Klotho and smoking--An interplay influencing the skeletal muscle function deficits that occur in COPD.

BACKGROUND: Klotho is an 'anti-ageing' hormone and transmembrane protein; Klotho deficient mice develop a similar ageing phenotype to smokers including emphysema and muscle wasting. The objective of this study was to evaluate skeletal muscle and circulating Klotho protein in smokers and COPD patients and to relate Klotho levels to relevant skeletal muscle parameters. We sought to validate our findings by undertaking complimentary murine studies. METHODS: Fat free mass, quadriceps strength and spirometry were measured in 87 participants (61 COPD, 13 'healthy smokers' and 13 never smoking controls) in whom serum and quadriceps Klotho protein levels were also measured. Immunohistochemistry was performed to demonstrate the location of Klotho protein in human skeletal muscle and in mouse skeletal muscle in which regeneration was occurring following injury induced by electroporation. In a separate study, gastrocnemius Klotho protein was measured in mice exposed to 77 weeks of smoke or sham air. RESULTS: Quadriceps Klotho levels were lower in those currently smoking (p = 0.01), irrespective of spirometry, but were not lower in patients with COPD. A regression analysis identified current smoking status as the only independent variable associated with human quadriceps Klotho levels, an observation supported by the finding that smoke exposed mice had lower gastrocnemius Klotho levels than sham exposed mice (p = 0.005). Quadriceps Klotho levels related to local oxidative stress but were paradoxically higher in patients with established muscle wasting or weakness; the unexpected relationship with low fat free mass was the only independent association. Within locomotor muscle, Klotho localized to the plasma membrane and to centralized nuclei in humans and in mice with induced muscle damage. Serum Klotho had an independent association with quadriceps strength but did not relate to quadriceps Klotho levels or to spirometric parameters. CONCLUSIONS: Klotho is expressed in skeletal muscle and levels are reduced by smoking. Despite this, quadriceps Klotho protein expression in those with established disease appears complex as levels were paradoxically elevated in COPD patients with established muscle wasting. Whilst serum Klotho levels were not reduced in smokers or COPD patients and were not associated with quadriceps Klotho protein, they did relate to quadriceps strength.

Structure of the carboxy-terminal PH domain of pleckstrin at 2.1 Angstroms.

Pleckstrin is an important intracellular protein involved in the phosphoinositide-signalling pathways of platelet activation. This protein contains both N- and C-terminal pleckstrin-homology (PH) domains (N-PH and C-PH). The crystal structure of C-PH was solved by molecular replacement and refined at 2.1 Angstroms resolution. Two molecules were observed within the asymmetric unit and it is proposed that the resulting dimer interface could contribute to the previously observed oligomerization of pleckstrin in resting platelets. Structural comparisons between the phosphoinositide-binding loops of the C-PH crystal structure and the PH domains of DAPP1 and TAPP1, the N-terminal PH domain of pleckstrin and a recently described solution structure of C-PH are presented and discussed.

Escherichia coli O157:H7 diarrhoea associated with well water and infected cattle on an Ontario farm.

A 16-month old female child living on an Ontario dairy farm was taken to hospital suffering from bloody diarrhoea. Escherichia coli O157:H7 was isolated from her stool. Initial tests of well water samples were negative for E. coli by standard methods but culture of selected coliform colonies on sorbitol-MacConkey agar led to isolation of E. coli O157:H7. E. coli O157:H7 was also isolated from 63% of cattle on the farm. The E. coli O157:H7 isolates from the child, the water and the cattle were phage type 14, produced verotoxins 1 and 2, and were highly related on analysis by pulsed field gel electrophoresis. The child did not have known direct contact with the cattle and did not consume unpasteurized milk. Hydrogeological investigation revealed the design and location of the well would allow manure-contaminated surface water to flow into the well. This investigation demonstrates that cattle farm well water is a potential source of E. coli O157:H7 which may not be identified by standard screening for E. coli in water.

Modified procedure for recovery of Giardia cysts from diverse water sources.

A compact battery-operated, portable field filtration apparatus was developed for efficient recovery of parasites, including Giardia lamblia cysts, from large volumes of water. In conjunction with modified filter elution and processing procedures, the filtration device was evaluated in field trials. To monitor performance of the procedure, a known number of Giardia lambia cysts were added during filtrations through a control injection assembly. This allowed calculation of cyst recovery at each test site, thereby allowing comparison of percentage recoveries from many diverse water sources. This apparatus and modified method now provide the tools to investigate further extraction and purification techniques to improve overall yield of Giardia lamblia, other parasites and potentially other organisms such as bacteria and viruses.

Bacillus cereus and Bacillus thuringiensis isolated in a gastroenteritis outbreak investigation.

During investigation of a gastroenteritis outbreak in a chronic care institution, Norwalk virus was found in stool specimens from two individuals and bacterial isolates presumptively identified as Bacillus cereus were isolated from four individuals (including one with Norwalk virus) and spice. Phage typing confirmed all Bacillus clinical isolates were phage type 2. All clinical isolates were subsequently identified as B. thuringiensis when tested as a result of a related study (L. Leroux, personal communication). Eight of 10 spice isolates were phage type 4. All B. cereus and B. thuringiensis isolates showed cytotoxic effects characteristic of enterotoxin-producing B. cereus. An additional 20 isolates each of B. cereus and B. thuringiensis from other sources were tested for cytotoxicity. With the exception of one B. cereus, all showed characteristic cytotoxic patterns.

Adrenal insufficiency in a man with non-classical 21-hydroxylase deficiency: consequence or coincidence?

Deficiency of the adrenal enzyme 21-hydroxylase, which is required for cortisol synthesis, appears in two forms: a rare classical variant with severe enzyme deficiency, usually presenting in neonates with ambiguous genitalia (from androgen overproduction) or adrenal crisis (from glucocorticoid and mineralocorticoid underproduction), and a common (1% of the general population) non-classical variant with mild enzyme deficiency, usually presenting in young adults with findings of androgen excess but without clinical evidence of decreased steroid hormone production. We describe a 22-year-old man who had clinical and biochemical findings consistent with adrenal insufficiency, including a favorable response to hydrocortisone replacement, in whom elevated serum levels of the cortisol precursor 17-hydroxyprogesterone were diagnostic of non-classical 21-hydroxylase deficiency and in whom no other cause of adrenal insufficiency could be identified. These findings raise the possibility that non-classical 21-hydroxylase deficiency, an extremely frequent disorder which is generally thought to be without significant morbidity, might cause or contribute to adrenal insufficiency in adults.

Rapid screening test for enterotoxin-producing Bacillus cereus.

Culture supernatants of 30 enterotoxin-producing Bacillus cereus isolates produced a characteristic progressive destruction of McCoy cell monolayers. Enterotoxin-negative B. cereus and other group 1 Bacillus spp. caused no monolayer disruption. The McCoy cell tissue culture system appears to provide a rapid screening assay for detection of enterotoxin-producing B. cereus.

Multiple typing techniques applied to a Clostridium perfringens food poisoning outbreak.

Twenty-one stool specimens obtained from persons implicated in two food poisoning outbreaks at the same institution in Smith Falls, Ontario, were examined for Clostridium perfringens. Ninety-two colonies of Cl. perfringens (3-5 per stool specimen) were typed with antisera, bacteriocins and by plasmid analysis. They were also tested for the in vitro production of bacteriocin and enterotoxin. Sixteen of the 21 stool specimens were tested directly for enterotoxin. This was detected in 13, five of which were from individuals listed as 'asymptomatic' food handlers. The predominant strain isolated from 15 of the 21 stool samples produced bacteriocin and enterotoxin in vitro, contained no plasmids, and was of a common bacteriocin type and serotype.

Symposium on microbiology update: old friends and new enemies. Bacillus cereus.

Bacillus cereus is an environmentally ubiquitous, Gram-positive, spore-forming bacillus responsible for 2 distinct foodborne disease syndromes as well as other manifestations of pathogenicity. The rapid-onset, "emetic," foodborne-disease syndrome is associated with an emetic toxin; the delayed-onset, "diarrheal" syndrome is associated with elaboration of enterotoxin. The majority of methods for detection of these toxins have relied on in vivo testing. More recent work on purification of enterotoxin facilitated the development of a rapid, specific, fluorescent immunodot assay and a tissue culture screening assay for enterotoxin. Work on characterization and detection of emetic toxin is ongoing.

The effect of exercise on bone growth and development in weanling horses.

Nineteen weanling horses (average age = 147 d) were divided into exercised (EX; n = 10) and nonexercised (NEX; n = 9) groups, with age, sex and breed represented as equally as possible. The EX group was exercised on an automatic walker at a medium trot for up to 20 min, 5 d each week. Both groups were fed to meet 100% of their protein and 110% of their energy requirements (NRC, 1978). The EX group's diet was supplemented, on exercise days, with corn starch to meet the additional energy requirements for exercise. The experiment was conducted over a 111-d period. Body weight was measured at 10-d intervals, and height at the withers and metacarpal circumference was measured at 20-d intervals. Radiographs of the distal radius, metacarpal joint and the proximal and distal ends of the third metacarpal were taken at 147, 218 and 255 d of age to determine bone density and to observe any possible bone abnormalities. There were no differences between groups in weight or wither height gain; however, gain in third metacarpal circumference was greater (P less than .01) in the EX group than in the NEX group from 167 to 215 d of age. Bone density in the EX group increased by a greater amount (P less than .06) than in the NEX group by the end of the trial. There were no lameness problems or bone abnormalities observed in either group. Exercise training of horses during the weanling to yearling age period was shown to improve the stress-bearing characteristics (radiographic bone density and metacarpal circumference) of the third metacarpal without affecting the quantity of body growth.

Development of a fluorescent immunodot assay for Bacillus cereus enterotoxin.

A fluorescent immunodot assay has been developed for rapid, specific detection of B. cereus enterotoxin. None of the other Bacillus species tested showed cross-reactivity in the assay with antiserum to purified B. cereus enterotoxin. The assay can detect greater than or equal to 50 ng of purified enterotoxin. Using this assay system, enterotoxin was found to be produced by 25 of 25 foodborne disease-related isolates and 22 of 25 isolates not related to foodborne disease (isolates from routine surveillance foods). Because of the apparent widespread ability of isolates to produce enterotoxin the assay may have potential as a rapid identification procedure for B. cereus. The substrate gel system described may have wider application in other immunoassay systems using a membrane solid phase.

Rapid fluorogenic assay for differentiation of the Candida parapsilosis group from other Candida spp.

A 30-min fluorogenic test was developed for differentiation of members of the Candida parapsilosis group from other Candida species commonly encountered in clinical material. The fluorogenic substrate, 4-methylumbelliferyl-beta-D-glucoside, was utilized to assay beta-glucosidase activity. A total of 50 C. parapsilosis isolates and 135 isolates of four other Candida species were tested. Assay sensitivity and specificity were 100 and 99.3%, respectively. The procedure was adapted for use with a spectrofluorometer.

The influence of high planes of nutrition on skeletal growth and development of weanling horses.

Effects of high dietary levels of energy, protein and Ca and deficient levels of Ca on skeletal growth and development in the equine were studied in two experiments. The weanlings were fed grain-based diets. In Exp. 1, Group 1 (Ctl) received all nutrients at NRC recommended levels for growth; Group 2 (HE) received 150% of their digestible energy (DE) requirement; Group 3 (LC) received 150% of DE and 35% of Ca requirements. In Exp. 2, Group 1 (Ctl) again received all nutrients at NRC recommended levels for growth; Group 2 (HEP) received 150% of DE and 275% of CP requirements; Group 3 (HEPC) received 150% of DE, 275% of CP and 500% of Ca requirements. In addition, all groups had access to pasture. The HE treatment resulted in greater (P less than .10) increases in BW and total cortical width. However, LC weanlings had lower (P less than .10) gains in third metacarpal length and radiographic bone density. Cumulative increases in BW and wither height were greater (P less than .10) for the HEP and HEPC weanlings than for the Ctl weanlings. Increases in third metatarsal length also were greater (P less than .10) for HEP weanlings than for Ctl weanlings. Higher planes of nutrition increased body weight gains and growth rate of several long bones. However, skeletal development (e.g., cortical area) may be compromised by a high rate of growth.

The influence of supplemental feed on growth and bone development of nursing foals.

Thirty foals of mixed breeding, from two consecutive years, were used in two 120-d experiments to evaluate the effects of supplemental feeding (creep feed) on growth in nursing foals. At 10 d postpartum, foals were randomly assigned either to a creep-fed group (CF) or an unsupplemented group (NCF). Initial measurements of body weight (BW), height at the withers (WH), third metatarsal length (MtIII) and third metacarpal length (McIII) were made at 10 d of age and at 30-d intervals thereafter. Medial and lateral cortical peak values for radiographic bone density, cortical width and cortical area at the midpoint of the third metacarpal were used to assess bone quality. Creep feed containing National Research Council (NRC, 1978) recommended levels for all nutrients was offered at 1.5% of BW per day. When compared with unsupplemented foals, supplemented foals had greater gains in BW (P less than .05), in WH (P less than .10) and in MtIII (P less than .05). Mean gains during the trial in BW, WH, MtIII and McIII for the CF foals were 133.3 kg, 22.8 cm, 2.1 cm and 1.9 cm and for the NCF foals were 117.6 kg, 21.2 cm, 1.5 cm and 1.9 cm, respectively. The mean value for the lateral cortical peak was slightly lower (P less than .10) for the CF foals. No differences (P greater than .10) were observed for the medial cortical peak, cortical width or cortical area of the third metacarpal. The results of this study indicate that a creep feeding program that supplies NRC-recommended nutrient levels can increase the rate of skeletal growth with little decrease in quality of bone.

Evaluation of the diagnostic application of an enzyme immunoassay for Clostridium perfringens type A enterotoxin.

The diagnostic application of an enzyme immunoassay for Clostridium perfringens type A enterotoxin was evaluated. Test results from 100 individuals associated with C. perfringens gastroenteritis outbreaks and 111 control individuals were included. The assay sensitivity was 93.7%, and the assay specificity was 98.7%.

Diagnostic importance of Clostridium perfringens enterotoxin analysis in recurring enteritis among elderly, chronic care psychiatric patients.

A series of Clostridium perfringens-related gastrointestinal outbreaks occurred over a period of several months among elderly, chronic care patients in a psychiatric hospital. Several serotypes of C. perfringens and many nontypeable isolates were found. The distribution of certain serotypes and the incidence of detection of enterotoxin in fecal extracts were related to wards on which patients were resident (six wards were involved). Several patients were reported to have chronic or recurring fecal incontinence or diarrhea or both. With a background of elevated spore counts of several serotypes and chronic diarrhea, only detection of enterotoxin could provide definitive evidence of C. perfringens etiology in gastoenteritis cases.

A double antibody sandwich enzyme-immunoassay for Clostridium perfringens type A enterotoxin detection in stool specimens.

A double antibody sandwich enzyme-immunoassay has been developed for detection of Clostridium perfringens enterotoxin. Anti-enterotoxin immunoglobulin G-alkaline phosphatase conjugates were prepared using a rapid minicolumn procedure. The assay can achieve a sensitivity of greater than or equal to 1 ng/ml with purified enterotoxin. Sensitivity for detection of cases of C. perfringens enteritis in a C. perfringens outbreak (86 individuals tested) was between 85.7 and 98.0 per cent depending upon stringency of criteria for defining positive cases. Specificity of the assay was demonstrated by the lack of positive results in 53 individuals involved in a gastroenteritis outbreak of unknown etiology.

The influence of dietary selenium levels on blood levels of selenium and glutathione peroxidase activity in the horse.

Twenty mature geldings, averaging 535 kg, were used to determine the influence of dietary selenium (Se) on the blood levels of Se and Se-dependent glutathione peroxidase (SeGSH-Px) activity in the horse. Horses were randomly assigned within breed to four treatments consisting of five horses each and fed a basal diet containing .06 ppm of naturally occurring Se. Diets were supplemented with .05, .10 and .20 ppm Se, as sodium selenite. Blood was drawn for 2 wk before, and for 12 wk following, the inclusion of supplement Se in the diets. Whole blood and plasma Se concentrations and plasma SeGSH-Px activities were determined from all blood samples. Selenium concentrations in plasma and whole blood increased linearly from wk 1 to wk 5 and 6, respectively, in Se-supplemented horses. After these times, no significant changes in Se concentration were observed in Se-supplemented or in unsupplemented horses throughout the remainder of the 12-wk trial. Plasma Se reached plateaus of .10 to .11, .12 to .14, and .13 to .14 micrograms/ml in horses supplemented with .05, .10 and .20 ppm Se, respectively. Whole blood Se reached plateaus of .16 to .18, .19 to .21, and .17 to .18 micrograms/ml in horses supplemented with .05, .10 and .20 ppm Se, respectively. Plasma SeGSH-Px activity was not significantly affected by dietary treatment. Therefore, this enzyme was not a good indicator of dietary Se in these mature horses.

Photosynthetic electron transport in guard cells of diverse species.

Guard cells of plants representing 18 species were assayed qualitatively for potential to conduct photosynthetic linear electron transport. These plants included C(3) pteridophytes, C(3) and C(4) monocots, and C(3), C(4), and Crassulacean acid metabolism dicots. By use of a microfluorospectrophotometer, guard cell samples in epidermal peels were isolated optically. Chlorophyll fluorescence was monitored from the onset of excitation light. For guard cells of all these species, fluorescence intensity increased during illumination. When samples were preincubated with 3-(3,4-dichlorophenyl)-1,1-dimethylurea, diuron, however, there was a more rapid increase in fluorescence. These results indicate that all tested guard cells conduct photosynthetic electron transport through the reaction center of photosystem II.