Effect of the level of dry matter and protein and degradation rate of starch on in vitro ruminal fermentation

Effects of starch type, feeding level and level of a mixed N source upon rumen fermentation were examined using an in vitro rumen simulation system (i.e., SIMCO) with a fluid volume of 1100 ml and liquid dilution rate of approximately 0.07/h. Two sources of starch, two feeding levels and two levels of N were examined in an experiment following a 2 × 2 × 2 factorial arrangement of treatments. The starch sources were raw slowly degrading (R), and cooked fast degrading (C), potato starch and constituted 450 g/kg diet dry matter (DM). The remaining was supplied in the form of a grass hay mixture. The feeding levels were 20 (DM20) and 40 (DM40) g DM/d and the diet N level was either low (N1) or high (N2), using peptone and ammonia salt additions in the buffer. Two simulations of 10 days each were completed. The amount of bicarbonate in buffer was varied according to feeding level, and pH (average 6.3) did not differ between treatments. An increased degradation rate of starch (R vs. C) depressed neutral detergent fibre (aNDFom) digestibility (0.46 vs. 0.36) and organic matter true digestibility (OMTD; 0.73 vs. 0.68), but there were no other treatment effects on digestibility. The VFA production efficiency (average 0.47 g VFA/g OMTD) was not affected by the main treatments, although an interaction between starch type and feeding level occurred. A decline in protozoa rating over the 10 day simulations was more apparent at the lower feeding level (DM20), indicating poor growth conditions for protozoa. Higher microbial efficiency (11.0 vs. 8.9 mg microbial N/g OMTD) and a higher proportion of propionate (272 vs. 207 mmol/mol VFA) occurred at DM20 compared to DM40. Increased degradation rate of starch (i.e., R vs. C) resulted in an improved microbial N efficiency (8.8 vs. 11.2 mg/g OMTD) and an increase in the proportion of propionate (226 vs. 253 mmol/mol VFA) at the expense of acetate (610 vs. 591 mmol/mol VFA). Increasing the level of N (i.e., N1 vs. N2) improved the microbial N efficiency (9.2 vs. 10.7 mg/g OMTD) and increased the proportion of propionate (230 vs. 249 mmol/mol VFA) and butyrate (100 vs. 119 mmol/mol VFA) at the expense of acetate (629 vs. 572 mmol/mol VFA). Interactions between starch type and N level upon VFA patterns were apparent. Results support earlier findings in that carbohydrate degradation rate is an important factor determining microbial growth rates and VFA distribution.     

The in vitro culture and tegumental dynamics of the plerocercoid of Ligula intestinalis (Cestoda: Pseudophyllidea)

Hoole D. and Arme C. 1985. The in vitro culture and tegumental dynamics of the plerocercoid of Ligula intestinalis (Cestoda: Pseudophyllidea). International Journal for Parasitology14: 609–615. An in vitro system for maintaining Ligula has been developed and used in an autoradiographic study on the tegumental dynamics. Parasite tissue was cultured in teleost Ringer, Eagle's medium, M199 medium and modified Leibovitz' medium. Small membrane-bound bodies within the microthrix border increased in number in all media, and abnormal mitochondria were found in the distal tegument in teleost Ringer, M199 and Eagle's. Autophagy occurred after 24 h culture in teleost Ringer. Ultrastructural and physiological (¹⁴C-D-glucose uptake) evidence suggests that parasite tissue maintained in modified Leibovitz' medium remains viable for at least 24 h. The turnover rate of the surface components of L. intestinalis in this medium is approx. 12–24 h.     

The in vitro effect of indomethacin on basal bone resorption, on prostaglandin production and on the response to added prostaglandins

Mouse calvaria were maintained in organ culture without serum additives. Basal active resorption, as measured by ⁴⁵Ca and hydroxyproline release, was significantly inhibited to 74% control levels by indomethacin (1.4 × 10⁻⁷ M). Prostaglandin F and prostaglandin E₂ production, determined by radioimmunoassay, were both significantly lowered by this concentration of indomethacin. DNA, protein and hydroxyproline synthesis, as indices of cell toxicity, were unaffected by low concentrations of indomethacin, while concentrations of 1.4 × 10⁻⁶M inhibited protein synthesis (p<0.005). In the presence of indomethacin (1.4 × 10⁻⁷M) both PGE₂ and PGF_2α stimulated resorption in a dose-dependent manner, with PGE₂ being the more potent. Neither prostaglandin affected hydroxyproline synthesis at low concentrations, but PGE₂ had a marked inhibitory action at a higher concentration (10⁻⁶M). In combination, the effects of PGE₂ and PGF_2α showed no evidence of synergism or any antagonistic action. The study shows that in vitro calcium and hydroxyproline resorption in the unstimulated mouse calvaria are inhibited by indomethacin at concentrations measured in serum during human therapy. The decreased PGF and PGE₂ production associated with this decreased bone resorption in the presence of non-toxic concentrations of indomethacin would suggest a role for these prostaglandins in maintaining the basal resorption of cultured bone.     

Effect of the proportions of neutral detergent fibre and starch, and their degradation rates, on in vitro ruminal fermentation

Effects of proportions of neutral detergent fibre (aNDFom) and starch, as well as their degradation rates, on rumen fermentation were tested using an in vitro rumen simulation system (SIMCO). The in vitro system was designed to simulate selective particle retention and had an average fluid volume of 1150 ml with a liquid dilution rate of approximately 0.07 h⁻¹. Two types of hay (aNDFom sources) and two types of starch were each included at two different levels in the diet and were examined in an experiment following a 2×2×2 factorial arrangement of treatments (eight diet combinations). The hay was either late-cut timothy (Phleum pretense L.) or early cut meadow grass (Poa pratensis L.), with ruminal in situ aNDFom digestion rates of 0.03–0.04 and 0.07–0.08 h⁻¹, respectively. The two starch types were raw (R) and cooked (C) potato starch with previously determined in vitro ruminal digestion rates of 0.04 and 0.20 h⁻¹, respectively. The starch levels were 300 and 600 g/kg diet dry matter (DM) with the remaining being hay (282–682 g/kg DM) and peptone (14–111 g/kg DM). The aNDFom level varied among the diets with different starch levels and hay types. The peptone acted as a source of peptides and, together with ammonia salts from buffer, was used to balance the N contents of the diets. The feeding level for each of the eight vessels was 28 g DM/d. Two 10-day simulations were made with the system. The average pH was higher (P<0.05) for all treatments with raw potato starch (6.19) versus cooked starch (6.07). Protozoa scores, on a qualitative scale, declined faster at the higher starch level. The aNDFom digestibility was, as expected, higher (P<0.001) for meadow hay (0.57) than timothy (0.32), and was also higher (P<0.001) at the lower starch level (0.54) versus the higher (0.35). Microbial protein production efficiency (mg microbial N/g organic matter truly digested) was higher for the faster degrading aNDFom (P<0.01) and starch (P<0.05) sources, but was not affected by starch level. Cooked starch resulted in a lower acetate proportion (449 mmol/mol versus 591 mmol/mol VFA; P<0.001) but higher proportions of propionate (297 mmol/mol versus 236 mmol/mol VFA; P<0.001), and butyrate (169 mmol/mol versus 127 mmol/mol VFA; P<0.01). Butyrate increased with starch level (127 mmol/mol versus 169 mmol/mol VFA; P<0.01), and was also higher for meadow hay versus timothy (168 mmol/mol versus 128 mmol/mol VFA; P<0.01). Interactions between the treatments demonstrate that the response in VFA pattern to starch level is dependent on starch and aNDFom sources. Substrates such as starch and aNDFom are fermented differently depending on their rates of ruminal degradation.     

机械拉伸对血管平滑肌细胞粘附及生长的影响

通过自行研制的“四点弯曲梁”实验装置对血管平滑肌细胞（VSMC）加载培养,并结合显微形态观察和计算机图像处理系统测量细胞铺展投影面积、微管吸吮实验系统检测细胞与表面的粘附力、α-肌动蛋白(actin)免疫组化试验,了解细胞骨架发育和排列取向、流式细胞仪检测细胞动力学以及细胞生长行为等认识VSMC对应变刺激的响应.发现VSMC粘附铺展与实验时间正相关,细胞粘附力、铺展面积、单位面积粘附力4 h后实验组与对照组无显著性差异.VSMC内α-actin发育随加载时间延长呈增加趋势.细胞动力学检测实验组加载24 h后VSMC增殖活动受到抑制.VSMC可能通过调节细胞铺展行为、胞内应力纤维发育等主动机制,实现对机械拉伸的适应性改建.应变刺激有利于体外培养的VSMC维持收缩表型.     

The effects of ghrelin on the in vitro spontaneous and sGnRH-A stimulated luteinizing hormone (LH) release from the pituitary cells of common carp (Cyprinus carpio L.)

In fish, like in mammals, ghrelin affects gonadotropin release acting at the level of the hypothalamus as well as directly on the pituitary gland. In the present study, enzymatically dispersed pituitary cells obtained from sexually mature male and female carp (Cyprinus carpio L.) were incubated in the presence of human ghrelin at the concentration of 10^− 7 or 10^− 6 M, salmon GnRH analogue (Des-Gly¹⁰, D-Arg⁶, Trp⁷, Leu⁸, Pro⁹)-LHRH (sGnRH-A) at the concentration of 10^− 8 M or the combination of ghrelin (both concentrations) and sGnRH-A. ELISA method was used for carp LH levels determination in the media collected after 10 or 24 h of incubation. Ghrelin at the concentration of 10^− 6 M caused the increase of the spontaneous LH secretion from female pituitary cells only. The combination of ghrelin (both concentrations) with sGnRH-A resulted in the significant elevation of LH levels in the incubations of both male and female pituitary cells in comparison with control incubations as well as with sGnRH-A alone treated cells. The results obtained in this study show that ghrelin functions as LH-stimulating hormone in common carp and that it acts directly on gonadotrophic cells, potentiating also the action of GnRH.     

Effects of dietary forage particle size and concentrate level on fermentation profile, in vitro degradation characteristics and concentration of liquid- or solid-associated bacterial mass in the rumen of dairy cows

This study investigated effects of dietary forage particle size (PS) and concentrate level (CL) on fermentation profiles of particle-associated rumen liquid (PARL) and free rumen liquid (FRL), in vitro degradation characteristics and concentration of bacterial mass attached to the solid or fluid rumen digesta phase in dairy cows. The experiment was a 4 × 4 Latin square design with four late-lactation dairy cows in four 23 day periods. Cows were restrictively fed (17 kg dry matter (DM)/d) one of four diets varying in the theoretical PS (6 and 30 mm) of grass hay and in the levels (approximately 200 and 550 g/kg, DM basis) of a cereal-based concentrate. Proportion of large particles (>6 mm) and the content of structural fibre in the diet increased by reducing dietary CL and, particularly, by increasing hay PS. This effect was not reflected by changes in mean total volatile fatty acid concentration or pH in the rumen. However, cows fed high concentrate diets had pH of 5.28 and 5.37 in PARL at 3 h after the last meal, when fine or long chopped hay was offered. The low pH may indicate a depression of the capacity of PARL to degrade fibre in vitro. Gas production in vitro of concentrate increased with the high concentrate diet at 12 h, suggesting that amylolytic capacity was affected only in early phases of fermentation. In addition, elevating dietary CL appeared to shift ruminal fermentation outputs from propionate to butyrate and valerate. Inclusion of coarsely chopped hay to a high concentrate diet does not appear to bring advantages due to increased structure in restrictively fed dairy cows. In addition, results suggest that the response of pH in PARL is more sensitive to dietary changes (i.e., forage PS and CL) than the response in FRL, and so PARL might be better to evaluate the risk of ruminal disfunction in dairy cows.     

The X protein of hepatitis B virus binds to the F box protein Skp2 and inhibits the ubiquitination and proteasomal degradation of c-Myc

The HBx protein of hepatitis B virus is involved in deregulation of cell cycle and development of hepatocellular carcinoma. Since c-Myc also plays an important role in cell proliferation and tumor development, we studied its regulation by HBx in a human hepatoma cell line. Co-expression of HBx and c-Myc resulted in increased stability of intracellular c-Myc. HBx blocked the ubiquitination of Myc through a direct interaction with the F box region of Skp2 and destabilization of the SCF(Skp2) complex. We suggest that sustained presence of c-Myc combined with mitogenic activity inherent to HBx may be associated with cell cycle deregulation and transformation.     

The α1-adrenergic receptor in human erythrocyte membranes mediates interaction in vitro of epinephrine and thyroid hormone at the membrane Ca-ATPase

Membrane Ca²⁺-ATPase activity was stimulated in vitro separately by T₄ (10⁻¹⁰ M) and by epinephrine (10⁻⁶ M). In the presence of a fixed concentration of T₄, additions of 10⁻⁸ and 10⁻⁶ M epinephrine reduced the T₄ effect on the enzyme. β-Adrenergic blockade with propranolol (10⁻⁶ M) prevented stimulation by epinephrine of Ca²⁺-ATPase activity, but did not prevent the suppressive action of epinephrine on T₄-stimulable Ca²⁺-ATPase. In contrast α₁-adrenergic blockade with unlabelled prazosin restored the effect of T₄ on Ca²⁺-ATPase activity in the presence of epinephrine. Like propranolol, prazosin prevented enhancement of enzyme activity by epinephrine in the absence of thyroid hormone. Neither prazosin nor propranolol had any effect on the stimulations by T₄ of red cell Ca²⁺-ATPase in the absence of epinephrine. Analysis of radiolabelled prazosin binding to human red cell membranes revealed the presence of a single class of high-affinity binding sites (K_d, 1.2 × 10⁻⁸ M; B_max, 847 fmol/mg membrane protein). Thus, the human erythrocyte membrane contains α₁-radrenergic receptor sites that are capable of regulating Ca²⁺-ATPase activity.