decreases the concentrations of multivalent cations via carbonate mineral formation, allowing phosphate to accumulate. Kinetic modeling of phosphate mineral dissolution suggests that geologically rapid release of P from seafloor weathering of a chondritic rocky core could supply millimoles of total dissolved P per kilogram of H₂O within 10⁵ y, much less than the likely age of Enceladus’s ocean (10⁸ to 10⁹ y). These results provide further evidence of habitable ocean conditions and show that any oceanic life would not be inhibited by low P availability.

The search for habitable worlds is usually guided by the presence of liquid water. Apart from Earth, water oceans also exist in the subsurface regions of some icy bodies (e.g., Enceladus, Europa, and Titan) in the outer solar system. The evidence for an ocean is strongest at Enceladus, where a water-rich plume erupts from a subsurface ocean (1, 2). Moreover, habitability is determined by other environmental factors, such as the availability of energy and CHNOPS elements (3). Data from the Cassini spacecraft indicate that Enceladus’s ocean likely satisfies most of these other requirements for life. For example, the abundance of molecular hydrogen that coexists with CO₂ in the plume suggests the availability of chemical energy that could support methanogens (4–6). Carbon dioxide and a variety of organic compounds, which can serve as carbon sources for microorganisms, are also abundant in the plume (4, 7–9). Detected ammonia (4) or organic amines (8), and tentatively detected hydrogen sulfide (9, 10), could provide biologically useable nitrogen and sulfur, respectively.However, a notable ingredient for habitability that has eluded detection on Enceladus is phosphorus. In terrestrial life, phosphorus in the form of orthophosphate (PO₄³⁻ and its conjugate acids) participates in biological and prebiological reactions. For example, it forms kinetically stable, charged linkages in genetic molecules and phospholipids and is used to transfer metabolic energy primarily via adenosine triphosphate (11). Phosphorus is essential and its availability is often a limiting factor for biological productivity (12, 13). Because phosphate is yet to be detected in the Enceladus plume or in Saturn’s E Ring (14, 15), geochemical modeling is necessary to constrain the availability of this critical nutrient in Enceladus’s ocean. Existing models (16–18) predict a very low concentration of phosphate [<10⁻¹⁰ mol/kg H₂O or molal, vs. ∼10⁻⁶ molal in Earth’s oceans (19)], which challenges the current perception of Enceladus’s ocean as a habitable environment. However, those models are not based on the latest understanding of the geochemistry of the ocean–seafloor system on Enceladus (20–22). In addition to phosphate, reduced forms of phosphorus could be present and might support prebiotic chemistry. Examples include phosphine (PH₃) acquired from the solar nebula (23) or phosphite (HPO₃²⁻ and its conjugate acids) formed from aqueous alteration of accreted chondritic rocks (24). Neither of these forms of phosphorus have been identified at Enceladus (10), and it is unknown how stable these species would be in its ocean.Here, we present thermodynamic and kinetic modeling results on the chemical speciation and solubility of phosphorus in Enceladus ocean water. Our results suggest the predominant stability of orthophosphate (vs. instability of reduced forms of P) and relatively high solubility in alkaline and carbonate-rich water.     

Genome-wide patterns of single-feature polymorphism in Arabidopsis thaliana

We used hybridization to the ATH1 gene expression array to interrogate genomic DNA diversity in 23 wild strains (accessions) of Arabidopsis thaliana (arabidopsis), in comparison with the reference strain Columbia (Col). At <1% false discovery rate, we detected 77,420 single-feature polymorphisms (SFPs) with distinct patterns of variation across the genome. Total and pair-wise diversity was higher near the centromeres and the heterochromatic knob region, but overall diversity was positively correlated with recombination rate (R(2) = 3.1%). The difference between total and pair-wise SFP diversity is a relative measure contrasting diversifying or frequency-dependent selection, similar to Tajima's D, and can be calibrated by the empirical genome-wide distribution. Each unique locus, centered on a gene, has a diversity and selection score that suggest a relative role in past evolutionary processes. Homologs of disease resistance (R) genes include members with especially high levels of diversity often showing frequency-dependent selection and occasionally evidence of a past selective sweep. Receptor-like and S-locus proteins also contained members with elevated levels of diversity and signatures of selection, whereas other gene families, bHLH, F-box, and RING finger proteins, showed more typical levels of diversity. SFPs identified with the gene expression array also provide an empirical hybridization polymorphism background for studies of gene expression polymorphism and are available through the genome browser http://signal.salk.edu/cgi-bin/AtSFP.     

Rate of progression of coronary atherosclerotic plaque in women.

OBJECTIVES: The purpose of this study was to determine the relationship between gender and the extent of coronary atherosclerosis assessed by intravascular ultrasound (IVUS) and its rate of progression in subjects treated with established medical therapies. BACKGROUND: It is uncertain whether the pathophysiology of coronary artery disease (CAD) differs between genders. METHODS: A systematic analysis was performed of 978 subjects who participated in serial studies of atheroma progression. Genders were compared with regard to the extent of coronary atheroma at baseline and subsequent change in response to use of established medical therapies. RESULTS: Women were more likely to have a history of hypertension and higher levels of body mass index, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol, triglycerides, C-reactive protein, and systolic and diastolic blood pressure. Despite this, women had less plaque in terms of percent atheroma volume (PAV) (33.9 +/- 10.2% vs. 37.8 +/- 10.3%, p < 0.001) and total atheroma volume (TAV) (148.7 +/- 66.6 mm3 vs. 194.7 +/- 84.3 mm3, p < 0.001). With medical therapy, the rate of change of PAV (0.7 +/- 0.6% vs. 0.7 +/- 0.5%, p = 0.92) and TAV (-2.3 +/- 3.2 mm3 vs. -1.9 +/- 2.9 mm3, p = 0.84) did not differ between genders. In the setting of intensive risk factor modification, there was no significant difference between genders with regard to the rates of plaque progression or regression. CONCLUSIONS: Despite the presence of more risk factors, the extent of atheroma in women with angiographic CAD is less than in men in subjects participating in clinical trials that employed serial assessments with IVUS. The finding that the rate of plaque progression or regression does not differ between genders in the setting of intensive risk factor modification supports the use of established medical therapies in women with CAD.     

Single-molecule transition-state analysis of RNA folding

How RNA molecules fold into functional structures is a problem of great significance given the expanding list of essential cellular RNA enzymes and the increasing number of applications of RNA in biotechnology and medicine. A critical step toward solving the RNA folding problem is the characterization of the associated transition states. This is a challenging task in part because the rugged energy landscape of RNA often leads to the coexistence of multiple distinct structural transitions. Here, we exploit single-molecule fluorescence spectroscopy to follow in real time the equilibrium transitions between conformational states of a model RNA enzyme, the hairpin ribozyme. We clearly distinguish structural transitions between effectively noninterchanging sets of unfolded and folded states and characterize key factors defining the transition state of an elementary folding reaction where the hairpin ribozyme's two helical domains dock to make several tertiary contacts. Our single-molecule experiments in conjunction with site-specific mutations and metal ion titrations show that the two RNA domains are in a contact or close-to-contact configuration in the transition state even though the native tertiary contacts are at most partially formed. Such a compact transition state without well formed tertiary contacts may be a general property of elementary RNA folding reactions.     

Sensitive cardiac troponin I predicts poor outcomes in pulmonary arterial hypertension

Circulating cardiac troponins are markers of myocardial injury. We sought to determine whether cardiac troponin I (cTnI), measured by a sensitive assay, is associated with disease severity and prognosis in pulmonary arterial hypertension (PAH). cTnI was measured in 68 patients with PAH diagnostic category 1 in a research-based sensitive immunoanalyser with a lower limit of detection of 0.008 ng · mL(-1). The associations between cTnI and PAH severity and clinical outcomes were assessed using Chi-squared and Wilcoxon rank sum tests, Kaplan-Meier analysis and Cox regression models. cTnI was detected in 25% of patients. Patients with detectable cTnI had more advanced functional class symptoms, a shorter 6-min walk distance, more pericardial effusions, larger right atrial area, and higher B-type natriuretic peptide and C-reactive protein levels. 36-month transplant-free survival was 44% in patients with detectable cTnI versus 85% in those with undetectable cTnI. cTnI was associated with a 4.7-fold increased risk of death related to right ventricular failure or transplant (hazard ratio 4.74, 95% CI 1.89-11.89; p<0.001), even when adjusted individually for known parameters of PAH severity. Elevated plasma cTnI, even at subclinically detectable levels, is associated with more severe disease and worse outcomes in patients with PAH.     

Super-resolution fluorescence imaging of organelles in live cells with photoswitchable membrane probes

Imaging membranes in live cells with nanometer-scale resolution promises to reveal ultrastructural dynamics of organelles that are essential for cellular functions. In this work, we identified photoswitchable membrane probes and obtained super-resolution fluorescence images of cellular membranes. We demonstrated the photoswitching capabilities of eight commonly used membrane probes, each specific to the plasma membrane, mitochondria, the endoplasmic recticulum (ER) or lysosomes. These small-molecule probes readily label live cells with high probe densities. Using these probes, we achieved dynamic imaging of specific membrane structures in living cells with 30–60 nm spatial resolution at temporal resolutions down to 1–2 s. Moreover, by using spectrally distinguishable probes, we obtained two-color super-resolution images of mitochondria and the ER. We observed previously obscured details of morphological dynamics of mitochondrial fusion/fission and ER remodeling, as well as heterogeneous membrane diffusivity on neuronal processes.