The extracellular ß-1,3-endoglucanase EngA is involved in autolysis of Aspergillus nidulans.

AIMS: To elucidate the roles of the ß-1,3-endoglucanase EngA in autolysis of the filamentous fungus Aspergillus nidulans and to identify the common regulatory elements of autolytic hydrolases. METHODS AND RESULTS: A ß-1,3-endoglucanase was purified from carbon-starving cultures of A. nidulans. This enzyme is found to be encoded by the engA gene (locus ID: AN0472.3). Functional and gene-expression studies demonstrated that EngA is involved in the autolytic cell wall degradation resulting from carbon starvation of the fungus. Moreover, regulation of engA is found to be dependent on the FluG/BrlA asexual sporulation signalling pathway in submerged culture. The deletion of either engA or chiB (encoding an endochitinase) caused highly reduced production of hydrolases in general. CONCLUSIONS: The ß-1,3-endoglucanase EngA plays a pivotal role in fungal autolysis, and activities of both EngA and ChiB are necessary to orchestrate the expression of autolytic hydrolases. The production of cell wall-degrading enzymes was coordinately controlled in a highly sophisticated and complex manner. SIGNIFICANCE AND IMPACT OF THE STUDY: No information was available on the autolytic glucanase(s) of the euascomycete A. nidulans. This study demonstrates that EngA is a key element in fungal autolysis, and normal activities of both EngA and ChiB are crucial for balanced production of hydrolases.

Opposite effect of Cu(II) and Se(IV) ions on the antibacterial-toxic action of mycotoxins.

The effect of Se(IV) and Cu(II) ions on the antibacterial activity of aflatoxins and ochratoxin A (mycotoxins) was studied in BioArena as a complex bioautographic system. In the presence of 0.23 and 0.46 mg/100 mL Se(IV) the inhibition zones of mycotoxins were decreased, however, lower concentration (0.046 mg/100 mL) increased the antibacterial effect of aflatoxin B1. Cu(II) (1.53 mg/100 mL) enhanced the toxicity of mycotoxins. The results supported the possible role of formaldehyde and its reaction products (e.g. 1O2, O3) in the antibacterial-toxic action of mycotoxins. Cu(II) can probably generate and mobilise the formaldehyde molecules and so it could increase the toxicity with its potential reaction products. It is possible that the enzymatic or spontaneous methylation of Se(IV) takes place through formaldehyde, which may cause partial formaldehyde depletion in the system. The enhanced antibacterial effect at low concentration Se(IV) is overlapping with the often experienced prooxidant effect in cases of natural antioxidants.

Formaldehyde and some fully n-methylated substances in boar seminal fluids. Short communication.

On the basis of recent observations it is supposed that seminal fluids may contain--mainly in hydroxymethyl groups--formaldehyde (HCHO) and quaternary ammonium compounds as potential HCHO generators, therefore, preliminary investigations were carried out for the identification of these compounds in pig seminal fluids using OPLC, HPLC and MALDI MS techniques. The fresh pig seminal fluid was frozen in liquid nitrogen, powdered and aliquots (0.25 g) were treated with 0.7 ml ethanolic dimedone solution. The suspension was centrifuged and the clear supernatant was used for analysis by OPLC or after dilution with HPLC or MALDI MS technique. After OPLC separation of formaldemethone the fully N-methylated compounds which are stayed on the start point were separated by OPLC using an other eluent system. It has been established that the HCHO is really a normal component of the pig seminal fluid, as well. It can be isolated and identified in dimedone adduct form. The measurable amount of HCHO depended on the concentration applied of dimedone. According to OPLC and MALDI MS investigations L-carnitine is the main quaternary ammonium compound in pig seminal fluid which can generate a protection of the sperm cells against environmental and other influences. Considerable differences have been found among individuals concerning concentrations of quaternary ammonium compounds in the seminal fluid of pigs.

Influence of subcutaneous injection of essential fatty acids on the stress-induced modifications of rat platelet aggregation and membrane lipid composition.

Membrane lipids play an important role in the function of blood platelets but the mechanisms by which the lipid composition of the platelet membrane is adjusted remain unclear. It has been shown that stress and poly-unsaturated fatty acids modified the lipid composition of blood plasma and platelet lipids, but very little is known about the effect of stress and fatty acids on membrane platelet lipid composition. The purpose of the present investigation was to study the influence of the essential fatty acids: linoleic, linolenic and arachidonic acids on the composition of the platelet membrane lipids of rats assigned to heat and restraint stress. It was shown that injections of polyunsaturated fatty acids decrease or suppress the stress-induced increase in platelet aggregation, suppress the stress-induced modification of the composition of the platelet membrane lipids and modify the fatty acid composition of the platelet membrane phospholipids.

Interactions of adolescent mothers and their 1-year-old children.

It is unclear why the school-aged children of adolescent mothers have more cognitive and behavioral problems than those of adult mothers. To clarify why these children have problems and when during their lives they develop, the relationship between adolescent maternal age and the nature of the behavioral interaction between mothers and their children was studied in the laboratory. Thirty lower socioeconomic status mothers who were 15.5 years to 20 years of age and their 9- to 12-month-old children were videotaped for 20 minutes. Rating scales were developed to score the videotapes. There were significant correlations indicating that younger mothers tended to show less acceptance (r = .63; P less than .001), less cooperation (r = .57; P less than .001), less accessibility (r = .51; P less than .003), less sensitivity (r = .46; P less than .006), and more negative verbal communication (r = .32; P less than .047) than older adolescent mothers. Younger maternal age was also associated with more overall negative interaction between mother and child (r = .35; P less than .032) and with less child-initiated social contact with the mother (r = .32; P less than .050). We conclude that over the relatively narrow age range younger adolescent maternal age is related to less favorable mothering behaviors in the laboratory when the children are 9 to 12 months of age.

Primary DNA damage in human blood lymphocytes exposed in vitro to 2450 MHz radiofrequency radiation.

Human peripheral blood samples collected from three healthy human volunteers were exposed in vitro to pulsed-wave 2450 MHz radiofrequency (RF) radiation for 2 h. The RF radiation was generated with a net forward power of 21 W and transmitted from a standard gain rectangular antenna horn in a vertically downward direction. The average power density at the position of the cells in the flask was 5 mW/cm(2). The mean specific absorption rate, calculated by finite difference time domain analysis, was 2.135 (+/-0.005 SE) W/kg. Aliquots of whole blood that were sham-exposed or exposed in vitro to 50 cGy of ionizing radiation from a (137)Cs gamma-ray source were used as controls. The lymphocytes were examined to determine the extent of primary DNA damage (single-strand breaks and alkali-labile lesions) using the alkaline comet assay with three different slide-processing schedules. The assay was performed on the cells immediately after the exposures and at 4 h after incubation of the exposed blood at 37 +/- 1 degrees C to allow time for rejoining of any strand breaks present immediately after exposure, i.e. to assess the capacity of the lymphocytes to repair this type of DNA damage. At either time, the data indicated no significant differences between RF-radiation- and sham-exposed lymphocytes with respect to the comet tail length, fluorescence intensity of the migrated DNA in the tail, and tail moment. The conclusions were similar for each of the three different comet assay slide-processing schedules examined. In contrast, the response of lymphocytes exposed to ionizing radiation was significantly different from RF-radiation- and sham-exposed cells. Thus, under the experimental conditions tested, there is no evidence for induction of DNA single-strand breaks and alkali-labile lesions in human blood lymphocytes exposed in vitro to pulsed-wave 2450 MHz radiofrequency radiation, either immediately or at 4 h after exposure.

Blockade of a cardiac K+ channel by tacrine: interactions with muscarinic and adenosine receptors.

The centrally acting anticholinesterase drug tacrine has been shown to block K+ channels in guinea pig left atrium. It competitively blocks the negative inotropic effects of adenosine, 2-chloroadenosine and carbachol. Ka values obtained from dose ratio plots were 2.5, 3.5 and 2.9 microM respectively. It was also able to antagonize the shortening of the action potential due to these compounds. Doses of tacrine ranging from 1 to 4 microM restored the AP configuration close to control values. Tacrine also antagonized the binding of 1-quinuclidinyl[phenyl-4-3H]benzilate ([3H]QNB) to membranes derived from the atrium and cerebral cortex. Ki values of 1.8 +/- 0.33 and 1.3 +/- 0.47 microM were obtained respectively. Tacrine was a weak competitor of [3H]phenylisopropyladenosine ([3H]L-PIA) binding in brain membranes. Its diverse pharmacological effects may be relevant to its use in Alzheimer's disease.

Binding of some organophosphorus compounds at adenosine receptors in guinea pig brain membranes.

The organophosphorus compounds, sarin, tabun and soman are known to be potent inhibitors of cholinesterase. It was claimed that these agents also interact at neurotransmitter receptor sites. We were unable to detect any interaction at the acetylcholine muscarinic sites; we now focus our attention on the adenosine receptors. Binding studies showed that all three anticholinesterases inhibited the binding of [3H]L-phenylisopropyladenosine ([3H]L-PIA) to the brain adenosine receptors in a dose-dependent manner until receptor concentration became limiting. Soman was found to be 5 and 9 times more effective than tabun and sarin respectively in inhibiting [3H]L-PIA binding. These results suggested that these organophosphorus anticholinesterases could interact directly at the A1 adenosine receptors which could subsequently mediate changes in K+ permeability of synaptic membranes.

Development of sensitive colorimetric capture elisas for Clostridium botulinum neurotoxin serotypes A and B.

Sensitive and specific enzyme-linked immunosorbent assays were developed to detect Clostridium botulinum neurotoxin serotypes A (BoNT A) and B (BoNT B) in assay buffer and human serum. The assay is based upon affinity-purified horse polyclonal antibodies directed against the approximately 50 kDa C-fragments of each toxin. Standard curves were linear over the range of 0.1-10 ng mL. Detection was possible at 0.2 ng mL (20 pg/well) and accurate quantitation at 0.5 ng/mL (50 pg well) in assay buffer and 10% human serum. Variations between triplicates was typically 5-10%. Less than 1% cross reactivity occurred between other serotypes when each assay was performed against serotypes A, B and E. When tested against toxins complexed to their associated nontoxic proteins, interference was absent (BoNT B) or < 25% (BoNT A). These assays demonstrate sensitivity close to that of the mouse bioassay without the use of animals and in a much simpler format than other reported assays of similar sensitivity.

Subsets in psoriatic arthritis formed by cluster analysis.

The aim of the study was to create subgroups among psoriatic arthritis patients on the basis of dermatological features, clinical pattern of arthritis, and laboratory, immunological and radiological findings. Data on 100 patients were expressed in a standardised form and entered into hierarchical cluster analysis according to Ward's method. Seven subgroups were created. Fifty-six patients with mild psoriasis were sorted into a 'polyarticular group'. Two 'RA-like groups' were formed, differing from each other serologically and in axial involvement. In an 'oligoarticular group' (18 patients) serious skin disease and female gender predominancy were found to be characteristic. Eight patients with polyarticular arthritis were assigned to an 'erythrodermal group', in which polyarticular arthritis, mutilating, severe arthritis and a history of erythroderma were characteristic. Close to this group on the dendrogram eight women were sorted into a 'distal form'. Sausage fingers were frequent, and nail dystrophy was present in every case. In a 'pustular group' (three patients) the different type of skin involvement was considered and nail dystrophy was common. In the newly created subgroups not only the arthritic status, but also the type of the skin disease, played a determining role.

Effect of nickel deficiency on biochemical variables in serum, liver, heart and kidneys of goats.

Nickel deficiency was induced in 2- to 4-year-old goats by feeding 0.1 mg Ni/kg dry matter with a semisynthetic diet. The control group consumed 5.0 mg Ni/kg d.m. Activity of several enzymes (SDH, LDH, HBDH, AST, ALT, ALD, CK, CHE) was determined in the serum, liver, heart and kidneys. Serum urea-N level was also measured and transmission electron microscopic (TEM) examinations were performed. Signs characteristic of nickel deficiency (retarded growth, increased mortality of dam and offspring, parakeratosis of the skin) appeared in the low-nickel group. The activity of SDH and ALD, as well as the level of urea-N was significantly lower in the serum of Ni-deficient animals than in the control. Ni-deficient animals also had significantly lower enzyme activities in the heart (SDH, HBDH, AST, ALT, ALD and CK), liver (SDH and CHE) and kidneys (HBDH and CK). Electron micrographs showed degeneration of cardiac and skeletal muscle in the Ni-deficient animals. Ni deficiency elicited changes primarily in the heart and these resulted in depressed activity of several enzymes.

Activities of some serum enzymes in halothane reacted and non-reacted pigs.

Eight- and nine-week-old Hungarian Landrace pigs were tested with halothane as described by Laky et al. (1985). Immediately after the test blood samples were taken for determination of the activity of serum creatine kinase (CK), creatine kinase MB (CK-MB) isoenzyme, aldolase (ALD), lactate dehydrogenase (LDH) and alpha-hydroxybutyrate dehydrogenase (alpha-HBDH). Elevated creatine kinase, creatine kinase MB isoenzyme and aldolase activities indicating enhanced susceptibility to stressors were found in 92% of the halothane reacted and 16% of the halothane non-reacted animals. In these individuals the activities of lactate dehydrogenase and alpha-hydroxybutyrate dehydrogenase were also high. Data of the literature show a close relationship between enhanced susceptibility to stressors and halothane reaction in pigs. It was suggested, therefore, that determination of the activity of appropriate serum enzymes might be used for detecting this enhanced susceptibility.

Economic losses caused by paratuberculosis in a dairy herd: case report.

The impact of clinical paratuberculosis in a 500 cow Jersey x Holstein dairy herd of loose housing system was followed up for three years. There was an increasing fall in annual milk production, namely 49, 474, and 1030 litres per cow, a decrease in the average age from 63.9 to 57.0 months, and of feed conversion from 60 to 39%. The effect of certain management deficiencies, existing throughout the study, is discussed.

Effects of nickel supply on the fattening performance and several biochemical parameters of broiler chickens and rabbits.

Broiler chicken and rabbit experiments were carried out to study the effects of nickel (Ni) supplementation on growth performance and Ni metabolism. ROSS cockerels and New Zealand White female rabbits were fed a diet containing Ni in concentrations of 0, 50 and 500 mg/kg in dry matter (DM). Dietary supplementation of 50 mg Ni/kg slightly improved the body weight gain (BWG) and had a beneficial effect on the feed conversion efficiency (FCE) in broiler chickens. However, Ni added at a level of 500 mg/kg significantly (P < 0.05) reduced the BWG by 10% and resulted in significantly (P < 0.05) worse (2.3 +/- 0.2 kg/kg) FCE. The relative weight of the liver in cockerels was significantly (P < 0.05) decreased by Ni as compared to the control group (1.7 and 2.1% vs. 2.6%). The activity of AST and CHE enzymes was increased insignificantly by dietary supplementation of 500 mg Ni/kg, indicating damage of the liver parenchyma. The results of serum biochemistry were confirmed by a mild or moderate form of pathological focal fatty infiltration of the liver in broilers. Supplemental Ni of 50 mg/kg concentration resulted in non-significantly increased BWG in rabbits. Ni added to the diet at a level of 500 mg/kg reduced the digestibility of crude protein by 3-4% and that of crude fibre by 20-25% in rabbits. Approx. 98% of the ingested Ni was lost from the body via the faeces, 0.5-1.5% via the urine and approx. 1% was incorporated into the organs of rabbits. As a result of dietary supplementation of 50 and 500 mg Ni/kg, Ni accumulated in the kidneys (4.9 +/- 0.5 and 17.1 +/- 3.1 vs. 1.9 +/- 0.3 mg/kg DM), ribs (10.3 +/- 0.4 and 10.4 +/- 0.6 vs. 9.1 +/- 0.6 mg/kg DM), heart (1.4 +/- 0.2 and 2.5 +/- 0.4 vs. 1.0 +/- 0.1 mg/kg DM) and liver (1.3 +/- 0.1 and 2.2 +/- 0.2 vs. 0.9 +/- 0.05 mg/kg DM), as compared to the control animals. It can be stated that supplementation of the diet with 50 mg Ni/kg had slight but non-significant beneficial effects on the growth performance of broiler chickens and rabbits.

Study of the soil-plant (carrot)-animal cycle of nutritive and hazardous minerals in a rabbit model.

Carrots were grown on soils polluted by heavy metal salts. Each particular microelement reached a high concentration [molybdenum (Mo) 39.00, cadmium (Cd) 2.30, lead (Pb) 4.01, mercury (Hg) 30.00, and selenium (Se) 36.20 mg/kg dry matter] in the carrot. In a metabolic balance trial conducted with 15 male and 15 female New Zealand White rabbits, the control animals (n = 5) were fed ad libitum with concentrate as basal diet, while the other rabbits received the basal diet and carrots containing the particular microelement. Blood samples were taken to determine the activity of serum enzymes. To investigate the metabolism of Mo, Cd, Pb, Hg and Se, samples were taken from the heart, liver, lungs, kidneys, spleen, ovaries/testicles, entire digestive tract, adipose tissue, femur, hair, faeces and urine. Carrot had significantly higher digestibility for all nutrients than the rabbit concentrate. Carrot samples of high Pb content had the lowest digestibility of crude protein. The microelements differed in their rate of accumulation in the organs examined: Mo and Cd accumulated in the kidneys, Pb in the kidneys, liver, bones and lungs, Hg in the kidneys and liver, while Se in the liver, kidneys and heart. The proportions of microelements eliminated from the body either via the faeces and urine (Mo 80.18% and Se 47.41%) or via the faeces (Cd 37.86%, Pb 66.39%, Hg 64.65%) were determined. Pathohistological examination revealed that the rate of spermatogenesis was reduced in the Mo, Cd, Pb and Hg groups compared to the control. Lead, Cd and Hg intake resulted in a considerable decrease in gamma-glutamyltransferase (GGT) and in an increase of alkaline phosphatase (ALP) activity because of damages to the kidneys and bones. All experimental treatments decreased the activity of cholinesterase (CHE) because of lesions in the liver.

L-carnitine as essential methylated compound in animal metabolism. An overview.

Certain kinds of N-methylated compounds proved to be essential for animals. L-Carnitine is a small-molecular-weight quaternary amine which occurs naturally in micro-organisms, plants and animals. Generally, plants contain little carnitine compared to animals where especially high levels may be found in heart and skeletal muscle. The main function of L-carnitine is the translocation of long-chain fatty acids from the extramitochondrial space to the mitochondrial space. It also facilitates the removal from mitochondria of short-chain and medium-chain fatty acids that accumulate as a result of normal and abnormal metabolism. This pathway provides a means to regenerate the intramitochondrial free coenzyme A under conditions where short-chain acyl-CoA esters are produced at a rate faster than they can be utilized. L-Carnitine is synthetized by most animals but its supplementation can be beneficial under certain condition including insufficient carnitine synthetic enzyme activity, metabolic abnormalities, dietary deficiencies or malnutrition. Several studies on pigs, fish, foal, quail and broiler chickens demonstrate a growth improvement by feeding extra dietary L-carnitine. It has been also found that L-carnitine supplementation resulted in lowered muscle and liver lipid contents. Formaldehyde is involved in metabolism, methylation-demethylation processes of these compounds.

Effects of dietary L-carnitine supplementation and protein level on performance and degree of meatness and fatness of broilers.

Feeding high-protein diets to broilers is a known means for improving performance and carcass composition and quality, however, the combined effects of dietary protein (CP) level concurrent with L-carnitine (CN) supplementation are not known. Performance and carcass traits of broilers fed CN-supplemented diets of different CP-levels were studied from 18 to 53 days of age. Three isocaloric diets containing 18, 20 or 22% CP were formulated, with or without added CN (50 mg/kg), and used. Apart from CP level, supplemental CN attained significantly higher body weight gain (BWG) and feed conversion (FC), means of BWG were 351 vs. 332 and 371 vs. 353 g, and those of FC were 1.25 vs. 1.33 and 1.73 vs. 1.84 for the first and second week of the experimental period, respectively. Amount and percentage of abdominal fat and ether extract (EE) contents of breast meat of 53-day-old broilers were significantly reduced (means of AF were 43.5 vs. 51.5 g, equivalent to 1.98 vs. 2.39%, those of EE were 1.82 vs. 2.24%) in response to CN supplementation. A significant interaction between added CN and CP level was noted on BWG and FC of broilers from 18 to 32 days of age.

Biochemical and physiological effects of long-term sublethal T-2 toxin feeding in rabbits.

Effects of 4-7-week feeding of naturally contaminated wheat grains containing 0.284 mg T-2 toxin/kg were investigated on the health, certain serum biochemical parameters and reproductive status of sexually mature, virgin female rabbits. Three of the ten contaminated animals died before the end of the experiment (acute, fibrinous-purulent peritonitis and pneumonia). Hepatic damages are suggested by significant serum alanine aminotransferase and slight aspartate aminotransferase, gamma-glutamyl transferase, malate dehydrogenase activity increases, as well as by cholinesterase activity decrease as compared to control animals. The damage of kidney function is indicated by significantly higher creatinine level, as compared to the control. The T-2 toxin feeding also impaired ovarian functions, reflecting by unaltered progesteron concentration, macro- and microscopical pictures after GnRH-stimulation.

Influence of dietary L-carnitine on performance and carcass quality of broiler chickens.

Effects of dietary L-carnitine supplementation on the performance, carcass yield and components, abdominal fat and composition of meat of broiler chickens were investigated. Ross broiler chicks, 18-day-old were used in this experiment. Four experimental diets were formulated, by adding three levels of supplemental L-carnitine (50, 100 or 150 mg/kg) to a basal diet and used from 18 to 46 days of age. Apart from the level of supplementation, L-carnitine (CN) addition resulted in significant increases (P < 0.05) in body weight gains (BWG) of broilers (means were 397 vs. 365 and 410 vs. 377 g, for the first and second weeks of the experimental period, respectively). Abdominal fat (AF) contents of 46-day-old broilers were significantly decreased, both as absolute weights (P < 0.05) and as percentages of body weight (P < 0.01), by added CN (means of AF were 36.8 vs. 44.8 g, equivalent to 1.87 vs. 2.32%). Level of added CN had no effect on BWG or AF of broilers. It was concluded that the effectiveness of supplemental L-carnitine for improving BWG and/or decreasing AF of broilers may depend on the age at which L-carnitine is added. Under the conditions of this study, a supplementation of 50 mg L-carnitine per kg diet proved to be effective.