Biomedical subjects
Publications and source records attributed to K J Cheng.
Two 3(2H)-benzofuranones and three chromanes were isolated from the mycoparasitic fungus Coniothyrium minitans. Their structures and absolute stereochemistry were determined by spectroscopic methods.
A cellulase from the ruminal fungus Orpinomyces joyonii cloned in Escherichia coli was purified 88-fold by chromatography on High Q and hydroxyapatite. N-terminal amino acid sequence analyses confirmed that the cellulase represented the product of the cellulase gene Cel B2. The purified enzyme possessed high activity toward barley beta-glucan, lichenan, carboxymethyl cellulose (CMC), xylan, but not toward laminarin and pachyman. In addition, the cloned enzyme was able to hydrolyze p-nitrophenyl (PNP)-cellobioside, PNP-cellotrioside, PNP-cellotetraoside, PNP-cellopentaoside, but not PNP-glucopyranoside. The specific activity of the cloned enzyme on barley beta-glucan was 297 units/mg protein. The purified enzyme appeared as a single band in SDS-polyacrylamide gel electrophoresis and the molecular mass of this enzyme (58000) was consistent with the value (56463) calculated from the DNA sequence. The optimal pH of the enzyme was 5.5, and the enzyme was stable between pH 5.0 and pH 7.5. The enzyme had a temperature optimum at 40 degrees C. The K(m) values estimated for barley beta-glucan and CMC were 0.32 and 0.50 mg/ml, respectively.
The effects of protozoa on the degradation of plant cell walls (CW) during different growth stages of the fungus Anaeromyces mucronatus have been investigated. Since fungi show a marked lag in their in vitro cultures and many protozoa rapidly die during a prolonged incubation time, the effects of protozoa may vary according to the growth phase of the fungi. Therefore, the approach adopted was (i) to inoculate CW with fungus monoculture, (ii) to inoculate CW with fungus-protozoa coculture, or (iii) to sequentially inoculate fungal cultures that had been grown in CW for 24 (initial stage of growth), 48, and 72 h (late stage of growth) with mixed protozoa. When a fungus was associated with protozoa, a growth phase dependent effect was observed. Ruminal protozoa adversely affected the growth and activity when introduced in the initial growth stage of A. mucronatus, but a synergetic interaction was detected when added to late growth stage cultures. Although there is no immediate explanation for these results, the data suggested that protozoa can engulf the fungal zoospores, which are in ruminal fluids and (or) attached to small feed particles, but cannot engulf the fungal thallus that is tightly attached to feed particles by a rhizoidal system. Our data indicated that the protozoa did not influence cellulolysis by the fungi in exponential and (or) stationary phase, but they had a marked inhibitory effect on fungi that were in lag phase. Inhibition during lag phase could result from the protozoal predation of fungal zoospores that had failed to attach to substrates.
The catalytic domain of a xylanase from the anaerobic fungus Neocallimastix patriciarum was made more alkalophilic through directed evolution using error-prone PCR. Transformants expressing the alkalophilic variant xylanases produced larger clear zones when overlaid with high pH, xylan-containing agar. Eight amino acid substitutions were identified in six selected mutant xylanases. Whereas the wild-type xylanase exhibited no activity at pH 8.5, the relative and specific activities of the six mutants were higher at pH 8.5 than at pH 6.0. Seven of the eight amino acid substitutions were assembled in one enzyme (xyn-CDBFV) by site-directed mutagenesis. Some or all of the seven mutations exerted positive and possibly synergistic effects on the alkalophilicity of the enzyme. The resulting composite mutant xylanase retained a greater proportion of its activity than did the wild type at pH above 7.0, maintaining 25% of its activity at pH 9.0, and its retention of activity at acid pH was no lower than that of the wild type. The composite xylanase (xyn-CDBFV) had a relatively high specific activity of 10128 micromol glucose x min(-1) x (mg protein)(-1) at pH 6.0. It was more thermostable at 60 degrees C and alkaline tolerant at pH 10.0 than the wild-type xylanase. These properties suggest that the composite mutant xylanase is a promising and suitable candidate for paper pulp bio-bleaching.
OBJECTIVE: To determine the effect of a clinic-based literacy intervention on the language development of preschool children. METHODS: A convenience sample of families presenting to 2 urban pediatric clinics for well-child care met the following criteria: the family was Latino or black and English- or Spanish-speaking; the child was 2 to 5.9 years old, with no neurodevelopmental disability, at a gestational age of 34 weeks or more, and not attending kindergarten. Participants at the first clinic (intervention group) were exposed to a literacy support program, based on Reach Out and Read (ROR), during the previous 3 years. At the second clinic (comparison group), a similar program started 3 months before the study. Parent-child reading activities were measured using the READ Subscale of the StimQ. Language development was measured using the One-Word Expressive and Receptive Picture Vocabulary Tests, and was performed in the child's primary language. RESULTS: A total of 122 study participants (49 interventions and 73 comparisons) met inclusion criteria and completed all measures. Intervention and comparison families were similar for most sociodemographic variables. Intervention families reported reading together with their children approximately 1 more day per week. Intensity of exposure to ROR (measured by total number of contacts with the program) was associated with increased parent-child reading activities, as measured by the StimQ-Read Subscale (r = 0.20). Intervention children had higher receptive language (mean: 94.5 vs 84.8) and expressive language (mean: 84.3 vs 81.6). After adjusting for potential confounders in a multiple regression analysis, intervention status was associated with an 8.6-point increase (95% confidence interval [CI]: 3.3, 14.0) in receptive language (semipartial correlation [SR]coefficient = 0.27), and a 4.3-point increase (95% CI: 0.04, 8.6) in expressive language (SR = 0.17). In a similar multiple regression, each contact with ROR was associated with an adjusted mean 0.4-point increase (95% CI: 0.1, 0.6) in receptive score, and an adjusted mean 0.21-point increase (95% CI: 0. 02, 0.4) in expressive score. CONCLUSIONS: ROR is an important intervention, promoting parental literacy support and enhancing language development in impoverished preschool children. Integration of literacy promoting interventions such as these into routine pediatric health care for underserved populations can be recommended.
Experimentation at the Lethbridge Research Center in Alberta, Canada using cross-polarized transmitted light to photographically record a staining technique on zymograms has proved to be successful with both color and black and white films. It has been possible to obtain the desired visible contrast without compromising the intensity of the enzyme activity bands. Increasing numbers of such PAGE gels are being submitted for photographic recording when it is believed that the image will be used for records, publication, scientific posters or AV presentations.
The localization of phytase (myo-inositol-hexaphosphate phosphohydrolase) in the ruminal bacteria, Selenomonas ruminantium JY35 and Mitsuokella multiacidus 46/5(2), was determined with transmission electron microscopy. Phosphate produced from the enzymatic dephosphorylation of the calcium salt of phytic acid is precipitated as calcium phosphate. The calcium is then replaced with lead to produce electron-dense lead phosphate. This deposition of lead phosphate localized phytase in S. ruminantium JY35 and M. multiacidus 46/5(2) to the outer membrane, and confirmed intracellular expression of the enzyme in Escherichia coli pSrP.2, the recombinant clone which possesses the gene (phyA) encoding phytase (phyA) in S. ruminantium.
The effects of supplying increasing ruminal doses of exogenous polysaccharide-degrading enzymes (EPDE) on rumen fermentation and nutrient digestion were studied using eight ruminally cannulated heifers, four of which were also duodenally cannulated, in a replicated Latin square. The heifers were fed a diet of 85.5% rolled barley grain and 14% barley silage (DM basis), and once daily they were given intraruminal doses of 0 (Control), 100, 200, or 400 g of a preparation containing polysaccharide-degrading enzymes. Enzyme treatment decreased ruminal pH (linear, P<.001) and increased ammonia N (quadratic, P<.001) concentration. The ruminally soluble fraction and effective degradability of feed DM in situ were increased (quadratic response, P<.001) by enzyme treatment. Ruminal administration of EPDE increased ruminal fluid carboxymethylcellulase and xylanase activities linearly (P<.001) and beta-glucanase activity quadratically (P<.01), decreased (quadratic response, P<.05) ruminal fluid viscosity, and did not affect (P>.05) ruminal fluid amylase activity. Elevated levels of fibrolytic activities in the rumen resulted in increased (quadratic, P<.001) carboxymethylcellulase, xylanase, and beta-glucanase (P<.01) activities in duodenal digesta. Duodenal amylase activity and reducing sugar concentration were also increased (quadratic responses, P<.001 and P<.05, respectively) by EPDE. Xylanase activity of fecal DM was increased linearly (P<.05) with increasing ruminal EPDE levels. Apparent digestibilities of DM, crude protein, and NDF were not affected by EPDE supplementation. Enzyme treatment did not affect (P>.05) urinary excretion of allantoin and uric acid, or concentrations of glucose and urea in blood.
Microbial enzymes extracted from mixed ruminal microorganisms were incubated for 2 h with casein and Tween 60 or Tween 80 at 10 concentrations ranging from 0 to 2.0% (vol/vol) to determine the effects of these nonionic surfactants on protease activation and thiol reactivity (unmasking of thiol groups). Rate and extent of protein adsorption to cellulosic substrate (barley straw) was measured in the presence of 0, 0.05, 0.10, 0.25, and 0.50% (vol/vol) Tween 80. Degradation of cellulose by a rumen bacterial fraction was measured over 48 h of incubation with and without Tween 60 or Tween 80 at 0.25% (vol/vol). Maximum accelerations of protease activity achievable with Tween 60 and Tween 80 (calculated from a Michaelis-Menten kinetics model) were 99.2 and 166.8%, respectively. Concentrations of Tween 60 and Tween 80 at which half the maximal velocities were attained were 0.28 and 0.20% (vol/vol), respectively. Tween 80 increased (P < 0.05) the rate and extent of adsorption of microbial protein to barley straw, and the effect was related to concentration of Tween 80 up to 0.10% (vol/vol). Initial rates of cellulose degradation with no surfactant, 0.25% Tween 60, or 0.25% Tween 80 were 0.60, 0.87, and 1.04 micrograms/ml per h, respectively. These nonionic surfactants were effective for enhancing rumen microbial protease and cellulase activities. Thus, further study is warranted to determine their potential for improving ruminant feeding.
OBJECTIVE: To assess the factors that increase or decrease the risk of pneumonia with particular attention to immunization with pneumococcal and influenza vaccines in a group of HIV-infected persons. DESIGN: A retrospective, case-control study based on information entered into a standard database and the medical record. SETTING: Patients attending a referral clinic specializing in AIDS/HIV care at a public hospital. PATIENTS: Among over 2000 subjects entered into a database in 8 years, 127 incidents of pneumonia were identified from the record. These cases were matched with 127 CD4 cell count matched, concurrent controls. INTERVENTIONS: None. MAIN OUTCOME MEASURE: The principal hypothesis was that chart review would find a decreased frequency of pneumococcal immunization in the pneumonia cases compared with matched controls. RESULTS: Pneumococcal immunization was associated with a reduction of the risk of pneumonia by nearly 70%. The effect was seen even when immunization was given with a CD4 cell count of less than 100/mm3. Injection drug users and African-Americans had a twofold increased risk of pneumonia. CONCLUSION: The study provides data to support the current recommendation for pneumococcal immunization of all HIV-infected persons. Although this conclusion could lead to renewed enthusiasm for increasing pneumococcal immunization rates in HIV-infected persons, it must be recognized that the study is observational and ascertainment bias cannot be excluded.
This paper reports the isolation and characterization from bovine milk of two proteins: angiogenin-1, a recently discovered angiogenin, and lactogenin, a novel protein. Both proteins were adsorbed on and eluted closely from CM-Sepharose and Mono S. Lactogenin possessed a molecular weight (17 kDa) slightly higher than that of angiogenin-1 (15 kDa). Lactogenin had a higher ribonucleolytic (RNase) activity than angiogenin-1 towards yeast transfer RNA (tRNA). The Km values estimated for the RNase activities of angiogenin-1 and lactogenin were 51 microM and 40 microM respectively. Both were specific for poly C. The optimal pH for the RNase activities of angiogenin-1 and lactogenin was 7.75 and 7.5 respectively. Comparison of the amino acid sequences of cyanogen bromide fragments and the pyroglutaminase-treated N-terminal fragment of lactogenin with the sequence of bovine liver RNase (RNase BL4) revealed identity in residues 3-22, 24, 26-27, 37, 41-44, 46-50, 54, 56, 63, 72-80, and 83. Considerable similarity to the N-terminal sequence of angiogenin-2 was also noted. Both lactogenin and angiogenin-1 inhibited cell-free translation in a rabbit reticulocyte lysate system with an IC(50) below 100 nM.
Differential agar media for the detection of microbial phytase activity use the disappearance of precipitated calcium or sodium phytate as an indication of enzyme activity. When this technique was applied to the study of ruminal bacteria, it became apparent that the method was unable to differentiate between phytase activity and acid production. Strong positive reactions (zones of clearing around microbial colonies) observed for acid producing, anaerobic bacteria, such as Streptococcus bovis, were not corroborated by subsequent quantitative assays. Experimentation revealed that acidic solutions generated false positive results on the selected differential medium. Empirical studies undertaken to find a solution to this limitation determined the false positive results could be eliminated through a two step counterstaining treatment (cobalt chloride and ammonium molybdate/ammonium vanadate) which reprecipitates acid solubilized phytate. This report discusses the application of the developed two step counterstaining treatment for the screening of phytase producing ruminal bacteria as well as its use in phytase zymogram assays.
The growth and survival of Stomoxys calcitrans (L.) larvae on egg yolk medium inoculated with bacteria isolated from a colony of stable flies was evaluated. Five species of bacteria--Acinetobacter sp., Aeromonas sp., Empedobacter breve (Holmes & Owen), Flavobacterium odoratum Stutzer, and Serratia marcescens Bizio--were identified according to fatty acid profiles using a microbial identification system. Larvae failed to develop on uninoculated plates, confirming that bacteria are required to complete development. Larvae also failed to complete development on plates inoculated with Aeromonas sp. and S. marcescens, and died during the 1st instar. Larvae completed development on the remaining 3 bacterial species as well as on Escherichia coli (Migula). Survival was generally higher when larvae were reared on Acinetobacter sp. and F. odoratum compared with E. coli and E. breve. Egg density did not influence larval survival, although the variability in survival was lowest using 20 and 40 eggs per plate. Larval survival in mixed cultures of Acinetobacter and Flavobacterium averaged 22.7% lower than survival in the pure cultures, and averaged 21.6% higher in mixed cultures of Empedobacter and Flavobacterium compared with pure cultures. Larval survival in mixed cultures did not differ significantly from mean survival in pure cultures for combinations of Acinetobacter and E. coli, Acinetobacter and Empedobacter, E. coli and Empedobacter, and E. coli and Flavobacterium. Larval developmental time was faster on all mixed bacterial cultures compared with developmental time on pure bacterial cultures. Optimal sample sizes and egg numbers are presented for detecting specified differences in larval survival. This rearing procedure will be useful for studying insect-microbe interactions and evaluating mortality using bacterial agents.
A xylanase gene (xynC) isolated from the anaerobic ruminal fungus Neocallimastix patriciarum was characterized. The gene consists of an N-terminal catalytic domain that exhibited homology to family 11 of glycosyl hydrolases, a C-terminal cellulose binding domain (CBD) and a putative dockerin domain in between. Each domain was linked by a short linker domain rich in proline and alanine. Deletion analysis demonstrated that the CBD was essential for optimal xylanase activity of the enzyme, while the putative dockerin domain may not be required for enzyme function.
In a replicated 3 x 3 Latin square experiment, six heifers (443 +/- 6.1 kg) fed a 61% barley grain:39% alfalfa silage diet (DM basis) were given intraruminal doses of powdered Yucca schidigera (YS). Doses of 0 (control), 20, or 60 g/d were given at 0800 daily. Ruminal content was sampled 0, 2, 4, and 6 h after dosing. Acidity, concentrations of reducing sugars, free amino acids, and peptides in the rumen were not affected (P > .05) by YS. Relative to control, ruminal ammonia concentration was reduced (P < .05) 2 h after YS dosing. Ruminal propionate concentration was increased (P < .05) by YS. Protozoal numbers in the rumen were lower (P < .05) with YS than without. Yucca did not affect (P > .05) rate or extent of in situ DM degradability. Fibrolytic, amylolytic, and proteolytic activities in ruminal contents were similar among treatments (P > .05). Dry matter intake, apparent digestibilities of DM, NDF, and CP, nitrogen balance, and microbial protein synthesis in the rumen were not affected (P > .05) by treatment. The effect of YS on ruminal ammonia concentration likely resulted from a decreased concentration of protozoa and, presumably, from ammonia binding by YS. The effect on ruminal propionate was probably a result of a selective inhibitory effect of YS on rumen microbial species.
Ruminal and total tract digestion of the amino acids (AA) in full fat canola seed was studied in two in situ experiments with three nonlactating, ruminally and duodenally fistulated dairy cows. Whole, full fat canola seed was hand-cracked or micronized (an infrared heat treatment) for 90 s and then studied in that form or after grinding to pass a 1.25-mm sieve. In the first experiment, the four sample types were ruminally incubated in nylon bags for up to 96 h. In the second experiment, they were sealed in mobile nylon bags, incubated ruminally for 16 h, placed in acidified pepsin for 1 h, and then inserted into duodenal cannulas for passage through the intestine. Amino acids in the canola seed and in the residues from in situ incubations were analyzed by HPLC. Micronization reduced ruminal disappearance of total AA and essential AA from full fat canola seed. Degradation kinetics from Experiment 1 indicated reduced soluble fraction and increased slowly degradable fraction of both total AA and essential AA following micronization. Micronization reduced disappearances of total AA and essential AA from whole canola seed in the total digestive tract but did not affect total tract digestion of total AA or essential AA in ground seed. Intestinal disappearance of total AA and essential AA from both whole and ground full fat canola seed were increased by micronization. Micronizing canola seed may be of value in improving AA utilization in ruminants.
Ruminal lactic acid-producing bacteria were selectively isolated and enumerated using a one hour aerobic exposure prior to incubation on a semi-selective Lactobacillus medium, MRS, under anaerobic conditions. The technique allowed growth of pure cultures of ruminal Lactobacillus spp. and Streptococcus bovis without supporting the growth of pure cultures of any of the prominent ruminal bacterial species. In mixed cultures, the one hour aerobic pre-incubation inhibited the growth of the obligate anaerobic ruminal bacteria which can otherwise grow on the MRS medium, and the subsequent anaerobic incubation permitted maximal recovery of the weakly aerotolerant ruminal lactic acid-producing Lactobacillus spp. and Streptococcus spp. The efficacy of this technique in selecting exclusively for the lactic acid-producing bacteria was also demonstrated from populations of rumen bacteria from mixed culture end-point in vitro fermentation, continuous in vitro culture and isolations from fresh ruminal samples.
Phytase catalyses the release of phosphate from phytate (myo-inositol hexakisphosphate), the predominant form of phosphorus in cereal grains, oilseeds and legumes. The presence of phytase activity was investigated in 334 strains of 22 species of obligately anaerobic ruminal bacteria. Measurable activities were demonstrated in strains of Selenomonas ruminantium, Megasphaera elsdenii, Prevotella ruminicola, Mitsuokella multiacidus and Treponema spp. Strains isolated from fermentations with cereal grains proved to have high activity, and activity was particularly prevalent in S. ruminantium, with over 96% of the tested strains being positive. The measured phytase activity was found exclusively associated with the bacterial cells and was produced in the presence of approximately 14 mM phosphate. The most highly active strains were all S. ruminantium, with the exception of the one Mitsuokella multiacidus strain examined. Phytase activity varied greatly among positive strains but activities as high as 703 nmol phosphate released (ml culture)-1 were measured for a S. ruminantium strain and 387 nmol phosphate released (ml culture)-1 for the Mitsuokella multiacidus strain.