Construction and characterization of an auxotrophic ctxA mutant of O139 Vibrio cholerae.

Cholera caused by the O139 serogroup still remains a public health concern in certain regions of the world and the existing O1 vaccines do not cross-protect cholera caused by this serogroup. An aminolevulinic acid (ALA) auxotroph vaccine candidate against the O139 serogroup, designated as VCUSM2, was recently developed. It was found to be immunogenic in animal model studies but showed mild reactogenic effects due to the presence of two intact copies of Vibrio cholerae toxin (CTX) genetic element. In the present study we have modified the ctx operon by systematic allelic replacement methodology to produce a mutant strain, designated as VCUSM14. This strain has two copies of chromosomally integrated and mutated ctxA gene, encoding immunogenic but not toxic cholera toxin A subunit (CT-A). The amino acids arginine and glutamic acid at position 7th and 112th, respectively, in CT-A of VCUSM14 were substituted with lysine (R7K) and glutamine (E112Q), respectively. Two copies of the ace and zot genes present in the ctx operon were also deleted. Cholera toxin-ELISA using GM1 ganglioside showed that the both wild type CT and mutated CT were recognized by anti-CT polyclonal antibodies. VCUSM14 produced comparatively less amount of antigenic cholera toxin when compared to the VCUSM2 and Bengal wild type strain. VCUSM14 did not elicit fluid accumulation when inoculated into rabbit ileal loops at doses of 10(6) and 10(8) CFU. The colonization efficiency of VCUSM14 was one log lower than the parent strain, VCUSM2, which can be attributed to the ALA auxotrophy and less invasive properties of VCUSM14. VCUSM14, thus a non-reactogenic auxotrophic vaccine candidate against infection by O139 V. cholerae.

The optical mouse as an inexpensive region-of-interest position recorder in optical microscopy.

In optical microscopy, the microscopic features of interest typically have to be derived from regions that are spatially distributed over the sample. While the features to be analyzed may be minute, the regions from where they must be obtained from may be located quite far apart from one another. If the features are not distinct enough to allow easy visual discrimination, it would be tedious and time-consuming to attempt to recall and revisit these regions of interest. One method to overcome this difficulty would be to note and record the graduated markings on the microscope, or to use built-in position encoders. The former is tedious while the latter is generally expensive. Here, a region-of-interest position-recording implement based on the use of an optical mouse is presented. It is inexpensive and easily adaptable to the manual stage of any optical microscope.

The role of nitric oxide in ocular surface diseases.

For the first time, the current series of studies provide a possible pathophysiologic mechanism of NO-induced ocular surface disease. NO is present in tear and aqueous humor and is suspected of having an important physiological role in maintaining normal homeostasis of the ocular surface. NO concentrations are higher in aqueous humor compared to tears, though some variability exists between different species. When inflammation was induced by PTK wounding or LPS, three forms of NOS expression were seen in corneal cells. Each isoform of NOS was expressed uniquely according to the specific location of inflammation. When concentrations of NO peaked, the levels of iNOS were markedly increased in fibroblasts and inflammatory cells. The correlation between NO and inflammation was confirmed by treatment with NOS inhibitor, which abrogated the amount of both NO and inflammation. The tissue damage by NO was measured by nitrotyrosine formation. Damage was detected mainly in inflammatory cells, especially those localized in and around the limbal vessel. It is likely that expression of iNOS in limbal fibroblasts has other roles related to survival of limbal stem cells and fibroblasts as well. Because the main source of NO are fibroblasts, we were able to determine the effect of various concentrations of NO on cell viability using a fibroblast culture system. Cell viability increased in dose dependent manner from 10 microM to 500 microM of the NO generator SNAP, but decreased at concentrations above 1000 microM, suggesting that the in vivo mechanism of cell death was indirect, through specific biologic pathways. Therefore, the pathophysiological mechanism of NO action is bimodal with a toxicological component in ocular surface diseases. Furthermore, its concentration and interaction with other oxygen mediators appear to vary depending on the degree of inflammation.

Thermal inactivation kinetics and application of phospho- and galactolipid-degrading enzymes for evaluation of quality changes in frozen vegetables.

Lipid-acyl hydrolases (LAHases) play significant roles in lipid degradation during the storage of vegetables. In particular, spinach contains a large portion of galactolipids (59.5%) and phospholipids (22.4%) among its fat-soluble components, which are used as substrates for LAHases. Thermal inactivation of various LAHases, including phospholipases A, C, and D, phosphatase, and galactolipase, from spinach and carrot was investigated to optimize the blanching process prior to the frozen storage of vegetables. Thermostability of phospholipase C or galactolipase was greatest among the LAHases from both spinach and carrot. Galactolipase from spinach exhibited a D value of 3.39 x 10(2) s at 80 degrees C and a z value of 8.21 degrees C, whereas phospholipase C from spinach showed D(80) of 1.72 x 10(2) s with a z value of 9.26 degrees C. In the case of LAHases from carrot, the D(65) and z values of galactolipase were 6.66 x 10(2) s and 8.69 degrees C, respectively, whereas phospholipase C displayed D(85) of 3.12 x 10(2) s and a z value of 15.8 degrees C. Highly active and thermostable galactolipase and phospholipase C in spinach and carrot made it possible for them to be used as indicator enzymes for the determination of quality deterioration of the stored vegetables.

Cloning and expression of the limonene hydroxylase of Bacillus stearothermophilus BR388 and utilization in two-phase limonene conversions.

A 3.6-kb fragment of Bacillus stearothermophilus BR388 chromosomal DNA that confers growth on limonene to Escherichia coli has been sequenced, revealing a single open reading frame encoding a single subunit limonene hydroxylase containing 444 amino acid residues. This enzyme proved capable of limonene hydroxylation to a mixture of carveol and perillyl alcohol as well as dehydrogenation of these products to carvone and perillyl aldehyde. Oxygen, FAD, and NADH were found to stimulate the hydroxylation reaction in cell extracts, and NAD+ stimulated the dehydrogenase reaction. In two-phase bioconversions using viable E. coli cells over-expressing the limonene hydroxylase, perillyl alcohol and carvone were the principal products observed.

Structure, specificity and function of cyclomaltodextrinase, a multispecific enzyme of the alpha-amylase family.

Cyclomaltodextrinase (CDase, EC 3.2.1.54), maltogenic amylase (EC 3. 2.1.133), and neopullulanase (EC 3.2.1.135) are reported to be capable of hydrolyzing all or two of the following three types of substrates: cyclomaltodextrins (CDs); pullulan; and starch. These enzymes hydrolyze CDs and starch to maltose and pullulan to panose by cleavage of alpha-1,4 glycosidic bonds whereas alpha-amylases essentially lack activity on CDs and pullulan. They also catalyze transglycosylation of oligosaccharides to the C3-, C4- or C6-hydroxyl groups of various acceptor sugar molecules. The present review surveys the biochemical, enzymatic, and structural properties of three types of such enzymes as defined based on the substrate specificity toward the CDs: type I, cyclomaltodextrinase and maltogenic amylase that hydrolyze CDs much faster than pullulan and starch; type II, Thermoactinomyces vulgaris amylase II (TVA II) that hydrolyzes CDs much less efficiently than pullulan; and type III, neopullulanase that hydrolyzes pullulan efficiently, but remains to be reported to hydrolyze CDs. These three types of enzymes exhibit 40-60% amino acid sequence identity. They occur in the cytoplasm of bacteria and have molecular masses from 62 to 90 kDa which are slightly larger than those of most alpha-amylases. Multiple amino acid sequence alignment and crystal structures of maltogenic amylase and TVA II reveal the presence of an N-terminal extension of approximately 130 residues not found in alpha-amylases. This unique N-terminal domain as seen in the crystal structures apparently contributes to the active site structure leading to the distinct substrate specificity through a dimer formation. In aqueous solution, most of these enzymes show a monomer-dimer equilibrium. The present review discusses the multiple specificity in the light of the oligomerization and the molecular structures arriving at a clarified enzyme classification. Finally, a physiological role of the enzymes is proposed.

Transglycosylation of neohesperidin dihydrochalcone by Bacillus stearothermophilus maltogenic amylase.

Neohesperidin dihydrochalcone (NHDC), a sweet compound derived from citrus fruits, was modified to a series of its oligosaccharides by transglycosylation activity of Bacillus stearothermophilus maltogenic amylase (BSMA). Maltotriose as a donor was reacted with NHDC as an acceptor to glycosylate for the purpose of increasing the solubility of NHDC. Maltosyl-NHDC was a major transglycosylation product among the several transfer products by TLC analysis. The structure of the major transglycosylation product was determined to be maltosyl-alpha-(1,6)-neohesperidin dihydrochalcone by MALDI-TOF/MS and (1)H and (13)C NMR. Maltosyl-NHDC was 700 times more soluble in water and 7 times less sweet than NHDC.

Kinetics and inhibition of cyclomaltodextrinase from alkalophilic Bacillus sp. I-5.

The cyclomaltodextrinase from alkalophilic Bacillus sp. I-5 (CDase I-5) was expressed in Escherichia coli and the purified enzyme was used for characterization of the enzyme action. The hydrolysis products were monitored by both HPLC and high-performance ion chromatography analysis that enable the kinetic analysis of the cyclomaltodextrin (CD)-degrading reaction. Analysis of the kinetics of cyclomaltodextrin hydrolysis by CDase I-5 indicated that ring-opening of the cyclomaltodextrin was the major limiting step and that CDase I-5 preferentially degraded the linear maltodextrin chain by removing the maltose unit. The substrate binding affinity of the enzyme was almost same for those of cyclomaltodextrins while the rate of ring-opening was the fastest for cyclomaltoheptaose. Acarbose and methyl 6-amino-6-deoxy-alpha-d-glucopyranoside were relatively strong competitive inhibitors with K(i) values of 1.24 x 10(-3) and 8.44 x 10(-1) mM, respectively. Both inhibitors are likely to inhibit the ring-opening step of the CD degradation reaction.

A prospective study of infections with atypical pneumonia organisms in acute exacerbations of chronic bronchitis.

The objective of this paper was to study the incidence of 6 atypical pneumonia pathogens or atypical organisms in local patients admitted for acute exacerbation of chronic bronchitis. This is a prospective observational study. Over a period of 3 years (1995 to 1997), 90 patients admitted to a large general hospital in Singapore for acute exacerbation of chronic bronchitis were tested for the following infections: Legionella, Mycoplasma, Chlamydia, influenza A, influenza B and parainfluenza viruses, using paired serological examination. The antibiotic prescribing pattern by the attending physicians in these cases were also examined. Positive serologies were found in 31 patients (34%), of whom 26 patients (28%) had viral infections. The most common organism was influenza A with 18 positive serologies (20%). Five patients were tested positive for Legionella. There was no evidence of acute infections by Mycoplasma pneumoniae or chlamydia using serological tests.

Modes of action of acarbose hydrolysis and transglycosylation catalyzed by a thermostable maltogenic amylase, the gene for which was cloned from a Thermus strain.

A maltogenic amylase gene was cloned in Escherichia coli from a gram-negative thermophilic bacterium, Thermus strain IM6501. The gene encoded an enzyme (ThMA) with a molecular mass of 68 kDa which was expressed by the expression vector p6xHis119. The optimal temperature of ThMA was 60 degrees C, which was higher than those of other maltogenic amylases reported so far. Thermal inactivation kinetic analysis of ThMA indicated that it was stabilized in the presence of 10 mM EDTA. ThMA harbored both hydrolysis and transglycosylation activities. It hydrolyzed beta-cyclodextrin and starch mainly to maltose and pullulan to panose. ThMA not only hydrolyzed acarbose, an amylase inhibitor, to glucose and pseudotrisaccharide (PTS) but also transferred PTS to 17 sugar acceptors, including glucose, fructose, maltose, cellobiose, etc. Structural analysis of acarbose transfer products by using methylation, thin-layer chromatography, high-performance ion chromatography, and nuclear magnetic resonance indicated that PTS was transferred primarily to the C-6 of the acceptors and at lower degrees to the C-3 and/or C-4. The transglycosylation of sugar to methyl-alpha-D-glucopyranoside by forming an alpha-(1,3)-glycosidic linkage was demonstrated for the first time by using acarbose and ThMA. Kinetic analysis of the acarbose transfer products showed that the C-4 transfer product formed most rapidly but readily hydrolyzed, while the C-6 transfer product was stable and accumulated in the reaction mixture as the main product.

The obstructive sleep apnoea syndrome--experience of a referral centre.

OBJECTIVE: The aims of this study were: (i) to document the presence and severity of obstructive sleep apnoea (OSA) in patients who complained of heavy snoring and other symptoms suggestive of the OSA syndrome; (ii) to examine the correlation between the clinical and polygraphic findings, and (iii) to document the efficacy and compliance of nasal continuous positive airway pressure (CPAP) among these Asian snorers with OSA. METHODS: We analysed our clinical and nocturnal polysomnographic data between January 1986 and December 1995 for physician-referred patients who had complained of snoring and other symptoms suggestive of OSA. RESULTS: A total of 277 diagnostic studies were performed of which 145 (52%) were positive to OSA. For studies performed in the last 2 years (n = 125), 72 of the 125 were positive for OSA. Anthropometric data was not discriminative between the OSA positive snorers and the OSA negative snorers. We found that hypertension and choking were the most significantly related to OSA, conferring a 7 and 4 times relative risk respectively. Nasal CPAP eliminated snoring, apnoeas and oxygen desaturations completely in almost all cases and there were only minor mask-related side effects. CONCLUSION: OSA may not be uncommon among Asian snorers. Of the major traits for OSA risk among our local population, a history of hypertension and reports of nocturnal choking were the most significantly related. We have also shown that nasal CPAP is safe and effective among our local snorers and should be considered a first-line treatment for OSA.

Production of alpha-terpineol from Escherichia coli cells expressing thermostable limonene hydratase.

The genes encoding a thermostable limonene hydratase have been located on a cloned fragment in Escherichia coli conferring growth on limonene and production of the monoterpenes perillyl alcohol and alpha-terpineol. Whole cell bioconversion studies at elevated temperature employing both an aqueous phase and neat limonene phase demonstrated significant production of alpha-terpineol with additional production of carvone.

Transdiaphragmatic pressure in young adult Singaporean subjects--normal values and a comparison between different respiratory manoeuvres.

BACKGROUND: Most published normal values for transdiaphragmatic pressure (Pdi) have been from Caucasian subjects and there is no universal agreement regarding the most appropriate manoeuvre for assessing Pdi. AIMS: The aims of our study were to obtain normal values and to compare the different manoeuvres used to assess Pdi in normal young Singaporean adults. METHODS: Twenty-four normal subjects (23 Chinese, one Indian) were studied by measuring Pdi during maximal sniffs from functional residual capacity (sniff Pdi), maximal inspiration to total lung capacity (Pdi TLC) and maximal static inspiratory efforts from residual volume (Pdi PImax). RESULTS: Mean values +/- SD for sniff Pdi, Pdi TLC and Pdi PImax were 101.8 +/- 31.7, 46.8 +/- 26.4 and 83.5 +/- 35.5 cm H2O respectively. Sniff Pdi was significantly higher than Pdi TLC (p < 0.001) and Pdi PImax (p = 0.005). Pdi PImax was significantly higher than Pdi TLC (p < 0.001). Males had significantly higher values for sniff Pdi (p = 0.026) and Pdi PImax (p = 0.022) than females. There was a significant correlation between the different methods of recording Pdi. Sniff Pdi had the highest values, least between- and within-subject variation and most consistent pattern of respiratory muscle recruitment with the lowest proportion of negative gastric pressure (Pg) values (p < 0.001). CONCLUSIONS: Therefore, sniff Pdi may be better than Pdi TLC and Pdi PImax in assessing diaphragm function. Also, our subjects seemed to have lower sniff Pdi and Pdi PImax, and higher Pdi TLC compared with Caucasian subjects.

The morphologic change of endothelial cells by ancrod-generated fibrin is triggered by alpha v beta 3 integrin binding and the subsequent activation of a G-protein coupled phospholipase C.

The mechanism of morphologic change of human cultured umbilical vein endothelial cells (HUVECs) caused by fibrin was investigated. Ancrod, a thrombin-like enzyme, did not cause morphologic alteration of HUVEC by itself at concentrations ranging from 0.01 to 10 U/ml. However, when 0.02 U/ml of ancrod was added to cultured HUVEC monolayers in the presence of citrated plasma, it caused pronounced morphologic change of HUVEC after 6-10 h incubation period. Gly-Pro-Arg-Pro (4 mg/ml), an inhibitor of fibrin polymerization, prevented the morphologic alteration, indicating that the morphologic alteration was caused by the polymerized fibrin. The morphologic change of HUVEC caused by ancrod-generated fibrin was not observed in the presence of an intracellular calcium mobilization inhibitor TMB-8 (50 microM), and the morphologic alteration was also less pronounced with BAPTA(15 microM)-loaded HUVECs and HUVECs pretreated with EGTA (1.2 mM). Ancrod (in Medium 199) itself did not stimulate phosphoinositide breakdown of HUVEC. However, when ancrod was present in plasma, it caused an increase of [3H]IP1 of HUVECs preloaded with [3H]myoinositol. This IP1 increment was inhibited by Gly-Pro-Arg-Pro. The increase of IP1 was significantly inhibited by the pretreatment of monoclonal antibodies 23C6 and 7E3 directed against alpha v beta 3 integrin. Neomycin (1 mM) and pertussis toxin (100 ng/ml), but not aspirin or mepacrine, blocked this enhanced phosphoinositide breakdown. The morphologic change was also prevented by the monoclonal antibodies, 23C6 and 7E3. These results suggest that both intra- and extra-cellular calcium participate in the event of morphologic change of HUVEC caused by ancrod-generated fibrin, and the morphologic change is mediated, at least in part, by fibrin binding to integrin alpha v beta 3 on HUVECs, causing the subsequent activation of the endogenous G-protein coupled phospholipase C.

Lung elastic recoil in normal young adult Chinese compared with Caucasians.

Chinese people have smaller total lung capacity (TLC) compared with Caucasians of similar age, sex and height. One possible reason would be a higher lung elastic recoil in Chinese. Most published values for lung elastic recoil viz static lung compliance (CLst), shape constant K, and maximal static transpulmonary pressure (PLmax) have been from Caucasian subjects. The aim of our study was to obtain values for lung elastic recoil in normal young adult Chinese subjects. Static expiratory pressure-volume (P-V) curves were studied in 22 healthy Chinese subjects (12 males and 10 females). The P-V curve was fitted using an iterative least mean squares regression on a computer, according to an exponential equation: V = A-Be-KP, where V is lung volume, P is transpulmonary pressure, and A, B and K are constants. Mean values +/- SD for K, CLst and PLmax were 0.12 +/- 0.04, 230 +/- 103 ml.cmH2O-1 and 27.5 +/- 7.5 cmH2O, respectively. The values of CLst and K were similar to that of normal Caucasian subjects, whereas values of PLmax were lower. We attributed the lower PLmax partly to weaker inspiratory muscles in Chinese compared with Caucasians. We conclude that lung elastic recoil in normal young adult Chinese is similar to that of healthy young adult Caucasians. Hence, lung elasticity is unlikely to explain the racial differences in static lung volumes.

Asthma in chemical workers exposed to aliphatic polyamines.

Following the identification of an index case of occupational asthma, we assessed the respiratory morbidity in 12 workers exposed to aliphatic polyamines in a chemical factory and in 60 unexposed workers in three other workplaces. Many amine workers reported symptoms of chronic cough (7/12), chronic phlegm (9/12), wheezing (4/12) and exertional breathlessness (3/12). These were significantly more frequent in exposed than in unexposed workers (P < 0.01). They also had significantly greater diurnal variation in peak expiratory flow rates (DV-PEFR) than the unexposed workers (P < 0.01). All four amine workers with wheezing reported onset of symptoms after employment at the factory, and all had DV-PEFR greater than 15%, compared to only one control worker (P < 0.01). A second case of occupational asthma was confirmed by bronchial challenge test. The study indicated a very high risk of asthma and chronic airways disease and a need for stringent measures to protect the health of industrial workers exposed to aliphatic amines in Singapore.

Case of occupational asthma due to glue containing cyanoacrylate.

Cyanoacrylates are a rare cause of occupational asthma, there being only six cases reported previously. High humidity has been implicated to be protective in the pathogenesis of the asthma. This is a case report of occupational asthma due to cyanoacrylate glue after working three years in a factory manufacturing doors. Diagnosis was made by history, serial peak flow measurements on and off work and specific inhalation challenge which elicited a delayed bronchoconstrictive response. An unusual feature of this case was the positive reaction to cyanoacrylate despite the high ambient humidity in Singapore.

Mechanisms of apnea termination in obstructive sleep apnea. Role of chemoreceptor and mechanoreceptor stimuli.

Previous work from our laboratory has indicated that mechanoreceptor feedback from the respiratory muscles may play an important role in arousal and apnea termination in obstructive sleep apnea (OSA). Other studies have pointed to a prominent role for chemoreceptor stimuli. We postulated that mechanoreceptor stimuli from the respiratory system are the primary determinant of apnea termination, and that chemoreceptor stimuli exert their effect indirectly through stimulation of ventilation and thus proprioceptive feedback. To test this, we measured the diaphragmatic tension-time index (TTdi) during obstructive sleep apneas in seven male subjects with severe untreated OSA. We compared the maximal TTdi values at end-apnea during administration of air, O2, and CO2. We reasoned that if mechanoreceptor stimuli mediate apnea termination, changing the degree of chemoreceptor stimulation during apneas should not alter the level of respiratory effort at end-apnea. O2 administration produced a significant increase in end-apneic arterial oxygen saturation (SaO2) and increased apnea duration. CO2 administration led to an increase in pre- and postapneic end-tidal carbon dioxide pressure (PETCO2), and tended to shorten apneas. However, the mean value for maximal end-apneic TTdi was 0.12 +/- 0.01 (SEM) during room air breathing and was unaltered by O2 (0.12 +/- 0.01) or CO2 (0.11 +/- 0.01) administration. The consistency of end-apneic TTdi values despite the varying chemical drive supports the hypothesis that apnea termination in OSA is mediated by mechanoreceptor feedback from the respiratory system, most likely from the respiratory muscles. The influence of chemoreceptor information may be mediated indirectly through an effect on ventilatory effort.

Nucleotide sequence and expression of a gentamicin resistance gene isolated from the R plasmid in Serratia marcescens.

The 1.0 kb fragment carrying a gentamicin resistance determinant was subcloned from the R plasmid in clinical isolates of Serratia marcescens and its nucleotide sequence was determined. It contains an open reading frame composed of 858 nucleotides, which shows 97% homology to the aacC2 gene in nucleotide sequence. Minicells carrying the 1.0 kb insert DNA on the plasmids produced a 30 kd protein that was consistent with the expected size of the deduced amino acid sequence from the open reading frame. A search for the promoter revealed a putative promoter of which the -35 region was provided by the right end of Tn3, suggesting that the expression of the gentamicin resistance gene is dependent on the Tn3 sequence.