Hydrophobic interaction electrokinetic chromatography for the separation of polycyclic aromatic hydrocarbons using non-aqueous matrices.

Capillary electrophoresis methodology is developed to provide a rapid, inexpensive and robust technique for screening polycyclic aromatic hydrocarbons (PAHs) in water using additive complexation. A series of conventional RPLC ion-pairing agents are investigated in three different totally non-aqueous separation solvents, and the relative role of hydrophobic interaction versus electrostatic association is evaluated. Methanol is found to provide optimal selectivity when coupled with the tetrahexylammonium cation providing total analysis times of approximately 15 min for the analysis of thirteen 2-7-ring PAH pollutants. Solid-phase microextraction is demonstrated to be an effective sample preparation technique for extraction/preconcentration of PAHs from water into methanol run buffer prior to injection.

Ethanol and glutamate effects on intracellular magnesium.

Acutely dissociated rat brain cells were loaded with the magnesium-sensitive fluorescent dye furaptra. Stimulation with varying concentrations of GLU + 1 microM GLY produced a concentration-dependent increase in free intracellular magnesium ([Mg2+]i) that was not dependent on the presence of extracellular Mg2+. Ethanol (50 mM) did not significantly inhibit GLU/GLY-stimulated increases in [Mg2+]i. However, ethanol alone significantly increased baseline [Mg2+]i.

Structural requirements for intracellular transport of pulmonary surfactant protein B (SP-B).

Human SP-B is synthesized by the alveolar Type II epithelial cell as a 381 amino acid preproprotein. The 79 residue mature SP-B peptide is extremely hydrophobic and flanked by propeptides of 200 and 102 amino acids at its NH2- and COOH-termini, respectively. The purpose of this study was to identify peptide domains of the SP-B proprotein necessary for trafficking of the mature peptide in the secretory pathway. To this end several constructs were generated, by subcloning the full length human SP-B (SP-B), COOH-terminally truncated SP-B (SP-B delta C, in which residues 201-381 were deleted), NH2-terminally deleted SP-B (SP-B delta N, in which residues 28-200 were deleted), NH2-terminal propeptide (SP-BN), mature SP-B (SP-BM) and COOH-terminal propeptide (SP-BC), into the mammalian expression vector pcDNA3. The resulting expression constructs were characterized by DNA sequencing and in vitro transcription/translation and subsequently transfected into Chinese hamster ovary cells. 48 h after transfection, cells were labeled with [35S]-met/cys and analyzed by immunoprecipitation, SDS-PAGE and autoradiography. Proteins encoded by SP-B, SP-B delta C, SP-BN and SP-BC constructs were secreted into media; in contrast, SP-B constructs lacking the NH2-terminal propeptide (SP-B delta N) remained in the endoplasmic reticulum (as assessed by endoglycosidase H sensitivity) and were rapidly degraded. We conclude that (1) 27 amino acids at the NH2-terminus of SP-B contain a functional signal peptide and (2) the NH2-terminal propeptide of the SP-B precursor is necessary and sufficient for intracellular trafficking of the mature peptide.

The testis isoform of the phosphorylase kinase catalytic subunit (PhK-gammaT) plays a critical role in regulation of glycogen mobilization in developing lung.

In order to identify the form of phosphorylase kinase catalytic subunit expressed in developing lung, degenerate polymerase chain reaction primers were designed based on conserved domains of the two known catalytic subunits, expressed primarily in muscle and testis. Amplification of cDNA from day 19 fetal rat lung followed by cloning and sequence analyses indicated that only the testis isoform of phosphorylase kinase (PhK-gammaT) was detectable in fetal lung. In situ hybridization analyses indicated that expression of PhK-gammaT RNA in developing lung tissue was widespread and not restricted to Type II epithelial cells; PhK-gammaT protein expression was temporally and spatially correlated with expression of PhK-gammaT RNA. PhK-gammaT RNA and protein expression was also characterized in the PhK-deficient glycogen storage disease (gsd) rat. PhK-gammaT RNA levels were similar in Type II cells isolated from wild type and gsd/gsd fetuses; in contrast, PhK-gammaT protein was virtually undetectable in gsd/ gsd Type II cells and enzyme activity was very low. These results suggest that PhK-gammaT plays a critical role in mobilization of glycogen during fetal lung development and that failure to catabolize glycogen in the gsd/gsd rat is related to an untranslatable PhK-gammaT RNA or unstable protein.

Characterization of a rat cDNA clone encoding calcium/calmodulin-dependent protein kinase I.

Two cDNA clones encoding calcium/calmodulin-dependent (CaM) protein kinase I were isolated. In contrast to the previously reported CaM kinase I cDNA, which encodes a protein with a mass of 37 kDa, the clones identified in this study encode a protein (10-1/CaM kinase I) with a predicted mass of 42 kDa; the size of 10-1/CaM kinase I was verified by hybrid-selected translation.

Expression of the cyclin-dependent kinase cdk4 in perinatal and adult rat lung.

The identification of numerous cyclin-dependent kinases (cdk) and G1 cyclins suggests that cell cycle progression through G1/S may be controlled in a tissue-specific manner by various cdk/cyclin complexes. In situ hybridization was used to characterize expression of the cyclin-dependent kinase cdk4 in prenatal and postnatal rat lung and other tissues and to determine whether cdk4 expression is limited to proliferating cells, identified by BrdU incorporation and cdk1 mRNA expression. cdk4 co-localized with cdk1 in proliferating cells of both prenatal and postnatal lung and other tissues, consistent with an SPF function that is not tissue-specific. The distribution of cdk1 and cdk4 expression was identical in fetal rat tissues and was detected in lung parenchyma and throughout the airway. Pulmonary cell proliferation declined with increasing postnatal age and could be found only in focal areas of day 21 terminal and respiratory bronchiolar epithelium. Proliferation was undetectable in adult lung. Postnatal cdk4 expression was not restricted to cells expressing cdk1: cdk4 was evenly distributed in bronchiolar epithelium and was present throughout the airway and alveolar septae of day 21 lung. Expression of cdk4 was also maintained in adult bronchiolar epithelium. These studies demonstrate that although the expression of cdk1 is tightly correlated with proliferative capacity, the expression of cdk4 is not limited to proliferating cells, suggesting that cdk4 may have additional cell-specific functions unrelated to cell cycle progression.

Cloning of the rat cyclin-dependent kinase 4 cDNA: implication in proliferation-dependent expression in rat tissues.

To identify protein kinases that may regulate fetal growth and differentiation, we used an oligonucleotide probe encoding the conserved sequence of serine/threonine kinases to screen a fetal lung cDNA library. Several clones were isolated and sequenced, one of which encodes the rat homolog of the 34 kilodalton cyclin-dependent kinase 4 (p34cdk4). Northern blot analyses of pre- and postnatal rat tissues show that rat cdk4 is expressed in a developmentally regulated pattern in all tissues examined. The mRNA is also significantly decreased in cells that are arrested in the G1 phase of cell cycle. The regulated expression of rat cdk4 is consistent with a proliferation-, rather than a differentiation-, dependent pattern.

A mouse Y chromosome pseudogene is related to human ubiquitin activating enzyme E1.

A 2041 bp DNA fragment isolated from the Sxr (sex reversed) region of the mouse Y Chromosome (Chr) was sequenced and characterized. The sequence, pY8/b, contains four exons that are highly similar to 525 contiguous bases from the cDNA of human ubiquitin activating enzyme E1. Two of the exons contain stop codons, indicating that pY8/b is not part of a functional gene. Sequences related to pY8/b were amplified from the Y Chr of the inbred mouse strain, C57BL/6J. These sequences may be portions of the recently discovered functional equivalent of pY8/b. Despite a high degree of similarity with the human E1 gene, the functional equivalent of pY8/b is not the mouse E1 gene, because unlike E1, the functional equivalent of pY8/b is expressed in a tissue-specific manner. These data are discussed with respect to theory on the evolution of the mammalian Y Chr, and in particular, to the prediction that functional genes on the Y Chr have a male-specific function.

Effect of soy fiber and soy protein on cholesterol metabolism and atherosclerosis in rabbits.

The effect of dietary fiber isolated from dehulled, defatted soybean seeds on cholesterol (CHOL) metabolism and atherosclerosis in rabbits was studied alone and in combination with isolated soy protein (ISP). Soy fiber (SF) contains both cellulosic and non-cellulosic dietary fiber. Based on the official AOAC method, soy fibers contains 75% total dietary fiber. Rabbits at 6 months of age were randomly assigned to 1 of 4 dietary treatments. All rabbits received either a casein or ISP-based diet with cellulose or SF as the only dietary fiber source for 36 weeks. Fasting blood samples and feces were collected and analyzed for lipids from individual rabbits. The entire aorta was removed and fixed, and sudanophilic stained lesions were examined visually. Rabbits consuming the SF and/or ISP diets had lower plasma CHOL levels and lower incidence of atherosclerotic lesions relative to the rabbits fed the casein-based cellulose diets. Rabbits consuming the SF and/or ISP diets also had a lower CHOL content in their liver and heart. Rabbits fed ISP-based diets had consistently increased fecal bile acid excretion, whereas rabbits fed diets containing SF had increased fecal and cholesterol concentration. These results suggest a complementary role for SF and ISP in preventing atherosclerosis in rabbits.

Prenatal allergic sensitization to helminth antigens in offspring of parasite-infected mothers.

Total and filaria-specific immunoglobulin E (IgE) levels were studied in cord blood from infants born in Madras, India, where filariasis and intestinal helminth infections are highly endemic. Increased total IgE levels were observed in 82% of 57 cord sera tested (geometric mean 12.6 ng/ml; range 1-1,900 ng/ml). 33 of these sera also contained IgE antibodies specific for filarial antigens as determined by solid-phase radioimmunoassay. Comparison of ratios of filaria-specific IgE to total IgE in paired maternal and cord sera suggested that cord blood IgE was derived from the fetus in most cases and not from transplacental antibody transfer. Our results suggest that prenatal allergic sensitization to helminth parasites occurs in the tropics. Such sensitization may contribute to the heterogeneity in host immune response and disease expression noted in filariasis and other helminth infections.