Cigarette smoking depletes cells spontaneously secreting Th(1) cytokines in the human airway.

Cigarette smoking may modify the immune balance in the airway since it alters the course of diseases in which immune system has an important role. This study examined whether cigarette smoking could affect the distribution of cells secreting Th(1) or Th(2) cytokines in the human airway. We utilized cytokine ELISPOT assay to detect and quantitate the frequencies of cells spontaneously secreting cytokines in bronchoalveolar lavage fluid (BALF). BALF was collected from six non-smokers and four heavy cigarette smokers without clinical airway symptoms. Cytokine ELISPOT assay was performed to quantitate cells secreting interleukin (IL-)2, IL-4 and interferon (IFN-)gamma with or without phorbor 12-myristate 13-acetate (PMA) stimulation. There were no cells spontaneously secreting IL-2 detected in all samples from smokers whereas most of non-smokers had detectable IL-2-secreting cells. The number of IFN-gamma-secreting cells was also extremely decreased in smokers. Mitogen-stimulated Th(1) cytokine-secreting cells were again significantly decreased in smokers' airways. The frequency of IL-2-secreting cells and CD4/CD8 ratio in BALF had a weak positive correlation. IL-4-secreting cells were not detected in any samples from both groups. These results show that cigarette smoking depletes Th(1) cytokine-secreting cells in the human airway. It may explain the susceptibility of smokers to certain airway disease conditions such as viral or mycobacterial infections and allergic diseases.

Structural requirement for the two-step dimerization of human immunodeficiency virus type 1 genome.

Generation of RNA dimeric form of the human immunodeficiency virus type 1 (HIV-1) genome is crucial for viral replication. The dimerization initiation site (DIS) has been identified as a primary sequence that can form a stem-loop structure with a self-complementary sequence in the loop and a bulge in the stem. It has been reported that HIV-1 RNA fragments containing the DIS form two types of dimers, loose dimers and tight dimers. The loose dimers are spontaneously generated at the physiological temperature and converted into tight dimers by the addition of nucleocapsid protein NCp7. To know the biochemical process in this two-step dimerization reaction, we chemically synthesized a 39-mer RNA covering the entire DIS sequence and also a 23-mer RNA covering the self-complementary loop and its flanking stem within the DIS. Electrophoretic dimerization assays demonstrated that the 39-mer RNA reproduced the two-step dimerization process, whereas the 23-mer RNA immediately formed the tight dimer. Furthermore, deletion of the bulge from the 39-mer RNA prevented the NCp7-assisted tight-dimer formation. Therefore, the whole DIS sequence is necessary and sufficient for the two-step dimerization. Our data suggested that the bulge region regulates the stability of the stem and guides the DIS to the two-step dimerization process.

A mini-protein designed by removing a module from barnase: molecular modeling and NMR measurements of the conformation.

A globular domain can be decomposed into compact modules consisting of contiguous 10-30 amino acid residues. The correlation between modules and exons observed in different proteins suggests that each module was encoded by an ancestral exon and that modules were combined into globular domains by exon fusion. Barnase is a single domain RNase consisting of 110 amino acid residues and was decomposed into six modules. We designed a mini-protein by removing the second module, M2, from barnase in order to gain an insight into the structural and functional roles of the module. In the molecular modeling of the mini-protein, we evaluated thermodynamic stability and aqueous solubility together with mechanical stability of the model. We chemically synthesized a mini-barnase with (15)N-labeling at 10 residues, whose corresponding residues in barnase are all found in the region around the hydrophobic core. Circular dichroism and NMR measurements revealed that mini-barnase takes a non-random specific conformation that has a similar hydrophobic core structure to that of barnase. This result, that a module could be deleted without altering the structure of core region of barnase, supports the view that modules act as the building blocks of protein design.

Type II 11beta-hydroxysteroid dehydrogenase expression in human colonic epithelial cells of inflammatory bowel disease.

Type IIbeta-hydroxysteroid dehydrogenase endows specificity on the mineralocorticoid receptor by metabolizing cortisol and regulates sodium absorption in renal and colonic epithelium. Altered expression of this enzyme may be associated with impaired sodium absorption often seen in colonic mucosa of inflammatory bowel disease. The aim of this study was to investigate possible abnormality of 11beta-hydroxysteroid dehydrogenase protein and mRNA expression in inflammatory bowel disease. In Crohn's disease, the colonic epithelium showed comparable levels of immunoreactivity and mRNA expression to those of control, except for the decreased immunoreactivity in severe inflamed lesions with deep ulcer. In contrary, a lack or decrease of immunoreactivity was relevant in ulcerative colitis regardless of the histological degree of inflammation. The mRNA expression was also significantly decreased in ulcerative colitis. This study demonstrates that abnormality of epithelial cells in ulcerative colitis includes the enzyme that regulates water and sodium absorption, which are physiologically essential.

Lipopolysaccharide exhibits synergistic enhancement of butyrate-induced and retinoic acid-mediated alkaline phosphatase activity on small intestinal epithelial cell line, IEC-6.

Although both lipopolysaccharide (LPS) and sodium butyrate are major bacterial products and bioactive chemicals with multiple functions on mucosal cells in the gut, their interaction effects on epithelial cells are not well understood. The purpose of the present study was to investigate whether LPS modulates butyrate-induced and retinoic acid-mediated alkaline phosphatase (ALP) activity of IEC-6 cells - a rat nontransformed small intestinal epithelial cell line. When cells reached confluency, various combinations of sodium butyrate, retinoic acid and LPS were added to the cultures. Cells were then harvested for the measurement of ALP activities. Sodium butyrate, but not retinoic acid or LPS alone, enhanced ALP activity. When LPS was additionally used with butyrate or retinoic acid, synergistic induction of ALP activities was demonstrated. No additive effect for ALP activity was observed when muramyl peptides or N-formylmethionyl-leucyl-phenylalanine was used with these acids. The present study clearly demonstrated that the specific combination of butyrate and LPS synergistically increased ALP activity, an epithelial differentiation-associated marker, of an intestinal epithelial cell line.

Prevalence of sleep-related respiratory disorders in 101 schizophrenic inpatients.

We studied the prevalence of sleep-related respiratory disorders (SRRD) in 101 schizophrenic inpatients (64 men and 37 women) and in 48 healthy volunteers (control group: 22 men and 26 women) using ambulatory pulse-oximetric devices. Those with a desaturation index (DI) > or = 5 were classified as having SRRD. The prevalence of SRRD in the schizophrenic patients (men 21.9%, women 13.5%) was not higher than that in the control group (men 30.7%, women 13.6%). Gender difference in the prevalence of SRRD was not observed in schizophrenic group. This was probably because the schizophrenic women took an increased amount of sleeping pills. Neuroleptics were shown to be least effected on SRRD.

Production of bioactive salmon calcitonin from the nonendocrine cell lines COS-7 and CHO.

To produce bioactive salmon calcitonin from the conventional nonendocrine cell lines, COS-7 and CHO, we devised a salmon calcitonin expression vector by combining the amino-terminus of human calcitonin precursor with a salmon calcitonin sequence, inserting the efficient furin-cleavable processing sequence Arg-X-Arg-X-Lys-Arg before salmon calcitonin, and deleting the carboxyl-terminal extension peptide. This chimeric calcitonin precursor terminates at glycine to easily receive an amidation reaction. COS-7 and CHO produced a high level of bioactive calcitonin by the resorption pit formation assay. Although amidating activity is highly expressed in CHO, but only a little in COS-7 cells, both cells produced a similar level of bioactive calcitonin. Thus, the engineered salmon calcitonin expression vector enables nonendocrine cells even with low amidation activity to produce bioactive calcitonin.

Modulation of neuronal function by intracellular pH.

Recent studies have revealed that excitation of specific nerve pathways can produce localized changes of pH in nervous tissue. It is important to determine both how these pH changes are generated and, even more importantly, how the excitability of neurons in the localized areas are affected. Evidence indicates that activation of both gamma-aminobutyric acid (GABA) and L-glutamate receptor channels in inhibitory and excitatory pathways, respectively, can raise extracellular pH (pHo) and lower intracellular pH (pHi). At the target location, it has been shown that several types of voltage-gated ion channels in neurons were modified by a change in pHi. These studies, taken together, enable us to hypothesize that intracellular hydrogen ions (H+) might function as neuromodulatory factors, like other types of intracellular second messengers. This hypothesis was tested by using horizontal cells enzymatically dissociated from catfish retina. We found that the high-voltage-activated (HVA) Ca2+ current, inward rectifier K+ current and hemi-gap junctional current are modulated by a change in intracellular H+ concentration, and that L-glutamate suppresses the HVA Ca2+ current by raising the intracellular H+ concentration. These observations support the hypothesis that intracellular H+, acting as a second messenger, governs neuronal excitability via modulation of ionic channel activity. This article reviews recent studies of ours and others on the effect of pHi upon neuronal function.

Effects of naloxone-HCl on cortisol levels in patients with affective disorder and normal controls.

Cortisol levels were measured before and after administration of naloxone-HCl in patients with affective disorder (n = 16) and normal control subjects (n = 8). On two consecutive days, 20 mg of naloxone-HCl or placebo was administered i.v. over 15 minutes in a double-blind crossover design. Blood samples were collected at 30, 15, and l minute(s) both before and after infusion. Cortisol rose from a mean baseline level of 14.8 microgram% to a mean peak level of 23.1 microgram% following the naloxone administration. Significant cortisol increases were found in both the 15- and 30-minute samples during the naloxone session. There were no differences between patient and normal subject samples or between diagnostic groups. A subgroup of manic patients who had responded to naloxone with a reduction of their manic behavior also had an attenuated cortisol response to naloxone. This proved to be an artifact secondary to variability in the cortisol response in these patients.

Lithium carbonate and ethanol induced "highs" in normal subjects.

The responses of twenty-three normal male subjects to a standardized dose of 95% ethanol (1.32 ml/kg of body weight) were compared after two weeks of placebo and two weeks of therapeutic serum lithium ion levels (mean 0.91 mEq/liter). The study was a placebo controlled, split-half crossover, double-blind design. Prealcohol and postalcohol responses were assessed by self-rating scales of affect and mood, independent rater observation, perceptual-motor, and cognitive performance tasks. Pretreatment by lithium carbonate neither blocked nor dampened an alcohol-induced subjective "high" in normal subjects. A complex reciprocal interaction may exist between the effects of lithium and alcohol upon other behavioral attributes. Alcohol was seen to reverse aspects of lithium-induced dysphoria and there is a suggestion that lithium may attenuate alcohol-induced cognitive inefficiency.

The effect of lithium carbonate on the cognitive functions of normal subjects.

The responses of 24 normal male subjects were compared after weeks of placebo administration and two weeks of lithium carbonate administration (mean serum lithium level, 0.9 mEq/liter) on a series of tasks of intellectual function, aesthetic judgement, and semantic creativity. This was a placebo-controlled, split-half crossover, double-blind design. There were no significant changes on semantic creativity or aesthetic perception measures following lithium carbonate maintenance. There were lithium carbonate-related performance deficits on three of five performance tasks concerned with cognitive and/or motor functions. The deficit is probably due to a lithium carbonate-induced slowing of performance, consistent with our previous report of subjective effects in normal subjects. The implications of slowing on possible behavioral mediating mechanisms by which lithium carbonate exerts its clinical effects are discussed.