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Background: Luminal fluid sequestration and diarrhea are early manifestations of mesenteric ischemia. This can be modeled in vitro with the use of T84 intestinal epithelia, where ischemia induces Cl(-) secretion with adenosine-mediated autocrine feedback. Protein kinase C (PKC) regulates epithelial transport and, in some organ systems, is involved in the response to ischemic stress. The purpose of this study was to define the role of PKC on epithelial transport during ischemia., Methods: By voltage-current clamp, short-circuit current (Isc) equals Cl(-) secretion. Ischemic conditions were simulated with the use of a well-established chemical hypoxia protocol., Results: Chemical hypoxia briskly activated Isc. Gö6850, an antagonist of novel and conventional PKC isoforms, markedly enhanced the ischemia-induced Isc response, although Gö6976 (which inhibits only conventional isoforms) had no effect. Rottlerin, a specific inhibitor of PKC delta, did not attenuate ischemic Isc. Both phorbol 12-myristate, 13-acetate and bryostatin-1, which selectively activate PKC epsilon in T84 cells, markedly attenuated the Isc response to ischemia. Both agents also inhibited the Isc response to exogenous adenosine., Conclusions: PKC (likely the novel epsilon isoform) in intestinal epithelia modulates ischemia-induced alterations in ion transport. Inhibition of PKC epsilon exaggerates the secretory response that is induced by ischemia and by authentic adenosine; conversely, augmented activation of PKC epsilon inhibits secretion. Manipulation of PKC epsilon could limit luminal fluid sequestration during mesenteric ischemia.

The pharmacokinetics of omeprazole delayed-release capsules and a simplified omeprazole suspension (SOS) were studied. Seven healthy volunteers randomly received either one 20-mg omeprazole delayed-release capsule or SOS (omeprazole 20 mg in 10 mL) for seven days before being crossed over to the opposite treatment for seven more days after a two-week washout period. On days 1 and 7, blood samples were drawn at intervals up to 360 minutes after drug administration. Plasma omeprazole concentrations were determined by a validated high-performance liquid chromatographic method, and pharmacokinetic values were determined. Area under the concentration-versus-time curve (AUC) from zero to six hours, AUC from time zero to infinity (AUC0-infinity), and maximum plasma concentration (Cmax) increased by 102%, 113%, and 85%, respectively, after seven days of treatment with the capsule. AUC0-infinity for SOS on day 1 was 58% of that for the capsule (p = 0.0141), and on day 7 it was 49% of that for the capsule (p = 0.0044). AUC0-infinity for SOS increased by 85% from day 1 to 7, but the difference was not significant. Cmax for SOS on day 1 was twice that for the capsule (p = 0.0014), but by day 7 the difference between the two formulations was negligible. Time to Cmax (tmax) for SOS on days 1 and 7 was shorter than for the capsule by 82% (p < 0.0001) and 70% (p < 0.0006), respectively. After one week of therapy, omeprazole absorption was faster and tmax was 70% shorter for SOS than for the capsule formulation, but AUC0-infinity was 49% lower for SOS.

Study Objective: To evaluate the effects of moderate, single-dose caffeine consumption on electrocardiographic variables: PR, QRS, QT, QTc, and RR intervals, and QT and QTc interval dispersion. Effects of caffeine on blood pressure and heart rate also were evaluated., Design: Randomized, double-blind, placebo-controlled, crossover study., Setting: University school of pharmacy., Patients: Ten healthy volunteers aged 17 years or older., Intervention: Participants abstained from caffeinated products for at least 2 days before the study began and were randomly designated to receive placebo or caffeine 400 mg on various days. For each of the study phases, a baseline 12-lead electrocardiogram (ECG) was performed and a subsequent 12-lead ECG performed 3 hours after ingesting the study drug. Blood pressure readings were taken with each ECG., Measurements and Main Results: No significant changes in any intragroup or intergroup electrocardiographic variables occurred. Caffeine increased blood pressure (systolic blood pressure [SBP]/diastolic blood pressure [DBP]) from 118+/-5/75+/-6 mm Hg to 128+/-8/77+/-7 mm Hg versus baseline (p=0.0022 and p=0.0368 for SBP and DBP, respectively). After drug dosing, SBP in the caffeine group was significantly higher than in the placebo group (128+/-8 mm Hg versus 119+/-7 mm Hg, p=0.0174)., Conclusion: Moderate caffeine consumption by healthy young adults does not acutely affect PR, QRS, QT, QTc, and RR intervals, or QT and QTc interval dispersion. Caffeine-naive subjects experienced persistent elevations in SBP and DBP 3 hours after caffeine ingestion, indicating that longer caffeine abstinence than that which is recommended is necessary for blood pressure determination in the clinical setting.

Objective: To review commonly used fibrinogen assay methods and the evidence demonstrating an association between fibrinogen and increased risk of coronary artery disease and to review the current literature to determine and assess the impact of hydroxymethylglutaryl coenzyme A (HMG-CoA) reductase inhibitors on fibrinogen., Data Sources: Primary and review articles identified from a MEDLINE search (1966-December 1999); references obtained from these publications were subsequently reviewed for additional relevant articles., Study Selection and Data Extraction: All articles were evaluated, and all relevant information was included in this review., Data Synthesis: The Clauss method is currently the preferred method for determining plasma fibrinogen concentrations, due to its high degree of accuracy and precision. Furthermore, unlike immunologic methods, its reliability is unaffected by change in triglycerides. The effects of four HMG-CoA reductase inhibitors (atorvastatin, lovastatin, simvastatin, pravastatin) on fibrinogen have been evaluated. Atorvastatin has been shown to induce significant increases in fibrinogen (22% increase; p <0.05) by using the immunonephelometric method. This method also demonstrated that lovastatin use was associated with a 24.4% increase (p < 0.0001) in plasma fibrinogen concentration. Simvastatin has been shown in multiple studies using the Clauss method to have a neutral effect on fibrinogen. The majority of studies have revealed significant decreases (7-19%) in fibrinogen following treatment with pravastatin., Conclusions: Future studies need to be performed evaluating the effects of HMG-CoA reductase inhibitors on fibrinogen, but using direct comparisons and clotting assay methodology.

The total synthesis of a demethoxy-egonol isolated from Styrax obassia, 5-(3-hydroxypropyl)-2-(3',4'-methylenedioxyphenyl)benzofuran (9), is described. The key steps involve the construction of a 2-arylbenzofuran skeleton 7 from methyl 3-(4-hydroxyphenyl)propionate with 2-chloro-2-methylthio-(3',4'-methylenedioxy)acetophenone (6) in the presence of ZnCl2 and successive desulfurization of the resulting product 7.

Lawsonia intracellularis is not culturable with a standard bacteriologic culture. One step PCR assay as a clinical diagnostic method was developed for the rapid detection of porcine proliferative enteritis (PPE) caused by L. intracellularis. Primers were designed based on the p78 DNA clone of L. intracellularis. The one step PCR resulted in the formation of a specific 210-bp DNA product derived from L. intracellularis. The nonspecific amplification product was not detected with swine genomic DNA or other bacterial strains causing similar symptoms to L. intracellularis infection. The one step PCR was as sensitive as 100 pg of L. intracellularis genomic DNA. We applied this method to field specimens diagnosed as PPE by macroscopic observation. Of 17 mucosal scraping specimens, 16 (94%) were identified as positive to PPE and 15 (88%) of 17 feces specimens. These results suggest that the one step PCR can be used as a rapid diagnostic method for L. intracellularis infection.

Over the past 4 years, six angiotensin II receptor antagonists (ARBs) were approved for treating essential hypertension. They differ with respect to dosing, metabolism, elimination, clinical efficacy, and investigational applications. Candesartan cilexetil is the only prodrug among the agents. Losartan is distinguished from other ARBs by cytochrome P450 (CYP) 3A4- and CYP2C9-mediated biotransformation to its active metabolite EXP-3174. No ARB requires dosage adjustment for renal impairment, but the initial dose of losartan should be reduced 50% in hepatically impaired patients. None of the drugs is significantly cleared by hemodialysis. Completion of continuing trials will elucidate the drugs' role in treating heart failure, cerebral stroke, and myocardial infarction.

Protein kinase C (PKC) and the actin cytoskeleton are critical effectors of membrane trafficking in mammalian cells. In polarized epithelia, the role of these factors in endocytic events at either the apical or basolateral membrane is poorly defined. In the present study, phorbol 12-myristate 13-acetate (PMA) and other activators of PKC selectively enhanced basolateral but not apical fluid-phase endocytosis in human T84 intestinal epithelia. Stimulation of basolateral endocytosis was blocked by the conventional and novel PKC inhibitor Gö-6850, but not the conventional PKC inhibitor Gö-6976, and correlated with translocation of the novel PKC isoform PKC-epsilon. PMA treatment induced remodeling of basolateral F-actin. The actin disassembler cytochalasin D stimulated basolateral endocytosis and enhanced stimulation of endocytosis by PMA, whereas PMA-stimulated endocytosis was blocked by the F-actin stabilizers phalloidin and jasplakinolide. PMA induced membrane-to-cytosol redistribution of the F-actin cross-linking protein myristoylated alanine-rich C kinase substrate (MARCKS). Cytochalasin D also induced MARCKS translocation and enhanced PMA-stimulated translocation of MARCKS. A myristoylated peptide corresponding to the phosphorylation site domain of MARCKS inhibited both MARCKS translocation and PMA stimulation of endocytosis. MARCKS translocation was inhibited by Gö-6850 but not Gö-6976. The results suggest that a novel PKC isoform, likely PKC-epsilon, stimulates basolateral endocytosis in model epithelia by a mechanism that involves F-actin and MARCKS.

The basolateral Na+/K+/2Cl(-) cotransporter (NKCC1) has been shown to be an independent regulatory site for electrogenic Cl(-) secretion. The proinflammatory phorbol ester, phorbol 12-myristate 13-acetate (PMA), which activates protein kinase C (PKC), inhibits basal and cyclic adenosine monophosphate (cAMP)-stimulated NKCC1 activity in T84 intestinal epithelial cells and decreases the steady state levels of NKCC1 mRNA in a time- and dose-dependent manner. The levels of NKCC1 protein also fall in accordance with the NKCC1 mRNA transcript and these levels are unaffected by 4alpha-phorbol, which does not activate PKC. Inhibition of maximal (cAMP-stimulated) NKCC1 functional activity by PMA was first detected by 1 h, whereas decreases in the steady state levels of NKCC1 mRNA were not detectable until 4 h. NKCC1 mRNA expression recovers toward control levels with extended treatment of cells with PMA suggesting that the PMA effects on NKCC1 expression are mediated through activation of PKC. Although NKCC1 mRNA and protein levels return to control values after extended PMA exposure, NKCC1 functional activity does not recover. Immunofluorescence imaging suggest that the absence of functional recovery is due to failure of newly synthesized NKKC1 protein to reach the cell surface. We conclude that NKCC1 has the capacity to be regulated at the level of de novo expression by PKC, although decreased NKCC1 expression alone cannot account for either early or late loss of NKCC1 function., (Copyright 1999 Wiley-Liss, Inc.)

Ammonia profoundly inhibits cAMP-dependent Cl- secretion in model T84 human intestinal crypt epithelia. Because colonic lumen concentrations of ammonia are high (10-70 mM), ammonia may be a novel regulator of secretory diarrheal responsiveness. We defined the target of ammonia action by structure-function analysis with a series of primary amines (ammonia, methylamine, ethylamine, propylamine, butylamine, pentylamine, hexylamine, heptylamine, and octylamine) that vary principally in size and lipid solubilities. The amine concentrations required for 50% inhibition of Cl- secretion in intact monolayers and 50% inhibition of outward K+ current (IK) in apically permeabilized monolayers vs. the logs of the respective amine partition coefficients give two plots that are strikingly similar in character. Half-maximal inhibition of short-circuit current (Isc) by ammonia was seen at 6 mM and for IK at 4 mM; half-maximal inhibition for octylamine was 0.24 mM and 0.19 mM for Isc and IK, respectively. The preferentially water-soluble hydrophilic amines (ammonia, methylamine, ethylamine) increase in blocking ability with decreasing size and lipophilicity. Conversely, the preferentially lipid-soluble hydrophobic (propylamine, butylamine, pentylamine, hexylamine, heptylamine, octylamine) amines increase in blocking ability with increasing size and lipophilicity. Ammonia does not affect isolated apical Cl- conductance; amine-induced changes in cytosolic and endosomal pH do not correlate with secretory inhibition. We propose that ammonia in its protonated ammonium form (NH4+) inhibits cAMP-dependent Cl- secretion in T84 monolayers by blocking basolateral K+ channels.

Background & Aims: Phenylimidazothiazoles have recently been shown to activate wild-type and mutant cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channels in transfected cells and were proposed as therapy for cystic fibrosis. The aim of this study was to investigate the effects of phenylimidazothiazoles on regulated transepithelial Cl- transport in intact epithelia., Methods: T84 intestinal epithelial cells grown on permeable supports and stripped human colonic mucosal sheets were studied by conventional current-voltage clamping. Selective permeabilization of apical or basolateral membranes with the monovalent ionophore nystatin was used to isolate basolateral K+ and apical Cl- channel activity, respectively. 86Rb+ uptake was assessed for Na/K/2Cl cotransporter and Na+,K(+)-adenosine triphosphatase activity., Results: In T84 monolayers and human colon, levamisole and its brominated derivative bromotetramisole failed to activate transepithelial secretion. In fact, these compounds dose-dependently inhibited secretory responses to the cyclic adenosine monophosphate agonist forskolin and the Ca2+ agonist carbachol. In permeabilized T84 monolayers, phenylimidazothiazoles weakly activated apical Cl- currents (consistent with their reported action on CFTR) and did not affect bumetanide-sensitive or bumetanide-insensitive 86+Rb+ uptake. Instead, they profoundly inhibited the basolateral Ba(2+)-sensitive and Ba(2+)-insensitive K+ currents., Conclusions: Phenylimidazothiazoles block K+ channels required for Cl(-)-secretory responses elicited by diverse pathways in model epithelia and native colon, an effect that outweighs their ability to activate apical Cl- channels.

For the rapid identification of noble genes in a specific tissue by computer analysis from the cDNA sequences determined by single-pass cDNA sequencing, clone redundancy was one of the major obstacles. To facilitate the efficiency in identification of noble genes, it was necessary to reduce the number of clones to be sequenced by eliminating the redundant clones for a rapid analysis. In order to increase the probability of isolating noble sequences from the cDNA clones of human fetal liver tissue origin, colony hybridization assay was adopted and redundant clones were efficiently removed. Four cDNA clones highly redundant in the human fetal liver cDNA libraries including alpha-globin, gamma-globin, serum albumin and H19 RNA sequences were selected as the probes. Two hundreds and sixty two cDNA clones were randomly selected and tested with the probes for hybridization properties. The identity of each cDNA clone giving positive or negative signals in the hybridization assay was determined by DNA homology search with the nucleic acid databases. Among the 76 clones giving positive signals, 57 clones (75%) were found to be identical to the probe sequences and could be eliminated by colony hybridization assay before neucleotide sequencing.

Objectives: The purpose of this study was to determine what risk factors play a role in the development of retinopathy of prematurity (ROP)., Study Design: Data were collected on 157 infants born and cared for in one institution between January 1991 and July 1994. Initially we evaluated all children enrolled in the study to determine potential risk factors for the development of ROP. We subsequently compared multiple variables for ROP positive singletons with ROP positive twins to determine ROP risk factors for each group and to determine if one group was more susceptible to a given risk factor., Results: Of the 157 infants examined, 72 infants (46%) developed ROP. Infants who developed ROP had a lower gestational age, a lower birth weight, a higher number of days on oxygen/ventilator, more days in the intensive care unit (ICU), a greater need for steroids and a higher incidence of sepsis when compared to infants who did not develop ROP. There was no significant difference noted between singleton and twin gestation infants that developed ROP when comparing gestational age, weight, ventilator time or length of ICU stay. Total number of days on oxygen therapy was higher in the singleton group and this difference did reach statistical significance., Conclusions: Several risk factors are associated with a higher incidence of ROP. These variables may not be independent risk factors but may be a sign of the increased severity of illness associated with those infants who are born earlier with a lower birth weight. Multiple gestational births do not appear to increase the risk of developing ROP when compared to a similar group of singleton birth infants.

We recently showed that ammonia profoundly inhibits cyclic nucleotide-regulated Cl- secretion in model human T84 intestinal epithelia but does not impair the secretory response to the Ca2+ agonist carbachol. Using transepithelial transport, fura 2 fluorescence, and radioisotopic efflux techniques, we further explored this dichotomy and arrived at a preliminary explanation for the inhibitory action of ammonia. The secretory response to the Ca(2+)-adenosinetriphosphatase inhibitor thapsigargin is unaffected by ammonia, which suggests that an increase in intracellular Ca2+ stimulates secretory pathways that are insensitive to ammonia. Surprisingly, Cl- secretion elicited by the Ca2+ ionophores ionomycin and A23187 is markedly blunted in monolayers pretreated with ammonia. However, ammonia posttreatment does not inhibit the secretory response to ionophore, which suggests that ammonia may interfere with the ability of these ionophores to increase intracellular [Ca2+]. This hypothesis is directly supported by fura 2 experiments. The inhibitory action of ammonia parallels the behavior of the K+ channel blocker Ba2+, and ammonia reduces the basolateral 86Rb+ efflux rate constant in forskolin- but not in carbachol-treated monolayers. Ammonia, which is present in high concentrations in the normal gastro-intestinal tract, may serve as a novel endogenous regulator of epithelial electrolyte transport by interfering with a Ba(2+)-sensitive basolateral K+ conductance distinct from the Ca(2+)-activated basolateral K+ conductance.

Five patients with systemic lupus erythematosus (SLE) were perfused through an extracorporeal shunt filled with DNA-immunoadsorbent (DNA immobilized on carbonized resin beads). High concentrations of anti-DNA antibodies (36.4-67.0%) (binding percentage with 125I-DNA) in the serum of SLE patients were reduced to 13.8-53.0%, respectively. The highest removal percentage was 62.1%. Although the decline levels varied, the symptoms of patients, i.e., long-term severe joint pain, severe edema, hydropericardium, and ascites were all relieved considerably. The immunoadsorbent showed satisfactory blood compatibility.

Large restriction fragments of genomic DNA from Micrococcus sp. Y-1 were separated by pulsed-field gel electrophoresis (PFGE). Since Micrococcus sp. Y-1 has a G+C content of approximately 70%, restriction fragments were obtained by digesting chromosomal DNA with endonucleases which recognize A+T-rich sequences. Five enzymes, SspI, SpeI, XbaI, HpaI and EcoRI, were used for generation of distinctly separated fragments in the size range 100-500 kb. No site for DraI was detected. In contrast, sites for 8-base-recognizing enzymes, but not for NotI and SfiI, were frequent. The genome size of Micrococcus sp. Y-1 was determined from restriction fragments separated by PFGE, and was estimated to be approximately 4061 kb. Partial digestion experiments revealed the order of the six SspI fragments on the chromosome.

The sequences of cDNA clones encoding the Bowman-Birk proteinase inhibitor (BBPI) and its isoinhibitors (D-II and C-II) have been identified. Nucleotide sequence homologies among these clones and between the two reactive domains in each clone were very high. These homologies suggest that the BBPI and its isoinhibitor genes may have evolved from a common ancestral inhibitor. Also, clone pB2 (D-II) had two identical reactive sites that inhibit trypsin only, and three repetitive regions that are about 81 bp and initiated with ATG. It was assumed from these results that isoinhibitor D-II was the most primordial type of these inhibitors.

Cytokines may be important in both the pathophysiology an ocular morbidity associated with treated and untreated uveal melanomas. In this study we used message amplification phenotyping to identify cytokines present in uveal melanoma samples. A number of cytokines were noted that have been associated with tumor progression, neovascularization, and inflammation in other malignancies. Interferongamma was commonly found in spindle cell tumors. Only one of 16 tumors had detectable levels of TNF-alpha and this occurred in one of two patients who developed metastatic disease.

To remove bilirubin from patients with hyperbilirubinemia, nonionic macroporous adsorbent NK-110 was tailor-made from styrene and divinyl benzene at Nankai University. Five patients were perfused through shunts filled with 500 ml of the above adsorbent. After 2 h of hemoperfusion, total bilirubin decreased within the range of 22-51%. During the treatment, no noticeable changes in electrolytes, urea nitrogen, serum glutamicpyruvic transaminase (SGPT), TTT, and amino acid concentrations were observed. Platelet depletion was satisfactory. Hemoglobin, white cell count, globulin, and albumin showed no remarkable changes. The nonionic adsorbent shows a high efficacy for bilirubin removal and has a great potential for future clinical applications.

To investigate the effect of acupuncture on the plasma levels of hormones, we have applied acupuncture in the meridian points. Two meridian points, B25 and B15, were selected for the hand-running acupuncture application. The plasma levels of immunoreactive atrial natriuretic peptide, aldosterone, and renin activity were measured by radioimmunoassay. Acupuncture in the meridian point B23 decreased plasma levels of immunoreactive atrial natriuretic peptide, while that in the B15 increased plasma levels of immunoreactive atrial natriuretic peptide. Acupuncture in the B15 decreased plasma levels of aldosterone while that in the B23 decreased plasma renin activity. These results suggest that acupuncture in the specific meridian points may have sites specific regulatory function for the hormone levels and that the meridian points, B23 and B15, may have control mechanism for the regulation in the body fluid and electrolytes balance.

A simple method for the molecular cloning of fragments of more than one hundred kilobase pairs of exogenous DNA, by the encapsulation of cells in agarose beads, was reported previously for the construction of a human genomic DNA library in a yeast artificial chromosome (YAC) vector (in situ YAC construction) [1]. The efficiency of this procedure is impaired by the step in which agarose beads that contain human DNA fragments are melted before transformation. The incomplete solubility of the ligated human DNA fragment-YAC vector often results in lower than desirable frequencies of transformation. To overcome this problem we have developed a new improved method that involves use of an agarose film. The technical manipulations involved in the construction of clones of very large segments of human DNA are discussed.

This investigation studied the preparation and clinical trial of DNA immunoadsorbent for hemoperfusion in systemic lupus erythematosus (SLE) therapy. DNA was complexed with blue tetrazolium (BT), then mixed with collodion, and finally adsorbed on macroporous spherical polymeric adsorbent. The percentage of DNA thus immobilized was 98-98.5%, and no release was detected during hemoperfusion. A patient with severe SLE was treated with the above adsorbent in hemoperfusion. The patient was in coma, with high anti-DNA antibody and abnormal immune complex levels, which fell sharply from 56.34% to 0.8% after 2.5 h of whole-blood hemo perfusion. There were no major clinical complications, and 2 weeks later, her proteinuria level became normal and immune complex levels were sustained. She was then discharged from the hospital and has been in good health for more than 1 year.

Virologic and seroepidemiologic studies were carried out during an epidemic of dengue fever on Hainan Island in 1980. Dengue 3 virus was isolated from 46 of 77 acute phase sera and from 1 of 10 pools of adult Aedes aegypti. Dengue 1 virus virus was isolated from a single acute phase serum. Seroepidemiologic investigations showed that 74% of healthy individuals in the epidemic area had antibody to dengue virus compared to 54% in an area where epidemic dengue had occurred in 1978, and less than or equal to 8% in nonepidemic areas. There were no significant differences in antibody prevalence for different sex and age groups.

Macroporous polystyrene and polyvinylpyridine beads with quaternary ammonium and tertiary groups were tailor-made for the adsorption of unconjugated bilirubin. In vitro studies in order to obtain high adsorption capacity, the synthesized macroporous resin should have an average pore size of about 160 A and with surface area as large as possible. As the crosslinking of the resin increased from 4%-40% the amount of bilirubin adsorbed increased from 0.17 mg/g to 2.0 mg/g. Resins containing strong basic groups have higher adsorption capacities than their corresponding weak base groups. Adsorption mechanism studies showed that both chemical and physical adsorption took place. Adsorption of bilirubin in plasma was some what lower than in buffer solution.