Optimal expression and purification of sapelovirus A structural protein VP1, and its immunogenicity in mice.

Sapelovirus A (SV-A) is a positive-sense single-stranded RNA virus which is associated with acute diarrhea, pneumonia and reproductive disorders. The virus capsid is composed of four proteins, and the functions of the structural proteins are unclear. In this study, we expressed SV-A structural protein VP1 and studied its antigenicity and immunogenicity. SDS-PAGE analysis revealed that the target gene was expressed at high levels at 0.6 mM concentration of IPTG for 24 h. The mouse polyclonal antibody against SV-A VP1 protein was produced and reached a high antiserum titer (1: 2,048,000). Immunized mice sera with the recombinant SV-A VP1 protein showed specific recognition of purified VP1 protein by western blot assay and could recognize native SV-A VP1 protein in PK-15 cells infected with SV-A by indirect immunofluorescence assay. The successfully purified recombinant protein was able to preserve its antigenic determinants and the generated mouse anti-SV-A VP1 antibodies could recognize native SV-A, which may have the potential to be used to detect SV-A infection in pigs.

[Comparison of molluscicidal cost and effectiveness between 5% niclosamide ethanolamine salt granules and 26% suspension concentrate of metalaldehyde and niclosamide ethanolamine salt].

OBJECTIVE: To compare the molluscicidal effects and cost-effectiveness of 5% niclosamide ethanolamine salt granules (NEG) and 26% suspension concentrate of metalaldehyde and niclosamide ethanolamine salt (MNSC) . METHODS: Two plots with high Oncomelania hupensis snail density were selected as research areas in Nanjing Chemical Industry Zone, and 5% NEG (40 g/m2) and 26% MNSC (40 g/m2) were used by the spraying method for snail control in the two plots, and their molluscicidal effects and cost-effectiveness were investigated and statistically analyzed. RESULTS: There was no significant difference between 5% NEG and 26% MNSC in the molluscicidal effects. The cost of 5% NEG was 1.25 times higher than that of 26% MN-SC per ten thousand square meters in snail control. CONCLUSIONS: The cost of 5% NEG is higher than that of 26% MNSC per ten thousand square meters in snail control. Their molluscicidal effects are similar.

Development of small intestinal enzyme activities and their relationship with some gut regulatory peptides in grazing sheep.

Growth depends on an animal's capacity to digest and assimilate ingested nutrients, and insufficient supply and impairment will constrain lamb growth. Eight groups of Alpine Finewool lambs were harvested on 0, 3, 7, 14, 21, 28, 42, and 56 d to measure pH and enzymatic activities in the duodenum, proximal jejunum, middle jejunum, distal jejunum, and ileum mucosa or digesta. From the duodenum to the ileum the pH of intestinal mucosa and digesta increased, whereas pH changed very little with age. The trypsin, chymotrypsin, lipase, lactase, and α-amylase activities observed at birth decreased by d 3, followed by a nonuniform enzymatic response in the small intestine. The trypsin activity increased from d 3 to peak, at d 21, followed by a decline. Chymotrypsin activity followed the same general trend but with smaller responses in activities. Trypsin demonstrated greater enzymatic activity than chymotrypsin at the same age. The lipase activity of small intestinal mucosa and digesta changed little with age. The lactase activity was high at birth, decreased by d 3, and then increased, followed by a decrease as lambs approached weaning. α-Amylase activity was similar in the small intestinal mucosa and digesta at birth but increased with age for the duodenum and proximal jejunum. Plasma concentrations of cholecystokinin (CCK), secretin, and gastrin were positively correlated ( < 0.05) with ileal mucosa lipase activity. Plasma concentration of CCK, secretin, gastrin, and gastric inhibitory polypeptide (GIP) were positively correlated ( < 0.05) with ileal mucosa lactase activity. Plasma concentration of pancreatic polypeptide (PP) was negatively correlated ( < 0.05) with lactase activity in the middle jejunum and ileal mucosa. Plasma concentrations of CCK, secretin, gastrin, and GIP were positively correlated ( < 0.05) with α-amylase activity in the ileal mucosa but negatively correlated ( < 0.05) with duodenum, prejejunum, and middle jejunum. Plasma PP concentrations were positively correlated ( < 0.01) with α-amylase activity of duodenum, middle jejunum, and postjejunum mucosa but not with the enzyme activity of postjejunum and ileal mucosa ( > 0.05). Small intestinal enzymatic activities exist and may be sufficient to enhance lamb growth via appropriate nutrient supplementation.

Developmental changes in fatty acid-binding protein (H-FABP) mRNA expression and intramuscular fat (IMF) content in Oula sheep.

Increasing meat consumption by Chinese people has created a focus for improving meat quality for increasing quality of life. Twenty-five Male Oula sheep were selected at 2, 21, 56, 84, and 112 d to investigate the developmental changes associated with age on the intramuscular fat (IMF) content of heart fatty acid-binding protein (H-FABP) mRNA expression in muscle. Longissimus dorsal muscle and biceps femoris muscle were sampled to measure IMF concentrations and total mRNA was extracted to measure H-FABP mRNA expression using real-time polymerase chain reaction (PCR). Growing male Oula sheep demonstrated that the IMF concentrations continuously increased with age and significant differences (P < 0.05) were detected among the age groups; 2. The IMF concentrations among tissues were different; 3. The development changes in H-FABP mRNA expression in longissimus dorsal muscle and biceps femoris muscle were similar with a decrease from 2 to 21 d, followed by continuously increasing concentrations being significant different (P < 0.05) among age groups; 4. The H-FABP mRNA expression in the longissimus dorsal muscle tissue was significantly (P < 0.05) higher compared to the biceps femoris muscle; 5. The muscle H-FABP mRNA expression concentration was positively correlated with IMF concentrations from d 21 to 112; 6. The correlation coefficients were significantly (P < 0.01)different between H-FABP gene mRNA expression in the longissimus dorsal muscle and IMF concentration of 0.815 compared to the biceps femoris muscle and IMF concentration of 0.787,which indicated that the H-FABP gene may be affecting the IMF concentrations in the early developmental stages of Oula sheep. These results support the hypothesis that H-FABP gene and its expression in muscle tissue is related to the IMF concentration of meat.

Point-of-care universal screening for meticillin-resistant Staphylococcus aureus: a cluster-randomized cross-over trial.

BACKGROUND: Meticillin-resistant Staphylococcus aureus (MRSA) is frequently endemic in healthcare settings and may be transmitted by person-to-person spread. Asymptomatic MRSA carriers are potential, unsuspected sources for transmission and some of them may be identified by admission screening. AIM: To assess whether rapid point-of-care screening (POCS) for MRSA at hospital admission may be associated with a reduction in MRSA acquisition rates when compared with slower laboratory-based methods. METHODS: A cluster-randomized cross-over trial was conducted in four admission wards of an acute London tertiary care hospital. Polymerase chain reaction-based POCS screening was compared with conventional culture screening. Patients were screened on ward admission and discharge, and the MRSA acquisition rate on the admission wards was calculated as the primary outcome measure. RESULTS: In all, 10,017 patients were included; 4978 in the control arm, 5039 in the POCS arm. The MRSA carriage rate on admission was 1.7%. POCS reduced the median reporting time from 40.4 to 3.7 h (P < 0.001). MRSA was acquired on the admission wards by 23 (0.46%) patients in the control arm and by 24 (0.48%) in the intervention arm, acquisition rates of 5.39 and 4.60 per 1000 days respectively. After taking account of predefined confounding factors, the adjusted incidence rate ratio (IRR) for change in trend for MRSA acquisition was 0.961 (95% confidence interval: 0.766-1.206). The adjusted IRR for step change for MRSA acquisition was 0.98 (0.304-3.162). CONCLUSION: POCS produces a significantly faster result but has no effect on MRSA acquisition on admission wards compared with culture screening. Where compliance with infection prevention and control is high and MRSA carriage is low, POCS has no additional impact on MRSA acquisition rates over the first one to four days of admission compared with conventional culture screening.

Comparison of the GlideScope® videolaryngoscope and the Macintosh laryngoscope for double-lumen tube intubation.

Intubation with a double-lumen tube is important for achieving one-lung ventilation and facilitating thoracic surgery. The GlideScope(®) videolaryngoscope (Verathon Inc., Bothell, WA, USA) is designed to assist tracheal intubation for patients with a difficult airway. We wished to compare the GlideScope and direct laryngoscopy for double-lumen tube intubation. Sixty adult patients requiring a double-lumen tube for thoracic surgery and predicted uncomplicated laryngoscopy were randomly assigned to a direct Macintosh laryngoscopy group (n = 30) or a GlideScope group (n = 30). The mean (SD) duration of intubation was longer in the Macintosh group (62.5 (29.7) s) than in the GlideScope group (45.6 (10.7) s; p = 0.007). There was no difference in the success of the first attempt at intubation (26/30 (87%) and 30/30 (100%) for Macintosh and GlideScope groups, respectively; p = 0.112). The incidence of sore throat and hoarseness was higher in the Macintosh group (18 (60%) and 14 (47%), respectively) than in the GlideScope group (6 (20%) and 4 (13%), respectively; p = 0.003 and 0.004). We conclude that double-lumen tube intubation in patients with predicted normal laryngoscopy is easier using the GlideScope videolaryngoscope than the Macintosh laryngoscope.

Study of carbon nanotube modified biosensor for monitoring total cholesterol in blood.

A carbon nanotube modified biosensor for monitoring total cholesterol in blood was studied. This sensor consists of a carbon working electrode and a reference electrode screen-printed on a polycarbonate substrate. Cholesterol esterase, cholesterol oxidase, peroxidase and potassium ferrocyanide were immobilized on the screen-printed carbon electrodes. Multi-walled carbon nanotubes (MWCN) were added to prompt electron transfer. Experimental results show that the carbon nanotube modified biosensor offers a reliable calibration profile and stable electrochemical properties.

Unusual thickness-dependent heat-capacity anomalies in free-standing hexatic liquid-crystal films.

High-resolution heat-capacity and optical-reflectivity measurements have been conducted near the smectic-A to hexatic-B transition in thin free-standing films of the liquid-crystal compound 64COOBC. We find an unexpected dependence on film thickness of the integrated magnitude of the heat-capacity anomalies as the films undergo layer-by-layer transitions. We measure the penetration depths of the ordering from the surface and next-to-surface layers which are pertinent to the highly correlated thermal behavior.

Unusual thickness-dependent thermal behavior and anticlinic coupling in chiral smectic free-standing liquid-crystal films.

We observe, in free-standing films of a chiral smectic liquid crystal, a series of discrete transitions in the relative orientation of the tilt of the interior and surface layers. These transitions include a remarkable reentrant synclinic-anticlinic-synclinic ordering sequence of the film surfaces in the presence of an electric field upon cooling. The profiles of the associated heat-capacity anomalies are found to be strongly thickness dependent and exhibit a novel crossover behavior in reduced dimensions. We measure the anticlinic coupling between tilted surface layers in the smectic- A phase.

Calcium influx inhibition: possible mechanism of the negative effect of tetrahydropalmatine on left ventricular pressure in isolated rat heart.

The active ingredient dl-tetrahydropalmatine (THP) isolated from the traditional Chinese herb Corydalis racemosa has been found to have antihypertensive effects. However, severe cardiac and neurological toxic effects were reported from using this herb for the treatment of pain. In an isolated perfused rat heart model, THP at the concentration of 100 microM was found to have a negative effect (-45%) on left ventricular pressure and this effect was produced concentration-dependently from concentrations lower than 50 microM. In isolated cardiomyocytes, radioactive calcium influx was also inhibited significantly by THP at the concentration of 100 microM and this effect was also in a concentration-dependent manner (-39%). In a patient with latent heart disease, the use of Corydalis should probably be detrimental, the toxic effect was probably due to calcium influx inhibition.

Immunological, hematological, and biochemical responses in immature white-footed mice following maternal Aroclor 1254 exposure: A possible bioindicator.

A number of hematological, immunological, and biochemical parameters were measured in Peromyscus leucopus pups born from dams exposed to a single dose (300 mg/kg body weight) of Aroclor 1254. To increase the chances of uncovering even modest consequences of the exposure, in one protocol the pups were weaned at 3 weeks and examined at 6 weeks of age, while in a second protocol the pups were kept with their mother for 4 weeks, at which time they were examined. The older pups showed significant decreases in body weight, ratio of spleen weight to body weight, numbers of peripheral white blood cells and lymphocytes, and number and percentage of monocytes. They also showed significant increases in the stimulation index in response to the mitogen phytohemagglutinin (PHA), percentage of peripheral blood neutrophils and liver EROD induction. Pups sacrificed at 4 weeks of age showed even more significant differences. Their body and liver weights, percentage and number of peripheral blood lymphocytes, and serum antibody titers were significantly lower than those of their controls, while spleen to body weight ratios, percent of neutrophils in their peripheral blood, and liver EROD, PROD, and BROD levels were significantly higher than those of the controls. The primary implication of this work is that white-footed mouse pups could be used as biomonitors of contaminated sites. Females could be captured at the sites and bred in captivity with normal males. The vulnerable parameters identified in this study could then be measured in the resulting offspring and compared with a database collected from normal pups.

Hematology values from clinically healthy Peromyscus leucopus.

Reported herein are hematocrit and total and differential WBCs obtained from 132 clinically healthy male and female white-footed mice (Peromyscus leucopus) of four different age groups: 4, 5-8, 9-20, and >32 wk old. Minimal differences were identified between the age groups. The number and percentage of neutrophils in the 4-wk-old mice were significantly higher than those in the other groups; the relative percentage of lymphocytes in the 4-wk-old mice was significantly lower than in the 9-20-wk-old mice. The only significant gender effects identified were higher numbers of WBCs and lymphocytes in females of the 4-wk-old group and higher hematocrits in males of the 5-8- and >32-wk-old groups.

A conventional myosin motor drives neurite outgrowth.

Neuritic outgrowth is a striking example of directed motility, powered through the actions of molecular motors. Members of the myosin superfamily of actin-associated motors have been implicated in this complex process. Although conventional myosin II is known to be present in neurons, where it is localized at the leading edge of growth cones and in the cell cortex close to the plasma membrane, its functional involvement in growth cone motility has remained unproven. Here, we show that antisense oligodeoxyribonucleotides, complementary to a specific isoform of conventional myosin (myosin IIB), attenuate filopodial extension whereas sense and scrambled control oligodeoxyribonucleotides have no effect. Attenuation is shown to be reversible, neurite outgrowth being restored after cessation of the antisense regimen. Myosin IIB mRNA was present during active neurite extension, but levels were minimal in phenotypically rounded cells before neurite outgrowth and message levels decreased during antisense treatment. By contrast, the myosin IIA isoform is shown to be expressed constitutively both before and during neurite outgrowth and throughout exposure to myosin IIB antisense oligodeoxyribonucleotides. These results provide direct evidence that a conventional two-headed myosin is required for growth cone motility and is responsible, at least in part, for driving neuritic process outgrowth.

Mechanisms of hyperproduction of TEM-1 beta-lactamase by clinical isolates of Escherichia coli.

The mechanisms of hyperproduction (defined as > or = 200 nmoles nitrocefin hydrolysed per minute per mg of protein) of TEM-1 beta-lactamase by 38 isolates of Escherichia coli were investigated. The copy numbers of TEM-encoding plasmids were determined for the hyperproducing isolates and for 39 TEM-1-producing isolates that did not hyperproduce the enzyme. Allele-specific PCR was used to determine if the promoter region of the TEM-1 gene was of the TEM-1 or TEM-2 type. Twenty three of the 38 hyperproducers had the TEM-1-type promoter but 15 had the more efficient TEM-2-type promoter; in contrast, 37 of the 39 isolates with lower activities had the TEM-1-type promoter and only two had the TEM-2-type promoter. Many of the TEM-1-hyperproducing isolates possessed small plasmids (< or = 20 MDal) with high copy numbers, in some cases together with large, low copy number plasmids; the average total copy number of TEM-encoding plasmids was 14; if only isolates with the TEM-1-type promoter were included, average total copy number was 22. Hyperproduction was attributed to high copy number (> or = 10) plasmids in 11 isolates; another seven had plasmids with moderately high copy numbers (4-9). The average total copy number for isolates that produced relatively small amounts of TEM-1 beta-lactamase (< or = 100 nmoles/min/mg protein) was 2.2, and for the 12 isolates with TEM-1 activities of 101-200 nmoles/min/mg protein it was 6.8. We conclude that both high copy number plasmids and a more efficient promoter are common causes of hyperproduction of TEM-1.

Incidence and mechanisms of resistance to the combination of amoxicillin and clavulanic acid in Escherichia coli.

Among Escherichia coli organisms isolated at St. Thomas's Hospital during the years 1990 to 1994, the frequency of resistance to amoxicillin-clavulanic acid (tested by disk diffusion in a ratio of 2:1) remained constant at about 5% of patient isolates (10 to 15% of the 41 to 45% that were amoxicillin resistant). Mechanisms of increased resistance were determined for 72 consecutively collected such amoxicillin-clavulanic acid-resistant isolates. MICs of the combination were 16-8 micrograms/ml for 51 (71%) of these and > or = 32-16 micrograms/ml for the remainder. The predominant mechanism was hyperproduction of enzymes isoelectrically cofocusing with TEM-1 (beta-lactamase activities, > 200 nmol of nitrocefin hydrolyzed per min per mg of protein) which was found in 44 isolates (61%); two isolates produced smaller amounts (approximately 150 nmol/min/mg) of such enzymes, and two isolates hyperproduced enzymes cofocusing with TEM-2. Eleven isolates produced enzymes cofocusing with OXA-1 beta-lactamase, which has previously been associated with resistance to amoxicillin-clavulanic acid. Ten isolates produced increased amounts of chromosomal beta-lactamase, and four of these additionally produced TEM-1 or TEM-2. Three isolates produced inhibitor-resistant TEM-group enzymes. In one of the enzymes (pI, 5.4), the amino acid sequence change was Met-67-->Val, and thus the enzyme is identical to TEM-34. Another (pI, 5.4) had the substitution Met-67-->Ile and is identical to IRT-I67, which we propose now be given the designation TEM-40. The third (pI, 5.2) had the substitution Arg-241-->Thr; this enzyme has not been reported previously and should be called TEM-41. The rarity and diversity of inhibitor-resistant TEM-group enzymes suggest that they are the result of spontaneous mutations that have not yet spread.

Effect of hyperproduction of TEM-1 beta-lactamase on in vitro susceptibility of Escherichia coli to beta-lactam antibiotics.

The susceptibility of 173 TEM-1-producing isolates of Escherichia coli was assessed by determination of MICs by the agar dilution method. MICs of amoxicillin, mezlocillin, cephaloridine, and, to a smaller extent, amoxicillin-clavulanic acid (but not cephalexin, cefuroxime, cefotaxime, ceftazidime, or imipenem) were higher for isolates that produced large amounts of beta-lactamase than for isolates that produced smaller amounts. The effect of fixed concentrations of clavulanic acid on resistance to amoxicillin was assessed for 34 selected TEM-1-producing isolates. Low concentrations of the inhibitor (0.5 to 1 microgram/ml) reduced the amoxicillin MICs substantially for almost all the isolates, although the reductions were not sufficient to render any of the isolates amoxicillin susceptible. Higher concentrations of clavulanic acid had progressively greater effects on amoxicillin MICs, but even at 8 micrograms/ml some of the isolates with high beta-lactamase activities remained resistant or only moderately susceptible to amoxicillin. All the isolates were inhibited by clavulanic acid (in the absence of amoxicillin) at concentrations of 16 to 32 micrograms/ml. TEM-1 beta-lactamase activity was inhibited in vitro by clavulanic acid, but not totally, with approximately 2% of the initial activity remaining at 2 micrograms/ml and 0.4% remaining at 8 micrograms/ml. These findings suggest that the amount of beta-lactamase activity is a major determinant of the degree of resistance to several beta-lactam antibiotics and can make the difference between susceptibility and resistance to some compounds, notably the combination of amoxicillin with clavulanic acid.

Developmental toxicity and teratogenicity of trichosanthin, a ribosome-inactivating protein, in mice.

The embryotoxic and teratogenic effects of trichosanthin (TCS), a protein isolated from tubers of Trichosanthes kirilowii (family Cucurbitaceae), were studied both in vivo and in vitro. The protein was administered i.p. to ICR mice on day 8.0 of pregnancy, and the animals were sacrificed 1 day before parturition. The fetuses were fixed and subsequently sectioned. At the highest TCS dose employed (7.5 mg/kg body weight), the viability of fetuses declined to 70.2%, compared with 96.5% in the saline-treated control group. The number of resorbed fetuses increased, and the crown-rump length of the surviving fetuses was reduced. At the doses of 5.0 and 7.5 mg TCS/kg body weight, 2.3% and 9.0%, respectively, of the surviving fetuses were found to be abnormal. The abnormalities observed included exencephaly, micromelia, and short tail. When mouse embryos at the early organogenesis stage were cultured with TCS at a dose of 200 micrograms/ml or above, a significantly larger number of embryos were found to be abnormal as compared with the controls. The abnormalities were observed in the head, trunk, and limb regions. Hence, TCS produced adverse effects on prenatal development both in vivo and in vitro.

Susceptibility of Porphyromonas gingivalis and P. asaccharolytica to the non-oxidative killing mechanisms of human neutrophils.

Neutrophils are essential for host defence against bacterial dental plaque and the pathogenic bacterial species within it, but in anaerobic environments such as the gingival crevice neutrophils can kill bacteria only with non-oxidative microbicidal compounds stored in their granules. Porphyromonas gingivalis W83, a pathogenic plaque species, and the avirulent non-oral type-strain P. asaccharolytica were incubated anaerobically with intact neutrophils and with compounds extracted from normal human neutrophil granules. The killing of bacteria and the inactivation of lysozyme, cathepsin G, elastase, bacterial-permeability increasing factor and defensins by culture supernatants were assayed. P. asaccharolytica but not P. gingivalis was killed under anaerobic conditions by intact neutrophils. P. gingivalis was also resistant to neutrophil granule compounds, its viability being reduced from a mean of 3.3 x 10(6) to 6.1 x 10(4) c.f.u/ml in 60 min by 400 micrograms/ml neutrophil granule extract, as compared to a reduction from 4.4 x 10(6) to 2.3 x 10(3) c.f.u/ml for P. asaccharolytica. P. gingivalis culture supernatant inactivated cathepsin G, elastase, bacterial-permeability increasing factor and defensins. Resistance to neutrophil non-oxidative killing mechanisms may be an important virulence factor for P. gingivalis.

Response of chemostat cultures of Pseudomonas aeruginosa to carbapenems and other beta-lactams.

Cultures of Pseudomonas aeruginosa were grown at different rates in a chemostat and challenged continuously or intermittently with ceftazidime, imipenem, meropenem or piperacillin. The killing rate was related to the bacterial growth rate; fast-growing cells being killed more rapidly than slow-growing ones. Mutants that were stably derepressed (i.e. constitutive) for chromosomal beta-lactamase expression were selected when a beta-lactamase inducible (i.e. typical) strain was challenged with ceftazidime or piperacillin. Addition of the beta-lactamase inhibitor tazobactam to piperacillin did not prevent selection. There was a lag of c. 24-48 h post-challenge before totally derepressed mutants were detectable. Once selected, the derepressed organisms were stable and were not outgrown by inducible cells if these were readded in the absence of selective antibiotics. Selection of resistant mutants was not observed with imipenem, despite the known tendency of this drug to select carbapenem-impermeable mutants of P. aeruginosa in vivo. Imipenem, but not ceftazidime or meropenem, caused a significant post-antibiotic effect after single or repeated dosage.