Effects of rumen-protected choline supplementation on milk production and choline supply of periparturient dairy cows.

The objective of the present study was to determine the effects of rumen-protected choline (RPC) supplementation on body condition, milk production and milk choline content during the periparturient period. Thirty-two Holstein cows were allocated into two groups (RPC group - with RPC supplementation, and control group - without RPC supplementation) 28 days before the expected calving. Cows were fed the experimental diet from 21 days before expected calving until 60 days of lactation. The daily diet of the RPC group contained 100 g of RPC from 21 days before calving until calving and 200 g RPC after calving for 60 days of lactation, which provided 25 g and 50 g per day choline, respectively. Body condition was scored on days -21, 7, 35 and 60 relative to calving. Milk production was measured at every milking; milk fat, protein and choline content were determined on days 7, 35 and 60 of lactation. Body condition was not affected by RPC supplementation. Milk yield was 4.4 kg higher for the group of cows receiving supplementary choline during the 60 days experimental period and 4% fat-corrected milk production was also increased by 2.5 kg/day. Milk fat content was not altered by treatment, but fat yield was increased by 0.10 kg/day as a consequence of higher milk yield in the RPC-treated group. Milk protein content tended to increase by RPC supplementation and a 0.18 kg/day significant improvement of protein yield was detected. Milk choline content increased in both groups after calving as the lactating period advanced. However, milk choline content and choline yield were significantly higher in the RPC group than in the control group. The improved milk choline and choline yield provide evidence that some of the applied RPC escaped ruminal degradation, was absorbed from the small intestine and improved the choline supply of the cows and contributed to the changes of production variables.

Establishment of the European College of Veterinary Clinical Pathology (ECVCP) and the current status of veterinary clinical pathology in Europe.

After 5 years of development, the European College of Veterinary Clinical Pathology (ECVCP) was formally recognized and approved on July 4, 2007 by the European Board of Veterinary Specialisation (EBVS), the European regulatory body that oversees specialization in veterinary medicine and which has approved 23 colleges. The objectives, committees, basis for membership, constitution, bylaws, information brochure and certifying examination of the ECVCP have remained unchanged during this time except as directed by EBVS. The ECVCP declared full functionality based on the following criteria: 1) a critical mass of 65 members: 15 original diplomates approved by the EBVS to establish the ECVCP, 37 de facto diplomates, 7 diplomates certified by examination, and 5 elected honorary members; 2) the development and certification of training programs, laboratories, and qualified supervisors for residents; currently there are 18 resident training programs in Europe; 3) administration of 3 annual board-certifying examinations thus far, with an overall pass rate of 70%; 4) European consensus criteria for assessing the continuing education of specialists every 5 years; 5) organization of 8 annual scientific congresses and a joint journal (with the American Society for Veterinary Clinical Pathology) for communication of scientific research and information; the College also maintains a website, a joint listserv, and a newsletter; 6) collaboration in training and continuing education with relevant colleges in medicine and pathology; 7) development and strict adherence to a constitution and bylaws compliant with the EBVS; and 8) demonstration of compelling rationale, supporting data, and the support of members and other colleges for independence as a specialty college. Formal EBVS recognition of ECVCP as the regulatory body for the science and practice of veterinary clinical pathology in Europe will facilitate growth and development of the discipline and compliance of academic, commercial diagnostic, and industry laboratories in veterinary clinical pathology. Future needs are in developing sponsorship for resident positions, increasing employment opportunities, increasing compliance with laboratory, training, and continuing education standards, and advancing relevant science and technology.

Evaluation of the effect of ketoprofen and carprofen on platelet function in dogs studied by PFA-100 point-of-care analyser.

The effect of two nonsteroidal anti-inflammatory drugs (carprofen and ketoprofen) on platelet adhesion and aggregation functions was evaluated by the PFA-100 analyser (Dade-Behring, CA, U.S.A.) using its collagen-adenosine diphosphate (ADP) and collagen-epinephrine (EPI) cartridges. The function of platelets was evaluated in 55 healthy dogs, in 7 dogs treated with ketoprofen and in 31 dogs treated with carprofen in a therapeutic dose for minimum 5 days. The therapeutic doses of carprofen had no effect on the closure time of PFA-100 (which is the marker of platelet function) but ketoprofen caused a significant increase when using collagen-EPI stimulation The closure times for both the healthy (control) and the treated dogs using EPI cartridges were often longer than the upper default cut-off point (300 sec) of the device. The PFA-100 analyser with collagen-ADP cartridges could be a useful tool for veterinary applications including the evaluation of platelet aggregation in dogs treated with NSAIDs. The upper cut-off point of PFA-100 might be extended.

Free radicals, lipid peroxidation and the antioxidant system in the blood of cows and newborn calves around calving.

The oxidative stress of birth in cattle (Bos taurus) was evaluated by measuring steady state concentration of free radicals in whole blood, rate of lipid peroxidation and activity of antioxidant enzymes in erythrocytes, antioxidant capacity of blood plasma in 14 calves at birth and four times after birth until 3 weeks of age and also in their mothers at calving. The same parameters were also measured in 58 dairy cows before calving, at parturition and after calving. Free radical concentration in the blood of newborn calves was higher than in cows confirming that birth means oxidative stress for calves. Red blood cell malondialdehyde in calves was the highest at birth and following the first solid feed intake at the third week. Superoxide dismutase activity increased in calves during the first three weeks of life. Ferric reducing ability of plasma was higher in calves at birth than in cows and decreased thereafter. Higher superoxide dismutase activity in red blood cells and lower ferric reducing ability of plasma in dairy cows was found at calving compared to the average of all pre- and post-calving results. We conclude that the blood of newborn calves is well prepared to deal with the oxidative stress of birth, and that such a stress is present even when some fingerprint markers of redox imbalance show no apparent alterations. Stress of calving has minor effects on the antioxidant system of cows.

Morphological evaluation of canine platelets on Giemsa- and PAS-stained blood smears.

The morphology of canine platelets (changes in size, shape, staining characteristics, degree of activation and clump formation, distribution of granules, appearance of vacuoles on Giemsa-stained smears) was investigated in 20 healthy control and 181 diseased dogs. In the group of the sick dogs 84 animals suffered from disorders affecting directly the haematological parameters or the haematopoietic organs such as bleeding, thymic haemorrhage, haemolytic disorders, lymphoma, immune-mediated thrombocytopenia, and other 97 dogs were affected by other diseases (hepatopathy, nephropathy, hepatic, splenic or intestinal neoplasm, skin diseases, diabetes mellitus, Cushing's syndrome, sepsis). The alterations found in platelet morphology were not specific for any disorder. The most common platelet abnormalities were polychromasia and the presence of giant platelets. These changes occurred in a high number in disorders accompanied by bleeding or haemolysis. Anisocytosis was the most frequent finding in hepatic, splenic or intestinal neoplasms and in certain endocrinopathies. Microcytosis was observed in immune-mediated thrombocytopenia, hepatic neoplasms and endocrine disorders. Extreme platelet activation was common in haemolysis, hepatopathies, neoplastic diseases and sepsis. Vacuolisation was present in thymic haemorrhage, pancreatitis, diabetes mellitus and Cushing's syndrome. A new morphologic phenomenon, i.e. a ring-like formation of granules, was described in the cytoplasm of the platelets both in healthy and diseased animals. In addition, two forms of pathologic granulation were also described for the first time in Giemsa-stained blood smears: the pseudonuclear and the spot-like formation of granules, which were observed especially in disorders affecting the blood cells. The granulation and morphological characteristics of platelets on smears stained by periodic acid-Schiff reaction (PAS) were also studied. Three localisations of granulation were observed, such as peripheral, eccentric and diffuse. The ratio of PAS-positive and -negative platelets was evaluated in several diseases. Our findings support the diagnostic value of platelet evaluation by light microscopy and help clinicians/clinical pathologists to understand why morphologic changes of thrombocytes might be expected in several diseases.

Effect of supplementation with methionine and different fat sources on the glutathione redox system of growing chickens.

The effect of supplementary methionine and fats of different saturation levels on the glutathione redox system of growing broiler cockerels was studied. The diet of three groups of chicks was supplemented with corn germ oil, beef tallow and fish oil at the levels of 30 g/kg and 50 g/kg of feed, respectively. The diet of further three groups was supplemented with methionine (5 g/kg of feed) in addition to the different fat sources. Control chicks were fed with a compound feed without methionine and fat supplementation. Reduced glutathione (GSH) and glutathione disulphide (GSSG) content as well as glutathione peroxidase activity in the liver were determined and GSH/GSSG ratio was calculated at day old and then at one and three weeks of age. Our results indicate that supplementary methionine stimulates both the synthesis of the glutathione redox system and glutathione peroxidase activity in growing chickens in the first period of postnatal life, when the risk of lipid peroxidation is high due to feeding unsaturated fats in the diet.

Percutaneous ultrasound-guided cholecystocentesis in dogs.

Percutaneous ultrasound-guided cholecystocentesis was performed on 13 healthy beagle dogs to determine whether percutaneous ultrasound-guided cholecystocentesis in the dog was a feasible and safe procedure. Clinical, laboratory and ultrasonographic examinations were done at 0 and 10 minutes, in the 2nd and 16th hour, and on the 7th day. They included a detailed physical examination of the mucous membranes, cardiorespiratory system and abdominal organs. Laboratory examinations of the blood consisted of a complete blood count, determination of packed cell volume (PCV), haemoglobin (Hb), total plasma protein (TPP), parameters of haemostasis including prothrombin time (PT), activated partial thromboplastin time (APTT), and enzyme activities reflecting hepatobiliary function, i.e. aspartate aminotransferase (AST), alanine aminotransferase (ALT), and gamma-glutamyltransferase (GGT). Ultrasonographic findings of the gallbladder (size, shape, wall, content) and appearance of the biliary tract and the surrounding cranial intraabdominal organs were also evaluated. Percutaneous ultrasound-guided cholecystocentesis was performed easily during the study, and dogs tolerated well the procedure performed without anaesthesia. All laboratory parameters of the blood remained within normal limits throughout the study. However, some follow-up values, i.e. PCV, TPP, APTT and ALT, demonstrated statistically significant differences when compared to baseline measurements, which might reflect the effect of 24-hour fasting before the experiment, as well as day-to-day metabolic fluctuations due to feeding and water supply during the study. There were no visible signs of bleeding from the liver, bile leakage from the gallbladder or accumulation of free peritoneal fluid during repeated ultrasonographic examinations. Percutaneous ultrasound-guided cholecystocentesis seems to be an important diagnostic procedure in canine gallbladder diseases and can be used safely and easily to gain gallbladder bile for diagnosis of bacterial cholecystitis or for investigating hepatobiliary function in the dog.

Relationship between concentration of citrate and ketone bodies in cow's milk.

The authors' hypothesis is that the members of the tricarboxylic acid cycle (TCA cycle) such as citrate decrease in association with increased ketone body formation. To prove this hypothesis the connection between ketone bodies and citrate formation of milk was studied. A fluorimetric method was used to determine citrate and a headspace sampling gas chromatographic (GC) method was developed for determination of ketone bodies. Under real conditions of milk sampling, transport and storage, preserved milk samples of 119 clinically healthy dairy cows obtained in the 48 hours after milking were investigated. A low level of acetoacetate (ACAC) was found in all samples. This fact can be explained by the spontaneous decarboxylation of acetoacetate during sample storage (previously decarboxylised acetoacetate = pdACAC) and, consequently, the majority of the amount of acetoacetate in the samples (AC + pdACAC) appeared in the measured acetone concentrations. Based on the measured acetone concentration of milk samples two groups were formed retrospectively: HA (high-acetone) group (n = 41) with an AC + pdACAC concentration of > 0.4 mmol/l and a LA (low-acetone) group (n = 78) with an AC + pdACAC level of < or = 0.4 mmol/l. In the milk of cows of Group HA a positive correlation (r = +0.623) and linear connection between acetone (AC + pdACAC) and beta-hydroxybutyrate (BOHB) levels was found [BOHB = 2.491 + 0.586 x (pdAC + ACAC)]. Furthermore, in this group a negative correlation between citrate and BOHB and AC + pdACAC was also established (r = -0.579). Focusing on the results of this group the authors found a significant drop of AC + pdACAC and citrate during the metabolically critical first 1-4 weeks of lactation. For this reason they suggest that simple, easy, automated methods (i.e. flow injection analysis, Fourier transformation infrared analysis) should be introduced for the simultaneous determination of acetone and citrate concentration in milk to make the evaluation of the energy status of high-producing dairy cows easier and more certain.

Effect of deferoxamine and L-arginine treatment on lipid peroxidation in an intestinal ischaemia-reperfusion model in rats.

This study investigated lipid peroxidation (LPO) changes during intestinal ischaemia-reperfusion with and without deferoxamine or L-arginine treatment. White Wistar rats were allotted into four groups as follows: sham-operated (Group SOP), ischaemia-reperfusion only (Group I/R), I/R with deferoxamine (Group D) or L-arginine (Group A) treatment. Concentration of thiobarbituric acid reactive substances (TBARS), overall concentration of malondialdehyde and 4-hydroxy-alkenals (LPO586), activities of superoxide dismutase (SOD) and glutathione peroxidase (GPX) of the jejunal homogenates were determined. The same analytes except LPO586 were assayed in RBC haemolysates. Measurements of ferric reducing ability (FRAP), total antioxidant status (TAS) and nitric oxide (NO) concentrations of plasma samples were also completed. The only significant change observed in the SOP group was an increased SOD activity after the ischaemic period. In the I/R group significant increase of intestinal LPO586 concentration was observed during hypoxia that was followed by similar changes in intestinal and RBC TBARS and plasma FRAP values upon reperfusion. In Group D the intestinal TBARS and LPO586 concentrations were significantly lower while FRAP and NO concentrations were significantly higher compared to the I/R group. At the same time RBC TBARS concentration and GPX activity significantly decreased within Group D. In Group A the intestinal LPO586 concentration was significantly lower than in the I/R group whilst RBC TBARS concentration showed a similar pattern. Plasma FRAP and NO concentration showed similar changes to those seen in Group D. It is concluded that I/R increased the LPO in the intestinal tissue and altered some parameters of plasma and RBCs, too. Deferoxamine treatment prevented these effects, while the usefulness of L-arginine remained doubtful.

Promoter recognition and discrimination by EsigmaS RNA polymerase.

Although more than 30 Escherichia coli promoters utilize the RNA polymerase holoenzyme containing sigmaS (EsigmaS), and it is known that there is some overlap between the promoters recognized by EsigmaS and by the major E. coli holoenzyme (Esigma70), the sequence elements responsible for promoter recognition by EsigmaS are not well understood. To define the DNA sequences recognized best by EsigmaS in vitro, we started with random DNA and enriched for EsigmaS promoter sequences by multiple cycles of binding and selection. Surprisingly, the sequences selected by EsigmaS contained the known consensus elements (-10 and -35 hexamers) for recognition by Esigma70. Using genetic and biochemical approaches, we show that EsigmaS and Esigma70 do not achieve specificity through 'best fit' to different consensus promoter hexamers, the way that other forms of holoenzyme limit transcription to discrete sets of promoters. Rather, we suggest that EsigmaS-specific promoters have sequences that differ significantly from the consensus in at least one of the recognition hexamers, and that promoter discrimination against Esigma70 is achieved, at least in part, by the two enzymes tolerating different deviations from consensus. DNA recognition by EsigmaS versus Esigma70 thus presents an alternative solution to the problem of promoter selectivity.

UP element-dependent transcription at the Escherichia coli rrnB P1 promoter: positional requirements and role of the RNA polymerase alpha subunit linker.

The UP element stimulates transcription from the rrnB P1 promoter through a direct interaction with the C-terminal domain of the RNA polymerase alpha subunit (alphaCTD). We investigated the effect on transcription from rrnB P1 of varying both the location of the UP element and the length of the alpha subunit interdomain linker, separately and in combination. Displacement of the UP element by a single turn of the DNA helix resulted in a large decrease in transcription from rrnB P1, while displacement by half a turn or two turns totally abolished UP element-dependent transcription. Deletions of six or more amino acids from within the alpha subunit linker resulted in a decrease in UP element-dependent stimulation, which correlated with decreased binding of alphaCTD to the UP element. Increasing the alpha linker length was less deleterious to RNA polymerase function at rrnB P1 but did not compensate for the decrease in activation that resulted from displacing the UP element. Our results suggest that the location of the UP element at rrnB P1 is crucial to its function and that the natural length of the alpha subunit linker is optimal for utilisation of the UP element at this promoter.

Mapping CooA.RNA polymerase interactions. Identification of activating regions 2 and 3 in CooA, the co-sensing transcriptional activator.

CooA is a CO-sensing protein that activates the transcription of genes encoding the CO-oxidation (coo) regulon, whose polypeptide products are required for utilizing CO as an energy source in Rhodospirillum rubrum. CooA binds to a position overlapping the -35 element of the P(cooF) promoter, similar to the arrangement of class II CRP (cAMP receptor protein)- and FNR (fumarate and nitrate reductase activator protein)-dependent promoters when expressed in Escherichia coli. Gain-of-function CooA variants were isolated in E. coli following mutagenesis of the portion of cooA encoding the effector-binding domain. Some of the mutations affect regions of CooA that are homologous to the activating regions (AR2 and AR3) previously identified in CRP and FNR, whereas others affect residues that lie in a region of CooA between AR2 and AR3. These CooA variants are comparable to wild-type (WT) CooA in DNA binding affinity in response to CO but differ in transcription activation, presumably because of altered interactions with E. coli RNA polymerase. Based on predictions of similarity to CRP and FNR, loss-of-function CooA variants were obtained in the AR2 and AR3 regions that have minimal transcriptional activity, yet have WT-like DNA binding affinities in response to CO. This study demonstrates that WT CooA contains AR2- and AR3-like surfaces that are required for optimal transcription activation.

Rate of lipid peroxidation in brain and liver tissues and the total antioxidant status of blood plasma in developing chicks.

Age-related changes of tissue lipid peroxidation (LPO) of liver and brain, as well as plasma antioxidant capacity of broiler chicken cockerels were investigated. Tissue LPO was characterised by the spectrophotometric assessment of thiobarbituric acid reactive substances (TBARS). Plasma antioxidant power was evaluated by the measurement of total antioxidant status (TAS). Newly hatched broiler chicks had similar TAS value (1.19 mmol/l) as newborns of mammalian species. Significant changes (p < 0.05) were observed in the time course of all parameters. Tissue TBARS concentration was higher in the brain than in the liver at hatching, while the latter organ was found to have more effective antioxidant defence during embryonic life. The concentration of TBARS increased up to the 10th day in the liver but only up to the 21st day in the brain, and the former was accompanied by an approximately 50% decrease of plasma antioxidant capacity. This suggests that the liver plays an important role in forming the antioxidant defence mechanisms of the blood plasma in broiler chicks.

Mechanism of regulation of transcription initiation by ppGpp. I. Effects of ppGpp on transcription initiation in vivo and in vitro.

To determine the role of ppGpp in both negative and positive regulation of transcription initiation during exponential growth in Escherichia coli, we examined transcription in vivo and in vitro from the growth-rate-dependent rRNA promoter rrnB P1 and from the inversely growth-rate-dependent amino acid biosynthesis/transport promoters PargI, PhisG, PlysC, PpheA, PthrABC, and PlivJ. rrnB P1 promoter activity was slightly higher at all growth-rates in strains unable to synthesize ppGpp (deltarelAdeltaspoT) than in wild-type strains. Consistent with this observation and with the large decrease in rRNA transcription during the stringent response (when ppGpp levels are much higher), ppGpp inhibited transcription from rrnB P1 in vitro. In contrast, amino acid promoter activity was considerably lower in deltarelAdeltaspoT strains than in wild-type strains, but ppGpp had no effect on amino acid promoter activity in vitro. Detailed kinetic analysis in vitro indicated that open complexes at amino acid promoters formed much more slowly and were much longer-lived than rrnB P1 open complexes. ppGpp did not increase the rates of association with, or escape from, amino acid promoters in vitro, consistent with its failure to stimulate transcription directly. In contrast, ppGpp decreased the half-lives of open complexes at all promoters, whether the half-life was seconds (rrnB P1) or hours (amino acid promoters). The results described here and in the accompanying paper indicate that ppGpp directly inhibits transcription, but only from promoters like rrnB P1 that make short-lived open complexes. The results indicate that stimulation of amino acid promoters occurs indirectly. The accompanying paper evaluates potential models for positive control of amino acid promoters by ppGpp that might explain the requirement of ppGpp for amino acid prototrophy.

Mechanism of regulation of transcription initiation by ppGpp. II. Models for positive control based on properties of RNAP mutants and competition for RNAP.

Strains containing ppGpp, a nucleotide whose synthesis is dependent on the RelA and SpoT proteins of Escherichia coli, display slightly lower rRNA promoter activity and much higher amino acid biosynthesis/transport promoter activity than deltarelAdeltaspoT strains. In the accompanying paper, we show that ppGpp directly inhibits rRNA promoter activity in vitro by decreasing the lifetime of the rrn P1 open complex. However, ppGpp does not stimulate amino acid promoter activity in vitro. We show here that RNA polymerase (RNAP) mutants, selected to confer prototrophy to deltarelAdeltaspoT strains, mimic the effects of ppGpp on wild-type RNAP. Based on the positions of the mutant residues that confer prototrophy in the structure of core RNAP, we suggest molecular models for how the mutants, and by analogy ppGpp, generally decrease the lifetime of open complexes. We show that amino acid promoters require higher concentrations of RNAP for function in vitro and in vivo than control promoters, and are more sensitive to competition for RNAP in vivo than control promoters. Furthermore, we show that the requirement of an amino acid promoter for ppGpp in vivo can be alleviated by increasing its rate-limiting RNAP-binding step. Our data are consistent with a previously proposed passive model in which ppGpp inhibits stable RNA synthesis directly by reducing the lifetime of the rrn P1 open complex, liberating enough RNAP to stimulate transcription from amino acid promoters. Our data also place considerable constraints on models invoking hypothetical factors that might increase amino acid promoter activity in a ppGpp-dependent fashion.

Chronic hepatitis in the dog--a review.

Chronic hepatitis is a heterogeneous group of inflammatory-necrotizing diseases of the liver. There is controversy in both human and veterinary medicine about the classification of chronic hepatitis and this is likely to remain until a classification based on aetiology rather than on morphology is introduced. Controversy exists as to whether chronic hepatitis in dogs is comparable to the human disorder. The aetiology of chronic hepatitis in dogs is poorly understood, whereas in humans an increasing number of viral causes have been found. Liver biopsy is essential for the diagnosis of chronic hepatitis both in dogs and in humans. Histopathological evaluation of the liver is required to make the diagnosis, which is based on the presence of liver cell necrosis and inflammatory reaction. The proposed criteria for the classification of hepatitis in dogs are then as follows: aetiology is the primary denominator (infectious, drug induced, autoimmune, or, if unknown, idiopathic). The other criteria are histopathological, with severity reflecting the severity of the necro-inflammatory activity (minimal, mild, moderate or severe) and chronicity reflecting the extent of fibrosis (none, mild, moderate, severe or cirrhosis).

Biochemical and antioxidant changes in plasma and erythrocytes of pentathlon horses before and after exercise.

Physical exercise in the horse induces a series of normal physiological and biochemical adaptations. Increasing metabolism and oxygen uptake may induce oxidative stress in various organs. The aim of this study was to examine exercise-induced changes in some plasma and RBC biochemical and antioxidant variables in pentathlon horses. Blood samples were taken from 14 horses before, immediately after, and 24 hours after competing in two 1-minute runs of intense exercise over jumps. The peak intensity periods were preceded by a 20-minute warm-up and separated by a 20-minute break. The following plasma biochemical analytes were determined: total protein, uric acid, and lactate concentrations, and lactate dehydrogenase (LDH) and creatine kinase (CK) activities. Total antioxidant status (TAS) and the ferric reducing ability of plasma (FRAP) also were measured. Thiobarbituric acid-reactive substances (TBARS), reduced glutathione (GSH), and total protein concentrations, and glutathione peroxidase (GSHPx) and superoxide dismutase (SOD) activities were determined in RBC hemolysates. Significantly increased concentrations of total protein, lactate, and FRAP, and increased activities of CK and LDH were observed immediately postexercise compared with pre-exercise samples (P<0.05). All results returned to approximately initial values after 24 hours of rest. RBC GSH and TBARS concentrations did not change immediately after exercise, but decreased after 24 hours of rest (P<0.05). Plasma uric acid and FRAP values were positively correlated in a linear model (r = 0.78). In summary, the type of exercise applied in this study, which can be considered quite usual for pentathlon horses, caused detectable biochemical and lipid peroxidative changes in plasma and RBCs. FRAP and TAS values changed in opposite directions, indicating that when antioxidant capacity is assessed using different methods, highly different results may be obtained.

UPs and downs in bacterial transcription initiation: the role of the alpha subunit of RNA polymerase in promoter recognition.

In recent years, it has become clear that promoter recognition by bacterial RNA polymerase involves interactions not only between core promoter elements and the sigma subunit, but also between a DNA element upstream of the core promoter and the alpha subunit. DNA binding by alpha can increase transcription dramatically. Here we review the current state of our understanding of the alpha interaction with DNA during basal transcription initiation (i.e. in the absence of proteins other than RNA polymerase) and activated transcription initiation (i.e. when stimulated by transcription factors).

Regulation of rRNA transcription is remarkably robust: FIS compensates for altered nucleoside triphosphate sensing by mutant RNA polymerases at Escherichia coli rrn P1 promoters.

We recently identified Escherichia coli RNA polymerase (RNAP) mutants (RNAP beta' Delta215-220 and beta RH454) that form extremely unstable complexes with rRNA P1 (rrn P1) core promoters. The mutant RNAPs reduce transcription and alter growth rate-dependent regulation of rrn P1 core promoters, because the mutant RNAPs require higher concentrations of the initiating nucleoside triphosphate (NTP) for efficient transcription from these promoters than are present in vivo. Nevertheless, the mutants grow almost as well as wild-type cells, suggesting that rRNA synthesis is not greatly perturbed. We report here that the rrn transcription factor FIS activates the mutant RNAPs more strongly than wild-type RNAP, thereby compensating for the altered properties of the mutant RNAPs. FIS activates the mutant RNAPs, at least in part, by reducing the apparent K(ATP) for the initiating NTP. This and other results suggest that FIS affects a step in transcription initiation after closed-complex formation in addition to its stimulatory effect on initial RNAP binding. FIS and NTP levels increase with growth rate, suggesting that changing FIS concentrations, in conjunction with changing NTP concentrations, are responsible for growth rate-dependent regulation of rrn P1 transcription in the mutant strains. These results provide a dramatic demonstration of the interplay between regulatory mechanisms in rRNA transcription.

Dog feeding test for assessing the nutritional adequacy of practical diets.

The nutritive value of dog foods declared by the manufacturer as nutritionally complete and balanced can be best assessed by feeding trials with dogs. A protocol of a feeding trial has been developed and tested with working dogs fed two different commercial complete and balanced diets for 8 weeks. The parameters used for evaluating the effect of diets were general health status, body and hair coat condition, change of body weight, haematological parameters (white blood cell (WBC) count, red blood cell (RBC) count, haemoglobin, packed cell volume), and biochemical parameters in blood serum (alanine aminotransferase, urea, albumin). The trial protocol proved to be appropriate to monitor the dogs' nutritional status and to reveal differences between diets. This method of evaluation is recommended for use in supporting the nutritional claims (labelling) of dog foods.