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G Breves

Publications and source records attributed to G Breves.

At least 37 records · Page 2Linked to original sources
PubMed2000

Comparative studies on Na-dependent Pi transport in ovine, caprine and porcine renal cortex.

In contrast to monogastric species, renal excretion of inorganic phosphate (Pi) in ruminants is low and this could be attributed to an almost complete tubular Pi reabsorption. However, the functional and regulatory basis for this phenomenon has not yet been clarified. Therefore, it was the aim of the present study to characterize the kinetic parameters of the tubular Pi reabsorption system as affected by P or Ca depletion using preparations of renal cortex brush border membrane vesicles (BBMV) from goats and sheep and to compare the data with respective parameters of porcine preparations. Na-dependent Pi uptake into renal cortex BBMV as a function of Pi concentration showed typical Michaelis-Menten kinetic and respective Scatchard plot analysis of the specific Pi uptake revealed linearity indicating the predominant presence of a single type of Pi transporters in the preparations. Under control conditions Vmax values of Na-dependent Pi uptake into BBMV were highest in goats and sheep and lowest in pigs (1.98, 1.39 and 0.95 nmol x mg(-1) protein x 10s(-1), respectively). Km values were not different between goats and sheep under all feeding conditions and ranged from between 0.34 mmol x l(-1) and 0.55 mmol x l(-1) which was three- to five-times higher than that found in pigs (0.11 mmol x l(-1)). Oligonucleotides derived from rat kidney cortex type IIa Na/Pi cDNA were used for reverse transcriptase-polymerase chain reaction (RT-PCR) in goat, sheep and pig kidney cortex. The products isolated were 768 bp for sheep and pigs and 765 bp for goats, with the respective amino acids sequences, representing a segment of approximately 40% in length of the entire transporter, exhibiting an at least 92% sequence homology between different species. From the results, involvement of type IIa Na/Pi cotransport in tubular Pi reabsorption in small ruminants can be postulated. However, it should not be considered that a potential role of other Pi transport systems be completely be excluded. Interestingly, neither P nor Ca depletion caused significant effects on Na-dependent Pi transport capacities and affinities in goats and sheep. From this, parathyroid-hormone independent regulatory pathways of tubular Pi reabsorption can be assumed.

Animals↗
PubMed2000

Application of the colon-simulation technique for studying the effects of Saccharomyces boulardii on basic parameters of porcine cecal microbial metabolism disturbed by clindamycin.

AIMS: The present study analyzed the effects of Saccharomyces boulardii on the biochemical parameters of microbial hindgut metabolism disturbed by clindamycin. METHODS: The experiments were carried out under in vitro conditions using the semicontinuous colon-simulation technique. This technique is standardized for quantitatively measuring parameters of microbial hindgut metabolism. The fluid and particle phase of pig hindgut contents were used for the in vitro incubations. The 5-day control period was followed by clindamycin exposure alone (312.5 mg/day for 5 days) or by a combined treatment of clindamycin and S. boulardii (400 mg/day for 5 days). RESULTS: Clindamycin resulted in significant decreases in production rates of short-chain fatty acids (SCFAs) which were associated with substantial changes in molar SCFA proportions at the expense of butyrate. These effects could at least partly be compensated for by S. boulardii, in particular by enhancements of acetate and propionate fermentation to control levels. In contrast, butyrate fermentation could not be reconstituted. In a second experiment the potential use of S. boulardii as a substrate for hindgut microbial metabolism was studied by comparing living and autoclaved yeast. Propionate and butyrate fermentation rates were unaffected whereas acetate fermentation tended to be higher in the presence of living yeast. CONCLUSIONS: S. boulardii can be effective to compensate for changes in microbial fermentation in response to antibiotic treatment. Despite the lack of statistical significance it might be concluded that the increase in fermentation end products can only partly be explained by the utilization of the yeast as a substrate for microbial metabolism.

Analysis of Variance↗
PubMed2000

[Computer simulation programs as an alternative for classical nerve, muscle and heart experiments using frog tissues].

Courses in Physiology include different methodical approaches such as exercises with living animals, experiments using organs or tissues from killed or slaughtered animals, application of diagnostic techniques in humans and theoretical seminars. In addition to these classical approaches computer programs for multimedia simulation of nerve, muscle and heart physiology are now a regular component of courses in Physiology at the School of Veterinary Medicine in Hannover. It is the aim of the present paper to give the first experiences about these new components.

Animal Testing Alternatives↗
PubMed1999

Functional expression and characterization of the wild-type mammalian renal cortex sodium/phosphate cotransporter and an 215R mutant in Saccharomyces cerevisiae.

The wild-type and an R215E mutant of the rat renal cortex sodium/phosphate cotransporter type 2 (NaPi-2) were functionally expressed in the yeast Saccharomyces cerevisiae strain MB192, a cell line lacking the high-affinity endogenous H+/P(i) cotransporter. The expression of the mRNA molecules and corresponding proteins was confirmed by Northern and Western blot analysis, respectively. As detected by indirect immunofluorescence and antibody capture assay, both wild-type and mutant NaPi-2 proteins are expressed in the yeast plasma membrane in comparable amounts. In the presence of 5 microM phosphate, Na+ promotes phosphate uptake into yeast cells expressing the wild-type NaPi-2 with a K(0.5) of 5.6 +/- 1.1 mM. The maximum uptake of phosphate (649 +/- 30 pmol/10 min) is approximately 8-fold higher than the uptake obtained with nontransformed cells (76.8 +/- 8 pmol/10 min). Yeast cells expressing the R215E mutant of NaPi-2 accumulate 213 +/- 9 pmol of phosphate/10 min under the same conditions. The K(0.5) for the stimulation of phosphate uptake by Na+ is 4.2 +/- 0.8 mM for the R215E mutant and thus not significantly different from the value obtained with cells expressing the wild-type cotransporter. The reduced level of accumulation of phosphate in yeast cells expressing the R215E mutant is probably due to a reduction of the first-order rate constant k for phosphate uptake: while cells expressing wild-type NaPi-2 accumulate phosphate with a k of 0.06 min(-1), the rate for phosphate uptake into cells expressing the R215E mutant (k) is 0.016 min(-1) and therefore about 4-fold lower. In comparison, the rate for phosphate uptake into nontransformed cells (k) is 0.0075 min(-1). Phosphate uptake into yeast cells that express the wild-type NaPi-2 in the presence of 150 mM NaCl is promoted by extracellular phosphate with a K(0.5) of 45 +/- 4 microM. A phosphate-dependent phosphate accumulation is also observed with cells expressing the R215E mutant, but the K(0.5) is twice as high (86 +/- 5 microM) as that obtained with the wild-type cotransporter. We conclude that the yeast expression system is a useful tool for the investigation of structure-function relationships of the renal sodium/phosphate cotransporter and that (215)R, although not involved in Na+ recognition, is a part of the structure involved in phosphate recognition and considerably influences the rate of phosphate uptake by the NaPi-2 cotransporter.

Animals↗
PubMed1999

In vitro studies on active calcium absorption from ovine rumen.

From various in vivo and in vitro studies it has been shown that the rumen represents a significant site of Ca2+ absorption in sheep and goats. It was the aim of the present study to further characterize the underlying mechanisms. Unidirectional flux rates of Ca2+ across rumen wall epithelia of sheep were measured in vitro by applying the Ussing-chamber technique in the absence of electrochemical gradients. Under these conditions, significant Ca2+ net flux rates (Jnet) clearly indicate the presence of active mechanisms for Ca2+ transport. Short chain fatty acids (SCFAs) caused highest stimulation of Ca2+ Jnet (6.3 +/- 1.9 nmol.cm-2.h-1) when used as a mixture of acetate, proprionate and butyrate in physiological proportions (36, 15, 9 mmol.l-1, respectively). The effect of 30 mmol.l-1 butyrate (3.2 +/- 0.6 nmol.cm-2.h-1) was higher than respective amounts of propionate and acetate (0.6 +/- 0.8 nmol.cm-2.h-1 and 0.9 +/- 0.8 nmol.cm-2.h-1, respectively). Eliminating SCFAs resulted in Ca2+ Jnet of 0.4 +/- 1.1 nmol.cm-2.h-1. Addition of Ca channel blocker verapamil (mucosal 1 mmol.l-1) had no significant effect on SCFA-stimulated Jnet of Ca2+, whereas application of Na+/H- inhibitor amiloride (mucosal 1 mmol.l-1) further enhanced the Ca2+ Jnet by > 65%. The Ca(2+)-pump inhibitor vanadate had no significant effect on Jnet of Ca2+. Dietary Ca depletion enhanced calcitriol plasma concentrations but had no effect on active Ca2+ absorption across the rumen wall of sheep. In addition, no effect on active Ca2+ absorption could be observed during early lactation. In conclusion, there is clear evidence for the rumen as a main site for active Ca2+ absorption in sheep. Our results suggest the presence of a Ca2+/H+ exchange mechanism in the apical membrane of rumen epithelial cells which depends on SCFA absorption and which does not seem to be under the control of calcitriol. Basolateral Ca2+ extrusion occurs independently from Ca(2+)-pump activity and may be accomplished via Na+/Ca2+ exchange.

Absorption↗
PubMed1999

Characteristics of dipeptide transport in pig jejunum in vitro.

Characteristics of dipeptide transport in pig jejunum were investigated in vitro by applying the Ussing-chamber technique and mucosal uptake studies. Addition of both glycyl-L-glutamine and glycyl-L-sarcosine (20 mmol.l-1) to the mucosal buffer solution significantly increased the short-circuit current by 2.60 +/- 0.15 and 1.57 +/- 0.20 mu eq.cm-2.h-1, respectively. Concentration-dependent changes in short-circuit current followed Michaelis-Menten kinetics with similar affinity constants for both dipeptides. From unidirectional flux rates for radiolabelled glycyl-L-sarcosine, a net flux rate for glycyl-L-sarcosine of 49.8 +/- 6.7 nmol.cm-2.h-1 was calculated. In mucosal uptake experiments, the apical influx of 14C-labelled glycyl-L-sarcosine into isolated porcine mucosa was pH dependent and significantly inhibited by glycyl-L-glutamine. Moreover, RT-PCR studies with primers derived from rabbit PepT1 identified two PCR fragments of identical size to rabbit PepT1 from pig intestinal mRNA preparations. In conclusion, our studies revealed key features of mammalian intestinal peptide transporters and give evidence for a PepT1-like transporter in the pig jejunum that could significantly contribute to the overall amino acid absorption from the gut.

Animals↗
PubMed1999

Influence of dietary phosphorus depletion on central pathways of intermediary metabolism in rats.

Studies on P depleted rats compared with control animals kept under pair fed conditions were carried out. Dietary P depletion led to reduced weight gain and a decreased food conversion ratio in comparison with pair fed control animals. Higher N retention, higher urea concentrations in plasma, liver and kidney tissues and a significant reduction of renal glutamate dehydrogenase--a central enzyme of amino acid degradation in kidney mitochondria--in P depleted animals indicated changes in N utilization and N excretion. While lipid metabolism was not affected by P depletion, carbohydrate metabolism was substantially changed in the kidney: Activity of fructose diphosphatase was significantly reduced, implicating a reduced gluconeogenesis in P depletion. Possible mechanisms of these metabolic effects in P depletion are discussed.

Animals↗
PubMed1999

Morphological and functional development of the rumen in the calf: influence of the time of weaning. 1. Morphological development of rumen mucosa.

The objective of this study was to determine whether the nutritional regimen of rearing calves would influence the morphometric and histological development of rumen mucosa. Twelve male Holstein calves 7 d of age were assigned to three groups of 4 animals each: milk group (I), early weaned (6 weeks) group (II) and late weaned (9 weeks) group (III). All animals received additional solid feed. Animals of group I were slaughtered after 6 weeks of age, whereas those in groups II and III were slaughtered after 9 weeks of age. At slaughter, the ruminal digesta amounted to 2035 g (milk group), 3092 g (late weaned group) and 5374 g (early weaned group). The differences in the ruminal molar percentage of SCFA were not significant. There was a trend for lower pH and higher SCFA concentrations in the order late weaned, early weaned and milk fed animals (pH: 6.4, 6.6 and 6.7, respectively; SCFA: 96, 87 and 77 mmol/l, respectively). The mean length (1.07 mm in milk group, 1.45 mm in late weaned group and 1.87 mm in early weaned group), width (0.43, 0.58 and 0.71 mm, respectively) and surface of papillae (190, 232 and 241 mm2/cm2 mucosa, respectively) increased with both the age of the animals and the elevated intake of solid feed, whereas the number of papillae (210, 140 and 92 per cm2 mucosa, respectively) decreased. In both milk-fed groups type A and B corneal cells were present in the Stratum corneum, whereas in the earlier weaned calves type C-cells could be also seen. These findings indicate a more advanced stage of development of the rumen epithelium in the earlier weaned calves fed higher amounts of concentrate and hay.

Animal Feed↗
PubMed1998

Analysis of parotid and mixed saliva in Roe deer (Capreolus capreolus L.).

In ruminants, different functions have been ascribed to the different salivary glands according to the feeding type. In this context, possible adaptations of salivary functions were investigated regarding the secretion of various proteins by different types of salivary glands. To yield uncontaminated parotid saliva in large quantities, a non-surgical method has been developed. Parotid gland secretions were collected via endoscopic placement of guide wires into each parotid duct, which were subsequently used for placement of collection catheters. Salivary flow was stimulated by intra-glandular administration of the parasympathomimetic compound pilocarpine-hydrochloride into the parotid gland. Mixed saliva (excluding parotid saliva) was collected into sterile tubes by normal outflow during the sampling of parotid saliva. The total flow volume, flow rate and the content of proteins as well as of several ions (Na+, K+, Ca2+, inorganic phosphate) of both types of saliva were measured in sheep, fallow deer and roe deer. Roe deer secreted the highest amount of total salivary proteins relative to body mass [mg/kg body mass] and the highest relative volume [ml/10 min/kg body mass], both in parotid and mixed saliva, of all ruminant species examined. Additionally, the protein profile and the tannin-binding properties of parotid and mixed saliva in roe deer were investigated. Parotid saliva bound almost twice as much tannin as mixed saliva, underlining the importance of yielding uncontaminated parotid saliva for tannin-binding studies.

Adaptation, Physiological↗
PubMed1998

Renal phosphate excretion in goats.

It was the aim of these experiments to characterize further the role of salivary glands and kidneys in phosphate (Pi) homeostasis of small ruminants. Renal clearance experiments were performed in five female, non-pregnant adult goats during infusions of physiological NaCl or isotonic phosphate solution. Basal plasma Pi concentrations of 1.6 +/- 0.3 mM (NaCl infusion) were associated with a renal resorption of the filtered Pi by 98.5-99.3% and Pi was excreted in the urine at a rate of 1.2-2.8 mumol/min. Elevating the plasma Pi levels up to 7.2 +/- 1.7 mM resulted in significant increases of Pi excretion. The mean plasma threshold for renal Pi excretion of these animals was high (4.3 +/- 1.0 mM) and thus substantially higher than the physiological range of plasma Pi. The maximal tubular resorption rate for Pi in goats was found to be higher than the respective values in monogastric animals. In relation to the enhanced Pi concentrations in plasma the salivary Pi concentrations increased simultaneously and reached a maximum level of about 66.3 mM at plasma levels between 4-6 mM. Indications for hormonal regulation mechanisms via parathyroid hormone (PTH) and calcitriol could not be detected during the infusion periods.

Animals↗
PubMed1998

Duodenal Ca2+ absorption is not stimulated by calcitriol during early postnatal development of pigs.

The role of calcitriol in stimulating intestinal active Ca2+ absorption during postnatal life was studied in newborn, suckling, and weaned control (Con) piglets and piglets suffering from inherited calcitriol deficiency (Def piglets). In addition, a group of Def piglets was treated with vitamin D3 (Def-D3 piglets), which normalized plasma calcitriol levels. Regardless of age, duodenal calbindin-D9k concentrations ranged between 1,839 and 2,846 microg/g mucosa in Con piglets, between 821 and 1,219 microg/g mucosa in Def piglets, and between 2,960 and 3,692 microg/g mucosa in Def-D3 animals. In weaned animals, active Ca2+ absorption as calculated from in vitro 45Ca2+ flux rate measurements in Ussing chambers could be related to calbindin-D9k levels. Thus active Ca2+ absorption was completely absent in Def animals but was reconstituted in Def-D3 animals. In contrast, in newborn Def piglets active Ca2+ absorption functioned normally despite the low plasma calcitriol and mucosal calbindin-D9k levels and could not be affected by treatment with vitamin D3. Similar results were obtained from suckling Def piglets. The microtubule-disrupting agent colchicine caused significant inhibition of transepithelial net Ca2+ absorption in duodenal epithelia from newborn piglets without exerting an effect in suckling and weaned animals. Colchicine had no effect on Ca2+ uptake across the brush border membrane of mucosal enterocytes or on glucose-dependent electrogenic net ion flux rates in duodenal preparations from newborn Con piglets. In conclusion, our findings reveal intestinal active Ca2+ absorption during early postnatal life of pigs that involves calcitriol-independent mechanisms and that may include intact microtubule actions.

Aging↗
PubMed1998

Phosphate transport in pig proximal small intestines during postnatal development: lack of modulation by calcitriol.

The role of calcitriol in the intestinal absorption of inorganic phosphate (Pi) during postnatal development was studied in newborn [<1 week postpartum (pp)], suckling (3-4 weeks pp), and weaned (>6 weeks pp) control piglets (con) and piglets suffering from inherited calcitriol deficiency (def). In addition, a number of def piglets were treated with vitamin D3 (def-D3). Regardless of age, plasma calcitriol concentrations in def piglets were unphysiologically low (16-21 pg/ml) and differed significantly from those in respective con animals (60-69 pg/ml) and vitamin D3-treated def piglets (50-56 pg/ml). However, newborn and suckling def piglets had normal Ca (approximately 3.0 mmol/liter) and Pi (approximately 2.8 mmol/liter) plasma levels. Def piglets became hypocalcemic (1.9 mmol/liter) and hypophosphatemic (1.9 mmol/liter) between 4-6 weeks pp. Treatment with vitamin D3 significantly increased plasma Ca (3.2 mmol/liter) and Pi (2.7 mmol/liter) levels in weaned def animals. Regardless of calcitriol status, net Pi flux rates (active Pi absorption, as determined with the in vitro Ussing-chamber technique) from the upper small intestines was maximal at birth [170-224 nmol/(cm2 x h)] and decreased by approximately 80% during the first week of life before remaining constant [30-50 nmol/(cm2 x h)] during the following development. In weaned def piglets, net Pi flux rates were significantly lower by about 80% compared with those in con animals. Treatment of def piglets with vitamin D3 had no effect in newborn and suckling animals but reconstituted net Pi flux rates to normal values at weaning age. Age-dependent and calcitriol-mediated changes in net Pi flux rates were paralleled by respective maximum velocity values of Na+-dependent Pi uptake across the brush border membrane of the enterocytes (newborn piglets, 1.9-2.2 nmol/(mg protein 10 sec); suckling piglets, 0.4-0.6 nmol/(mg protein x 10 sec); weaned piglets, 0.7, 0.3, and 0.7 nmol/(mg protein x 10 sec) in con, def, and def-D3 animals, respectively). These findings suggest that the apical Pi uptake represents the major rate-limiting step of the overall transepithelial Pi transport. At weaning, Na+/Pi transport across the intestinal brush-border membrane is clearly stimulated by calcitriol, but no significant effects of age or calcitriol on the Km values (0.5-0.7 mmol/liter) were observed. In conclusion, our findings reveal calcitriol-independent mechanisms for active intestinal Pi absorption during the neonatal and suckling periods. The onset of the classical calcitriol-dependent mechanism for active intestinal Pi absorption does not occur until weaning.

Animals↗
PubMed1997

In vitro studies on calcium absorption from the gastrointestinal tract in small ruminants.

Unidirectional flux rates of Ca2+ across gastrointestinal tissues from sheep and goats were measured in vitro by applying the Ussing-chamber technique. Except for the sheep duodenum, mucosal to serosal Ca2+ flux rates (Jms) exceeded respective flux rates in the opposite direction (Jsm) in both species and in all segments of the intestinal tract. This resulted in net Ca2+ flux rates (Jnet = Jms-Jsm) ranging between -2 and 9 nmol.cm-2.h-1 in sheep and between 10 and 15 nmol cm-2.h-1 in goats. In sheep, only Jnet in jejunum, and in goats, Jnet in duodenum and jejunum were significantly different from zero. Using sheep rumen wall epithelia, significant Jnet of Ca2+ of around 5 nmol.cm-2.h-1 could be detected. Since the experiments were carried out in the absence of an electrochemical gradient, significant net Ca2+ absorption clearly indicates the presence of active mechanisms for Ca2+ transport. Dietary Ca depletion caused increased calcitriol plasma concentrations and induced significant stimulations of net Ca2+ absorption in goat rumen. Jnet of Ca2+ across goat rumen epithelia was significantly reduced by 1 mmol.l-1 verapamil in the mucosal buffer solution. In conclusion, there is clear evidence for the rumen as a main site for active Ca2+ absorption in small ruminants. Stimulation of active Ca2+ absorption by increased plasma calcitriol levels and inhibition by mucosal verapamil suggest mechanistic and regulatory similarities to active Ca2+ transport as described for the upper small intestines of monogastric species.

Animals↗
PubMed1997

In vitro studies on transport and metabolism of short-chain fatty acids in pig hindgut.

An analytical method based on alkaline freeze drying, ultracentrifugation, and quantitative gas chromatography was established to differentiate between mucosal uptake, tissue accumulation, and serosal release of SCFA in pig hindgut. It was shown that serosal release of SCFA was substantially lower than mucosal uptake and tissue accumulation, indicating substantial degradation and/or metabolism during transepithelial movement.

Animals↗
PubMed1997

[Suffering and misfortune--the beginning of comprehension].

Pain, suffering and damage are important items of the animal protection law and thus the basis for its accomplishment. It is the aim of this paper to give a review on those methods which are available to record and to assess well-being or suffering of animals. Since suffering cannot be measured directly different indicators such as health, productivity, physiological and biochemical parameters and behaviour have to be used in order to obtain conclusive evidence of suffering or well-being. Since it has to be taken into account that the individual indicators are of limited value with respect to their reliability and accuracy the combination of these indicators has to be recommended.

Animal Welfare↗
PubMed1996

Role of calbindin-D9k in buffering cytosolic free Ca2+ ions in pig duodenal enterocytes.

1. The aim of the present study was to test whether the vitamin D-dependent Ca(2+)-binding protein calbindin-D9k could function as an important cytosolic Ca2+ buffer in duodenal enterocytes while facilitating transepithelial active transport of Ca2+ ions. For the investigations we used dual-wavelength, fluorescence ratio imaging, with fura-2 as the Ca(2+)-sensitive dye, to measure changes in cytosolic concentrations of free Ca2+ ions ([Ca2+]i) in isolated pig duodenal enterocytes affected by different cytosolic calbindin-D9k concentrations. 2. Epithelial cells were obtained from weaned piglets with normal calbindin-D9k concentrations (con-piglets), from piglets with low calbindin-D9k levels due to inherited calcitriol deficiency caused by defective renal 25-hydroxycholecalciferol D3-1 alpha-hydroxylase activity (def-piglets), and from piglets with reconstituted calbindin-D9k concentrations, i.e. def-animals treated with high doses of vitamin D3 which elevated plasma calcitriol levels by extrarenal production (def-D3-piglets). Basal levels of [Ca2+]i ranged between 170 and 205 nM and did not differ significantly between the groups. 3. After addition of 5 mM theophylline, the [Ca2+]i in enterocytes from con-piglets doubled during the 10 min incubation. This effect, however, was three times higher in enterocytes from def-piglets compared with those from con-piglets. Similar results were obtained after 4 min incubation of enterocytes from con- and def-piglets in the presence of 1 microM ionomycin. In preparations from def-D3-piglets, ionomycin-induced increases in [Ca2+]i were significantly lower compared with enterocytes from def-piglets and were not different from the control values. 4. From the results, substantial support is given for the hypothesis that one of the major functions of mucosal calbindin-D9k is the effective buffering of Ca2+ ions.

Animals↗