Quantitation of mouse and rat beta-actin mRNA by competitive polymerase chain reaction using capillary electrophoresis.
Explore the source record for details and available documents.
Actins↗
Biomedical subjects
M Funaba
Publications and source records attributed to M Funaba.
Increased cartilage and bone formation in spontaneously hypercholesterolemic rats.
Spontaneously hypercholesterolemic (SHC) rats are known to exhibit accelerated bone resorption. We compared endochondral bone formation induced by implantation of demineralized bone matrix (DBM) to 4-week-old SHC rats with that of age-matched Sprague-Dawley (SD) rats. When DBM prepared from adult SD rats was implanted, the cartilageous area enlarged, and C-propeptide of type II procollagen content on day 7 was higher in SHC rats. Alkaline phosphatase activity and calcium content on day 12 and tartrate-resistant acid phosphatase activity on day 19 were higher in SHC rats. These results suggest active chondrogenesis, with a subsequent increase in osteogenesis, and stimulated osteoclastic bone resorption in SHC rats. When DBM from 10-week-old SHC rats was implanted into SD or SHC rats, the levels of bone forming parameters on day 12 were reduced to one-third, suggesting inhibiting factor(s) for bone induction in bone matrix of SHC rats. In contrast, when DBM from 6-month-old SHC rats was implanted, although bone forming parameters in SD rats were comparable to the case of implantation of DBM from SD rats, the accelerated bone formation detected in SHC rats was blocked, indicating resistance to systemic bone inducing factor(s) of SHC rats in aged bone matrix. These results suggest that age-related decrease in responses to some systemic bone inducing factor may lead to the bone loss with advancing age.
Acid Phosphatase↗
Suppressed bone induction by follistatin in spontaneously hypercholesterolemic rat bone.
Bone inducing activity in demineralized bone matrix (DBM) of young spontaneously hypercholesterolemic (SHC) rats has been shown to be lower than that of aged SHC rats. This study examined the involvement of bone follistatin, an activin-binding protein, in bone induction. Immunoreactive follistatin was higher in DBM from 10-week-old SHC rats (DBM-10wk) than in DBM from 6-month-old SHC rats (DBM-6mo). When DBM without follistatin supplement was implanted, the C-propeptide of type II procollagen and calcium contents on day 12 in implants of DBM-6mo were 68% and 40% higher than those of DBM-10wk, respectively. In contrast, follistatin supplement to DBM decreased C-propeptide of type II procollagen and calcium contents in implants of both DBM-10wk and DBM-6mo, and the levels of these parameters were comparable between DBM-10wk and DBM-6mo, indicating reduced formation of cartilage and bone. These findings suggest that 1) follistatin content in bone matrix decreases with advancing age in SHC rats, and 2) the follistatin interferes with endochondral bone formation. We demonstrate that the lower bone induction of DBM from young SHC rats was partly due to the abundance of follistatin in bone matrix.
Aging↗
Immunolocalization of type I or type II activin receptors in the rat brain.
We have studied immunolocalization of activin receptors in the central nervous system using polyclonal antibodies (IgG) to type I (50-55 kDa, ActRI), type II (70-75 kDa, ActRII) or a subtype of type II known as type IIB (ActRIIB) receptors of activin. A total of 7 antisera to rat activin receptors was generated, i.e. 3 kinds of antisera to the extracellular domain (ActRI(81-89), ActRII(91-100), or ActRIIB(90-99)) and 4 antisera to the kinase domain (ActRI(323-333), ActRII(307-319), ActRII(407-420) or ActRIIB(306-319)). The region of aa 407-420 of ActRII is identical with that of ActRIIB. At first, we characterized these antibodies by Western blot analysis using ovarian proteins fractionated by preparative SDS-PAGE. All antibodies to ActRII and ActRIIB specifically reacted with 75 kDa-proteins which could also bind to activin-A. Anti-ActRII(91-100) antibody also reacted with 62 kDa-proteins which were capable of binding with activin-A. Although no positive reactions to anti-ActRI(81-89) antibody were seen in ovarian proteins, a positive reaction was detected at 52 kDa only when the proteins were deglycosylated. By use of these antibodies, immunolocalization of activin receptors was examined in the rat brain. The patterns of expression of activin type I and type II receptors were different. Positive reactions to anti-ActRII(91-100) antibody were detected in neurons of the cerebral cortex, hippocampus, medial amygdala and thalamus. In the hypothalamus, some neurons of the supraoptic nucleus were weakly stained, and widely scattered neurons of the lateral hypothalamic area were moderately stained. On the contrary, the most intense reactions to anti-ActRI(81-89) antibody were detected in neurons of the lateral hypothalamic area. In addition, many neurons of the cerebral cortex were also stained, but neurons of the hippocampus and the amygdala were not stained. These results suggest that activin may have physiological roles not only for hypothalamic neuroendocrinological and feeding-related systems as suggested previously but may also have functions in cortical and limbic pathways as a neuromodulator or for maintenance of neurons.
Activin Receptors↗
Quantitative RT-PCR for inhibin/activin subunits: measurements of rat hypothalamic and ovarian inhibin/activin subunit mRNAs during the estrous cycle.
Inhibins (alpha-beta(A) and alpha-beta(B)) and activins (beta(A)-beta(A), beta(A)-beta(B) and beta(B)-beta(B)) were originally isolated from ovarian follicular fluids as FSH secretion modifiers. Inhibin/activin subunits, alpha, beta(A) and beta(B), are widely distributed in several tissues, including gonads and brain, and inhibins and activins have been reported to be involved in ovarian or hypothalamic functions. In this study, we established and employed a competitive RT-PCR assay system for rat inhibin/activin subunits by capillary electrophoresis to determine rat hypothalamic and ovarian inhibin/activin subunit mRNA levels during the estrous cycle. Linearity of standards for alpha, beta(A), and beta(B) subunit assays were between 0.01-0.3 amol, 0.003-0.09 amol and 0.002-0.02 amol of each fragment DNA as a standard, respectively. Hypothalamic beta(A) subunit mRNA during the estrous morning (1000 h) tended to be increased compared with that of the proestrous evening (1700 h), although they were not significantly different. Ovarian alpha subunit mRNA levels tended to be increased during the proestrous morning (1000 h) and were significantly increased in the proestrous evening (1700 h), compared with diestrus and estrus (P < 0.05). Ovarian beta(A) subunit mRNA was also significantly higher in the proestrous evening, compared with diestrus and estrus (P < 0.05), but in the case of beta(B) subunit mRNA there was no difference among diestrus, proestrus and estrus. We thus established a sensitive competitive RT-PCR system for the measurement of inhibin/activin alpha, beta(A) and beta(B) subunits, and this assay system would be helpful for the study of inhibin/activin action in brain and other tissues where these factors are expressed at low levels.
Animals↗
Duodenal flow of microbial nitrogen estimated from urinary excretion of purine derivatives in calves after early weaning.
Duodenal flow of microbial N (MN) was estimated from urinary purine derivatives to examine age-related changes in MN in male Holstein calves. In Exp. 1, endogenous purine derivatives were determined by measurement of purine derivatives in five calves fed nucleic acid-free milk replacer alone. In Exp. 2, the ratio of urinary excretion as purine derivatives to purines administered via the reticular groove was determined in three calves weaned at 5 wk of age. As a result, endogenous purine derivatives were constant at 705 mumol/(kg BW.75.d), irrespective of the amount of milk replacer, and the ratios of purine derivatives to duodenal purines were estimated to be .549, .276, .363, and .466 at wk 1, 6, 11, and 20 after weaning, respectively. Using these variables and urinary purine derivatives, the duodenal flow of MN was estimated and its relation with N balance was examined in 15 calves weaned at 5 wk of age in Exp. 3. Digestible OM was lower at wk 1 after weaning and transiently higher at wk 6. The percentage of N absorbed to N intake, N absorbed, N retained, and estimated duodenal MN were also lower at wk 1, and rapidly increased for the first 6 wk. These findings suggest that the increases in N absorbed and N retained for the first 6 wk after weaning were due to augmentation of duodenal flow of MN and dietary N that escaped ruminal degradation.
Aging↗
Lysine deficiency in postweaned calves fed corn and corn gluten meal diets.
Holstein bull calves (n = 36) weaned at 6 wk of age were used in six trials to examine the response of N balance to postruminal administration of lysine with or without methionine in postweaned calves receiving diets based on corn and corn gluten meal. Calves were younger than 3 mo of age in Trials 1 and 2 but older than 3 mo in Trials 4 to 6. L-Lysine monohydrochloride was supplemented with or without DL-methionine twice daily through the reticular groove, except in Trial 4, in which N supplements were infused through duodenal cannulas. L-Glutamine was used as a nonspecific N source in every trial, and casein was a positive control in Trials 4 and 5. When daily CP intake from the diet was 3.9 g/kg BW, lysine was limiting for calves less than 11 wk of age (Trials 1 and 2) but not limiting for calves greater than 12 wk of age (Trial 3). No amino acid seemed to be limiting for calves greater than 20 wk of age (Trial 4) when daily CP intake was 4.1 g/kg BW, but lysine was limiting when CP intake was restricted to 3.0 g/kg BW when calves were more than 17 wk of age (Trial 5). However, lysine was not limiting above 18 wk of age (Trial 6) when CP intake was increased to 3.8 g/kg BW by adding urea to the diet. Results suggest that lysine may be limiting for corn and corn gluten meal diets only when ruminal microbial protein synthesis is restricted.
Aging↗
Bone growth rather than myofibrillar protein turnover is strongly affected by nutritional restriction at early weaning of calves.
Our previous study revealed that weaning of calves aged 5 wk (early weaning) resulted in transient nutritional restriction with subsequent repletion. The present study was conducted to examine age-related changes in nitrogen balance, bone growth and myofibrillar protein degradation after early weaning in calves. At weaning, calves used in Experiment 2 had more severe nutritional restriction than those used in Experiment 1 due to a shorter duration of the suckling period (3 vs. 4 wk), a lesser amount of daily milk replacer (500 vs. 600 g) and a shorter period when given solid feed (for the last week vs. throughout the suckling period). In both experiments, nitrogen and calcium retentions were lower at weaning and transiently increased at 6 wk after weaning. However, detrimental effects on nitrogen and calcium retention immediately after weaning were more evident, and compensatory increases of nitrogen and calcium retention after weaning were more pronounced and continuous in Experiment 2 than in Experiment 1. The changes in plasma intact osteocalcin concentrations and urinary hydroxyproline excretion after weaning were similar to those for nitrogen and calcium retention in Experiment 2, whereas fractional degradation rate of myofibrillar protein was constant throughout the study in both experiments. These results suggest that the extent of nutritional restriction at weaning affects subsequent enhanced utilization of nitrogen and calcium, and that age-related changes in nitrogen retention after early weaning partly reflect changes in bone growth regulated by both forming and resorbing activities, independent of myofibrillar protein degradation.
Aging↗
Unique recognition of activin and inhibin by polyclonal antibodies to inhibin subunits.
Inhibin-A is a glycoprotein composed of an alpha subunit containing a glycosylation site and a beta A subunit, whereas activin-A is a homodimer of two inhibin beta A subunits. We examined the recognition of activin-A and inhibin-A by several antisera to the alpha or beta A subunit, and factors affecting the recognition. A total of six polyclonal antibodies to inhibin subunits, i.e., two antisera to a peptide fragment of the alpha subunit [alpha (1-19) and alpha (1-26)], and four antisera to the beta A subunit [beta A (1-10), beta A (70-79), beta A (87-99), and beta A (94-105)], was generated. On Western blot analysis, the anti-beta A (87-99) and beta A (94-105) sera recognized recombinant human activin-A but not inhibin-A under non-reducing conditions. When inhibin-A was deglycosylated with N-glycosidase-F, inhibin-A could be recognized by the anti-beta A (87-99) and beta A (94-105) sera. In addition, when activin-A bound to a nitrocellulose membrane was pre-incubated with recombinant human follistatin, the recognition of activin-A by the anti- beta A (87-99) and beta A (94-105) sera was decreased. These results suggested that the lower affinity of follistatin to inhibin-A than to activin-A might be likely explained as reflecting a site associated with the glycosylation of inhibin-A. However, the exposure of amino acids 87-105 of the inhibin beta A subunit on the molecular surface through deglycosylation did not increase the affinity of inhibin-A for follistatin but rather resulted in poor binding with follistatin. The present data suggest that (1) amino acids 87-105 of the inhibin/activin beta A subunit are located on the molecular surface, although this region of inhibin-A is concealed by the carbohydrate chain of the alpha subunit, (2) the region responsible for follistatin binding within the activin beta A subunit is spanned by amino acids 87-105, and (3) the mode of binding of inhibin-A to follistatin is quite different from that of activin-A to follistatin, and the former may be influenced by glycosylation.
Activins↗
Follistatin and activin in bone: expression and localization during endochondral bone development.
The involvement of activin and follistatin, an activin-binding protein, in endochondral bone development was examined by sc implantation of demineralized bone matrix in rats. Immunoreactive follistatin was localized in proliferating chondrocytes and round osteoblasts, whereas it was not detected in hypertrophic chondrocytes and osteoblasts surrounding bone marrow. Western blot analysis also revealed that immunoreactive follistatin was higher during the initial stages of chondrogenesis (day 5) and osteogenesis (days 11 and 14) and lower during the conversion from cartilage to bone (day 9). These results suggest that follistatin is produced by proliferating cells, and the expression decreases with differentiation of the cells. Implants injected with follistatin on days 9 and 10 contained lower calcium levels on day 14 than those injected with rat albumin. Furthermore, the follistatin-injected implants were still mainly composed of cartilage, suggesting that the disappearance of follistatin is necessary for the conversion of cartilage to bone. In contrast, immunoreactive activin beta A (55-60 kDa) was continuously detected in implants on days 7-14. The content of C propeptide of type II procollagen was increased and cartilageous area was enlarged on day 7 by activin A injections on days 5 and 6, suggesting a chondrogenic effect of activin in the initial stage of cartilage formation. These results indicate that proliferating chondrocytes and round osteoblasts produce follistatin, and that the activity of activin is regulated by changes in the expression of follistatin at the stages of chondrogenesis and transition from cartilage to bone.
Activins↗
Effect of chronic high protein intake on magnesium, calcium, and phosphorus balance in growing cats.
The effects of high protein feeding on food and water intake, and the retention and urinary excretion of macrominerals (magnesium (Mg), phosphorus (P), and calcium (Ca)) were examined in growing cats. Seven female cats aged 4 months were fed diets containing 55% crude protein (n = 4) or 29% crude protein (n = 3) for 12 months on an ad libitum basis. Mineral balances were determined at 0.5, 2, 6, 10, and 12 months of feeding. The higher protein intake stimulated daily water intake and urine excretion throughout the study, although daily food intake was not affected by dietary protein levels. The urinary Mg concentration was decreased by the high protein intake, resulting from both increased urine volume and reduced excretion of urinary Mg. In contrast, the concentration and daily excretion of urinary P were increased by the high protein intake. The protein-induced increase in urinary P would not necessarily imply the increased excretion of PO4(3-), the anion responsible for struvite crystallization, because the dissociation of phosphate depends on urinary pH. Urinary Ca excretion was not affected by the dietary protein levels, but the high protein intake caused less retention of P and Ca as a result of enhanced urinary P excretion and lowered Ca absorption. The possibility of high protein feeding for the prevention of struvite crystallization in growing cats is discussed.
Animals↗
Anti-activin A antibody (IgY) specifically neutralizes various activin A activities.
Activin A (beta A beta A), originally isolated from ovarian follicular fluids as a follicule-stimulating hormone (FSH) secretion stimulator, has also been identified as an erythroid differentiation factor (EDF), a neuron survival factor and a mesoderm-inducing factor. Thus, activin A is a multifunctional factor, and further studies on its physiological function are important. However, it is very difficult to produce a specific antibody to neutralize the activity of activin A because of its highly conserved amino acid sequence across mammalian species. In this study, we succeeded in generating an antibody against activin A, which can neutralize several activities of activin A, such as the stimulation of FSH secretion from pituitary cells and the induction of the differentiation of erythrocytes in vitro. This antibody did not affect the activity of activin B (beta B beta B), which induces the differentiation of erythrocytes in vitro, and the activity of inhibin A (alpha beta A), which inhibits FSH secretion from pituitary in vitro, but slightly neutralized that of activin AB (beta A beta B). Western blotting analysis showed that this antibody recognized both dimeric and monomeric forms of the beta A subunit of activin and inhibin. These results suggest that this antibody recognizes the beta A subunit of activin and specifically neutralizes the activity of a dimer of the beta A subunit, activin A. Furthermore, by the addition of this antibody to the culture medium, the development of murine embryos was suppressed, suggesting that endogenous activin A plays an important role in murine development. These results indicate the usefulness of this antibody for studies of endogenous activin actions.
Activins↗
Effects of a high-protein diet on mineral metabolism and struvite activity product in clinically normal cats.
OBJECTIVE: To examine effects of high-protein diets (> 50% crude protein of dry matter) on urinary mineral excretion and struvite activity product ([Mg2+] x [NH4+] x [PO(4)3-]). ANIMALS: 14 clinically normal cats, 4 adult female and male cats for experiments 1 and 2, respectively, and 6 female kittens aged 4 months for experiment 3. PROCEDURE: Relations between dietary protein amount (25.9, 38.3, 51.4, and 65.4% crude protein [dry matter]) and urinary excretion of Mg, P, and Ca were examined in a 4 x 4-Latin square design (experiment 1). Struvite activity product, the index of solubility of struvite crystals, was determined when a high-protein diet (54.9%) was fed (experiment 2). Utilization of minerals in cats fed a high-protein diet long term was examined (experiment 3). RESULTS: Water intake and urine volume increased with increasing dietary protein concentration. Urinary Mg2+ excretion was not affected (experiment 1) or was decreased (experiment 3) by higher protein intake, leading to lower urine Mg2+ concentration in groups fed higher protein amounts. Urine pH was decreased by high-protein intake. As a result, PO(4)3- concentration was decreased by high-protein intake (experiment 2), although total daily urinary excretion of P was increased. Consequently, struvite activity product tended to decrease in cats fed high-protein diets, indicating increase in struvite solubility. High-protein intake decreased Ca and P retention by increasing their fecal and urinary excretions, respectively. CONCLUSION: As a consequence of the increase in urine volume and urine acidification, high-protein diets have potential ability to increase solubility of struvite crystals.
Animals↗
Effect of continuous infusion of lysine via different routes and hepatic vagotomy on dietary choice in rats.
The effect of continuous L-lysine (Lys) infusion on dietary choice between Lys deficient and protein-free diets in Sprague-Dawley rats was studied to determine the sensing site of Lys deficiency. After daily intake of each diet became constant, Lys was continuously infused for 11 days via intraperitoneal (IP), intragastric (IG) or intracerebroventricular (ICV) route, with an osmotic pump. Daily intake of each diet was measured. Intake of the Lys deficient diet compared with protein-free diet in either IP or IG Lys-infused group increased significantly (p < 0.001) vs. the intake in the baseline period. The selection of the Lys deficient diet was quite comparable between IP and IG groups. But the intake of the ICV group was unchanged. Hepatic vagotomy during IP infusion transiently delayed selection of the Lys deficient diet. These results imply the roles of postabsorptive mechanisms in sensing an amino acid deficiency, and possible involvement of the hepatic branch of the vagus in the sensing. However, sensing in the brain or indeed in the intestine was not excluded.
Aging↗
Characteristic relation between dietary metabolizable energy content and digestible energy content in laboratory cats.
This study was conducted to examine the relationship between dietary nitrogen (N)-corrected metabolizable energy (MEn) and dietary digestible energy (DE) in cats, in order to verify the reliability of the present metabolizable energy (ME) system for cats. Four adult female cats were fed diets containing four different levels of crude protein (CP) (24, 35, 49, and 62% as fed) 4 hours a day in 4 x 4 Latin square design to determine energy- and N-balance. Dietary CP levels had hardly any effect on daily food intake, but acid-ether extract (AEE) intake tended to increase and carbohydrate (CHO) intake tended to decrease, in response to increases in dietary CP levels. Apparent CP and AEE digestibility did not change, regardless of the experimental diet. In contrast, CHO digestibility tended to diminish as dietary CP levels increased. Although the ratio of urinary energy (UE) to urinary N (UN) was higher in cats fed the lowest CP diet, it was still much lower than in other mammals. Regression between UE/digestible crude protein (DCP) and N-balance indicated that dietary ME at N-equilibrium (i.e., MEn) could be expressed as DE -0.47 x DCP. MEn could also be estimated as DE -0.62 x DCP by using the average ratio of UE/(UN x 6.25). Both DCP coefficients were much lower than in other mammals, including dogs and pigs, suggesting a unique form of N metabolism in cats. Because ME values applied to practical feline feed ingredients have been either estimated in pigs or calculated according to the equation, DE -1.25 x DCP, similar to the method used for dogs, the present ME values for cats are believed to have been underestimated.
Animals↗
Dietary protein levels affect water intake and urinary excretion of magnesium and phosphorus in laboratory cats.
The effects of dietary protein levels on food and water intake, and urinary excretion of magnesium (Mg) and phosphorus (P) were examined in cats fed dry-type diets. Four adult female cats were used for trials in a 4 x 4 Latin square design, and fed diets with increasing protein content (25.9, 38.3, 51.4 or 65.2% in dry matter) daily from 9:00 to 13:00. While daily food intake was almost constant regardless of the dietary protein level, water intake and urine volume increased with increasing the dietary protein. Daily urinary excretion of P increased in response to the increase in dietary protein level. The urinary concentration of P was positively related to nitrogen (N)-intake. In contrast, daily urinary excretion of Mg was not affected by the dietary protein level, and the urinary concentration of Mg was negatively related to N intake. A dry-type diet with a high protein content might be effective in preventing the deposition of Mg salts in the urinary tract of cats under the meal-fed condition without affecting food intake because of both the lower concentration of urinary Mg resulting from the increase in urine volume and, probably, urinary acidification.
Animals↗
Validity of NRC method for estimating metabolizable energy value of laboratory dry canine diets.
An equation to estimate the metabolizable energy (ME) content of practical dry canine diets, [metabolizable energy (MENRC, kcal/g) = 3.50 x crude protein + 8.46 x acid ether extract + 3.50 x nitrogen-free extract] has been recommended by the National Research Council (NRC), which assumes fixed digestibility for each nutrient. This estimation method is much more convenient than that of nitrogen-corrected metabolizable energy (MEn) following the equation MEn = digestible energy-1.25 x digestible crude protein. This study aimed to assess the validity of MENRC through a comparison with MEn determined by using 11 diets with 4 mature male Beagle dogs. The relation between MENRC and MEn was expressed as a quadratic equation (MENRC = 0.83MEn2 - 5.43MEn + 12.36, r2 = 0.956, P < 0.01), suggesting that MEn is overestimated when the NRC method was applied to lower energy diets. It was also suggested that the strict estimation of MEn by means of fixed digestibility coefficients was impossible, because of the relatively wide variation in digestibility among dry canine diets.
Animal Feed↗
Changes in nitrogen balance with age in calves weaned at 5 or 6 weeks of age.
Three experiments were conducted to examine the changes of N utilization after early weaning in calves. In Exp. 1, eight male Holstein calves were weaned at 5 wk of age and N balance was determined on the 1st, 6th, 11th, and 19th wk after weaning. In Exp. 2, to examine the effect of weaning age on N utilization, three calves were weaned at 5 wk of age and the other three at 11 wk of age. The N balance of both groups was determined on the same weeks of age (i.e., the 1st, 6th, 8th, and 11th wk after weaning in the earlier-weaned group). In Exp. 3, to examine the effect of a higher content of CP (17.9%) and TDN (75%) in concentrate on N utilization, three calves were weaned at 6 wk of age and the N balance was determined on the 1st, 5th, 10th, and 20th wk after weaning. The intake of concentrate was low in wk 1 but rapidly increased in the next 5 wk (1.74 and 1.72 times in Exp. 1 and 2, respectively). Nevertheless, the TDN intake tended to be lower in calves weaned early just after weaning compared with that in suckling calves in Exp. 2. The apparent N digestibility steadily increased with age and reached a plateau on wk 11 and 6 after weaning in Exp. 1 and 2, respectively. Conversely, the ratio of N retained:N absorbed tended to be high up to wk 6 after weaning and thereafter decreased, leading to a transient high N retained:N intake ratio on wk 6 after weaning.(ABSTRACT TRUNCATED AT 250 WORDS)
Aging↗