Preliminary observations on the relationship between gas production and microbial protein synthesis in vitro.

The relationship between gas production and microbial protein synthesis was studied in vitro using the method of MENKE et al. (1979). 150 mg starch or cellulose or a mixture of 10% glucose, 40% starch and 50% cellulose was used as the carbohydrate source. The microbial protein synthesis and gas production occurring during 2 hrs after the 5th, 10th, and 23rd hr of incubation were studied. Total and net microbial synthesis were estimated using 32P as a microbial marker and by the net disappearance of NH3-N respectively. The data indicate that the type of carbohydrate and the rate of carbohydrate fermentation influence microbial protein synthesis per unit volume of gas produced. However, the relationship between total synthesis and cumulative gas production (up to 8 hrs incubation) with carbohydrate mixture as the substrate was linear. With reference to these observations, the possibilities and difficulties in using cumulative gas production as an index of microbial growth potential of the feedstuffs are discussed.

Chemical models for the chemical nature of endogenous digitalis.

The inability or the capacity to promote the phosphorylation of Na+/K(+)-transporting ATPase (Na/K-ATPase) from [32P]Pi is shown to differentiate between mechanistically digitalis-unlike and digitalis-like inhibitors of this enzyme known to be the receptor for all digitalis actions. A negative or positive response in the phosphorylation promotion assay introduced here appears thus to be suitable to diagnose the chemical species in the isolates of animal origin related to the putative endogenous digitalis. Various digitalis-congeneric C/D-cis steroids, progesterone-congeneric C/D-trans steroids and the Erythrophleum alkaloid cassaine promote the enzyme phosphorylation and show a similar pattern of discrimination between three Na/K-ATPase variants. Thus, their cyclopentanoperhydrophenanthrene or perhydrophenanthrene nuclei appear to serve as the minimal pharmacophoric lead structures for bimolecular recognition and to represent chemical models for the chemical nature of endogenous digitalis. Specifically, the hormonal C/D-trans steroids could provide the basic skeleton in endogenous digitalis biosynthesis.

Interaction of progesterone derivatives with the digitalis target enzyme: impact of glycosidation on inhibitory potency.

As a function of the structural modification of the steroid nucleus, the inhibitory interaction of 11 progesterone derivatives with human Na/K-ATPase (Na+/K(+)-transporting ATPase, EC 3.6.1.37), through C3-O-rhamnosylation, is either much decreased or weakly up to strongly increased, so that the rhamnosyl residue contributes to the complementary Gibbs energy of interaction, at the most, the same Gibbs energy increment as realized in ouabain. After C3 beta-O-rhamnosylation, the activity of some progesterone derivatives considerably surpasses that of 3 beta-O-rhamnosyl-chlormadinolacetate, which has been known to elicit positive inotropy in cats. The progesterone derivatives (aglycons and glycosides), that have been analysed more closely, produce their effects by the same molecular mechanism of interaction with Na/K-ATPase as characteristic for digitalis aglycons and glycosides. The results promise to pave the way for the identification of the chemical nature of endogenous digitalis and for the design of novel inotropic drugs.

Studies on in vitro degradation and in vivo digestion of a slow ammonia releasing urea product.

a slow ammonia releasing urea product (Uromalt) has been produced by autoclaving and drying of germinated barley and urea. In vitro degradability of nitrogen (IVDN, Raab et al., 1983) was in average 62 +/- 9% in 4 hours, compared to 76 +/- 8% with an autoclaved and dried mixture of urea and not-germinated barley. Mixtures of urea with Leucaena l. or Azadarichta indica (Neem cake) showed degradabilities of about 80 and 93%, respectively, when dried at 39 degrees C, but of 65% when dried at 100 degrees C. For comparison, degradabilities of proteins of ground nut, guar, sunflower, mustard, rape and sesame cake/meal have been determined. When growing lambs were fed a ration containing 15% Uromalt, faecal N-excretion was significantly higher on the expense of urinary N-excretion, compared with groups fed isonitrogenous rations containing soya bean meal with or without urea and molasses. N-retention was not significantly affected. It is concluded that this slow ammonia releasing urea product can be used in cases where molasses or other feedingstuffs rich in rapidly fermentable carbohydrates are not available.

5 Beta,14 beta-androstane-3 beta,14-diol binds to the digitalis receptor site on Na/K-ATPase.

5 beta,14 beta-Androstane-3 beta,-14-diol, the lead (minimum) structure in digitalis compounds, shows the same characteristics of interaction with Na/K-ATPase as ordinary digitalis compounds judged by the following six criteria: (I) shape of the concentration-inhibition curves, (II) species differences in affinity for the enzyme, (III) apparent competition with K+, (IV) competition with digitoxigenin for binding to the enzyme, (V) stabilization of phosphoenzyme formed from ATP, and (VI) enhancement of phosphorylation from orthophosphate.

Origin of differences of inhibitory potency of cardiac glycosides in Na+/K+-transporting ATPase from human cardiac muscle, human brain cortex and guinea-pig cardiac muscle.

The inhibitory potency of altogether 95 steroidal compounds (including cardenolides, bufadienolides and their glycosides) on the Na/K-ATPases (Na+/K+-transporting ATPases, EC 3.6.1.37) from human cardiac muscle, human brain cortex and guinea-pig cardiac muscle was compared to probe the complementary chemotopology of the inhibitor binding site areas on the three enzyme variants. The changes of potency, resulting from systematic variations of the geometry of steroid skeleton and the character as well as the structure of side chains at C3 or/and C17 of steroid backbone, allowed the following major conclusions. With the human cardiac and cerebral enzyme forms, the paired K0.5 (K'D) values for 77 steroid derivatives, covering seven orders of ten, were highly correlated. On an average, the total of compounds showed a 1.5-fold higher affinity to the cardiac enzyme. This tiny differentiation did not appear to be connected with an important difference in the chemotopology of the complementary subsites for steroid nucleus binding on the two enzyme forms. With the human and guinea-pig cardiac enzyme variants, the K0.5 values for 69 steroid derivatives, covering six orders of ten, were determined. For 41 5 beta, 14 beta-androstane derivatives only, the paired K0.5 values showed a close correlation. Here, the human enzyme variant exhibited 27-fold higher affinity. However, the paired K0.5 values determined on both enzymes for 28 steroid derivatives of differing structural features were but poorly correlated. Essentially, the geometries of the steroid nucleus determined the differential contributions of the side chains at C3 and C17 to the integral inhibitory potency on the two enzyme variants. Thus, the species differences in the potency of cardiac glycosides were traced to species differences in the complementarity of the steroid binding subsites. Hence, estimates of the potency of new steroidal compounds obtained on the guinea-pig cardiac enzyme can be neither quantitatively nor qualitatively easily extrapolated to the human cardiac enzyme. The extrathermodynamic analysis of the data opened major new insights in the structure-activity relationships concerning the role of C14 beta-OH, the character of the lead structure in cardioactive steroid lactones, and the significance of the configuration of A/B ring junction.

Evidence against parallel operation of sodium/calcium antiport and ATP-driven calcium transport in plasma membrane vesicles from kidney tubule cells.

The present study aimed to clarify the existence of a Na+/Ca2+ antiport device in kidney tubular epithelial cells discussed in the literature to represent the predominant mechanistic device for Ca2+ reabsorption in the kidney. Inside-out oriented plasma membrane vesicles from tubular epithelial cells of guinea-pig kidney showed an ATP-driven Ca2+ transport machinery similar to that known to reside in the plasma membrane of numerous cell types. It was not affected by digitalis compounds which otherwise are well-documented inhibitors of Ca2+ reabsorption. The vesicle preparation contained high, digitalis-sensitive (Na+ + K+)-ATPase activities indicating its origin from the basolateral portion of plasma membrane. The operation of a Na+/Ca2+ antiport device was excluded by the findings that steep Ca2+ gradients formed by ATP-dependent Ca2+ accumulation in the vesicles were not discharged by extravesicular Na+, and did not drive 45Ca2+ uptake into the vesicles via a Ca2+-45Ca2+ exchange. The ATP-dependent Ca2+ uptake into the vesicles became increasingly depressed with time by extravesicular Na+. This was not due to an impairment of the Ca2+ pump itself, but caused by Na+/Ca2+ competition for binding sites on the intravesicular membrane surface shown to be important for high Ca2+ accumulation in the vesicles. Earlier observations on Na+-induced release of Ca2+ from vesicles pre-equilibrated with Ca2+, seemingly favoring the existence of a Na+/Ca2+ antiporter in the basolateral plasma membrane, were likewise explained by the occurrence of Na+/Ca2+ competition for binding sites. The weight of our findings disfavors the transcellular pathway of Ca2+ reabsorption through tubule epithelium essentially depending on the operation of a Na+/Ca2+ antiport device.

Kinetic studies on interaction of (Na,K)-ATPase with Cibacron Blue F3GA as probe of the nucleotide fold.

Cibacron Blue F3GA (Cb) effectively and reversibly inhibits the activity of (Na,K)-ATPase. Its inhibitory effect does not occur through occupation of the ouabain binding site, but presumably results from Cb-occupation of one catalytic site not competitively attracting ATP. Cb also inhibits ouabain binding to (Na,K)-ATPase. Its inhibitory effect is competitively antagonized by ATP proving accommodation of Cb in the ATP binding site. - If one admits Cb as a suitable analytical tool for the detection of a supersecondary structure folding pattern, the findings suggest that the ATP binding site is lined by beta-pleated sheets flanked by alpha-helices thus providing an environment that funnels ATP to the catalytic site.

[Nutritive physiological effect of dietary fats in growing swine rations. 3. Effect of sunflower and coconut kernels on protein and fat retention, backfat fatty acid pattern and various blood parameters in piglets]. / Untersuchungen über ernährungsphysiologische Wirkungen von Futterfetten in Rationen für wachsende Schweine. 3. Mitteilung. Einfluss von Sonnenblumen- und Kokoskernen auf den Protein- und Fettansatz, das Fettsäurenmuster des Rückenspecks sowie auf einige Blutparameter bei Ferkeln.

Two groups, each of 9 castrated male piglets of the German Landrace breed, body weight 13-29 kg, were fed ad libitum for a 5 week period in order to determine the influence of the fatty acid composition of the dietary fat on protein and fat retention. The concentrations of urea, insulin, glucose, free glycerol and triglycerides were also determined. That fat in ration I (derived from 27% sunflower seed) contained 50% linoleic acid while that of ration II (derived from 12,5% coconut kernels) had a maximum lauric acid content of 41,8%. The relationship between digestible crude protein and ME-content was calculated to be similar in both rations. In addition protein quality was calculated to be similar in both rations. Protein deposition was determined from both nitrogen and comparative slaughter procedures. The results can be summarized as follows: The animals fed ration I had a higher daily ME-intake and growth rate than those fed ration II, although no great difference was observed in ME required per kg body weight gain. The efficiency of protein utilization was significantly better in group I and this was associated with a lower blood urea concentration. The concentrations of insulin and glucose in the blood, measured before as well as 20, 45, 60 and 120 minutes following feeding, showed little differences between the groups. The triglyceride concentration measured before and 1 and 5 hours following feeding was significantly higher (p less than 0,01) in group II. There was no significant difference in the protein and fat content of the body as well as in the daily protein and fat deposition. The fatty acid composition of the back fat was significantly influenced by the fatty acid pattern of the dietary fat. In group I the linoleic acid content was 3,6 times higher and the lauric- and myristic acid content 11,7 times lower compared to that of group II.

Rumen protein degradation and biosynthesis. I. A new method for determination of protein degradation in rumen fluid in vitro.

A method is described for the determination of protein degradation based on measurements of ammonia concentration and gas production (Menke et al. 1979) when a feedingstuff was incubated with rumen fluid in vitro. NH3 liberated during incubation is in part used for microbial protein synthesis. Production of carbon dioxide and methane can be regarded as a measure of energy available for protein synthesis. The ratio, gas production: incorporation of NH3-nitrogen was estimated by addition of starch to the substrate. The response in gas production was linear in the range 0-200 mg starch, when starch was added to 0-200 mg feedingstuff dry matter and 30 ml rumen fluid-medium mixture. Linear regression between NH3-N concentration (y, mg) and gas production (x, ml) yielded an intercept (bo) representing that amount of NH3-N which would be released when no fermentable carbohydrates were available and consequently no bacterial protein synthesis took place. The difference between this intercept bo and NH3-N content in the blank (rumen fluid without substrate added) indicated the amount of NH3 liberated from protein and other N-containing compounds of the feeding-stuff incubated. In vitro-degradable N (IVDN) was calculated as a proportion of total N by the equation: (formula; see text).

[Nutritional physiology effect of dietary fats in the diet of growing pigs. 1. Effect of an isocaloric exchange of carbohydrate energy compared with fat energy on protein retention, in vitro synthesis of fat from glucose and on several blood parameters in piglets]. / Untersuchungen über ernährungsphysiologische Wirkungen von Futterfetten in Rationen für wachsende Schweine. 1. Mitteilung. Einfluss eines isokalorischen Austausches von Kohlenhydratenenergie gegen Fettenergie auf den Proteinansatz, die in-vitro-Fettsynthese aus Glukose sowie auf einige Blutparameter bei Ferkeln.

Three groups of male, castrated piglets of the German Landrace breed, weight range 3-30 kg, were used to study the relation between fattening performance and blood parameters when feeding rations containing different amounts of fat. The fat content of the rations was either 5% (group I), 18% (group II) or 35% (group III). Concomitantly with the increased metabolizable energy (ME) content of the ration the content of digestible protein (DP) was increased and the amount of feed reduced in order to guarantee an equal intake of ME and DP in all three groups. The digestibility of the crude nutrients and protein retention of the subjects were determined in nine subsequent trials each lasting 7 days. At a body weight of 27 kg the blood concentrations of insulin, glucose, free fatty acids, neutral lipids and cholesterol were determined at different times after feeding. In addition, an oral glucose tolerance test was made and the in-vitro synthesis of fat from glucose was measured. The apparent digestibility of fat amounted, unexpectedly, to approximately 94% on the high rat rations II and III. In all groups a significant positive relation between body weight and digestibility of the fat was determined. Despite equal daily intakes of ME in all groups, in group III daily weight gain and protein retention were 7% (p less than 0.01) and 4% (p less than 0.01) higher than in group I, respectively. Blood urea levels of group III were 67% (p less than 0.01) lower than in group I. The mean daily nitrogen retention of the three groups rose gradually from 4 g at a body weight of 5 kg to 16-17 g at a body weight of 25 kg. The feed conversion of group I was lower by 22 and 36% as compared to groups II and III, respectively. Highly significant differences were observed among either groups (p less than 0.01). It has been calculated that in group III the consumption of ME/kg weight gain was about 8% (p less than 0.05) lower than in the two other groups. According to the increased fat percentage of the rations, body protein content diminished from 17,1% in group I to 16,5 and 16% in groups II and III, respectively. Only the difference between groups I and III proved to be significant (p less than 0.05). In group I feed intake resulted in an increase in the insulin level by 55% (p less than 0.05) whereas glucose did not change significantly.(ABSTRACT TRUNCATED AT 400 WORDS)

[Effect of maize-soya rations of various amino acid levels on utilization and endogenous loss of protein in piglets]. / Proteinverwertung und Proteingrundumsatz bei Verfütterung von Mais-Soja-Rationen unterschiedlicher Aminosäurenzusammensetzung an Ferkel.

The influence of different maize-soya rations of varying amino acid composition and at several levels of intake on the utilization and endogenous losses of protein in piglets.

[Behavior of trace elements copper, zinc and manganese in bovine rumen. 2. Passage rate and course of trace element concentration in rumen as influenced by copper sulfate administration]. / Verhalten der Spurenelemente Kupfer, Zink und Mangan im Pansen des Rindes. 2. Mitteilung. Passagerate und zeitlicher Verlauf der Spurenelementkonzentration im Pansen unter dem Einfluss von Kupfersulfat-Gaben.

In experiments on the distribution of copper, zinc and manganese in fractions of rumen liquor (Wetzel and Menke, 1977 (1)) changes in PEG and in trace element concentration during day-time (between 8 a.m. and 6 p.m.) were measured at 2-hour intervals. Rumen volume was 61 l and passage rate 108 and 128 l/day in the two animals used. Average daily passage was calculated to be 40 mg copper, 98 mg zinc and 371 mg manganese. These values were found to be 11% higher than copper intake and 44 and 68% lower than zinc and manganese intakes, respectively. The differences indicate a remarkable endogenous secretion of copper, and a high absorption of zinc and manganese within the rumen. Concentration (mcg/g dry matter) of copper, zinc and manganese in the bacterial fraction and in total rumen liquor decreased slowly in the first hours after feeding, reaching a minimum after 4 to 6 hours and showed a slight increase thereafter. After addition of copper sulphate the curves mostly became linear. The conclusion is drawn that 1.8 ppm copper in inorganic binding does not lead to changes in trace element concentration to an extent that might be harmful to bacterial fermentation.

[Behavior of the trace elements copper, zinc and manganese in bovine rumen. 1. Trace element content of different fractions of rumen fluid and the effect of copper sulfate administration]. / Verhalten der Spurenelemente Kupfer, Zink und Mangan im Pansen des Rindes. 1. Mitteilung. 1. Spurenelementgehalt in verschiedenen Fraktionen des Pansensaftes und seine Beeinflussung durch Kupfersulfat-Gaben.

Two adult, rumen-fistulated steers were fed, over four consecutive 3-week periods, Cu-deficient and normal hays with and without copper sulphate supplementation. Their rumen liquor levels of copper, zinc and manganese were determined correspondingly. The rumen liquor samples were split into 3 fractions by way of centrifugation, sodium dodecyl sulphate treatment of the sediment and filtration. The fractionation of the rumen liquor showed copper to be contained mainly in the water-soluble supernatant (68%), whilst zinc and manganese are more strongly linked to the microbial fraction (57 and 46%, resp.). The plant residue did not average but 9, 24 and 22% of copper, zinc and manganese, respectively. Copper sulphate supplementation to normal hay resulted in a significantly increased copper content in the supernatant and in the microbial fraction. At the same time, the zinc and manganese content values in the microbial fraction were found increased. However, the same copper sulphate supplementation to Cu-deficient hay was found to reduce highly significantly the copper level in the microbial fraction. It is supposed that the excessively high molybdenum content of this hay (2.5 ng/kg) has prevented any effect of the copper sulphate supplementation. The positive effects of copper sulphate supplementation on the zinc and manganese levels in the bacterial fraction were maintained even when feeding Cu-deficient hay rich in Mo.