Biomedical subjects

M Funaba

Publications and source records attributed to M Funaba.

At least 19 recordsLinked to original sources

PubMed2001

Fish meal vs. corn gluten meal as a protein source for dry cat food.

To compare the effects of two dietary protein sources, fish meal (FM) and corn gluten meal (CGM), fecal moisture content, nitrogen balance and urinary excretion were examined in adult cats. The dietary protein source did not cause a significant difference in daily food intake, water intake, urine volume, dry matter digestibility or urinary nitrogen excretion, but fecal moisture content was lower (P<0.02) in the CGM group. The HCl-insoluble fraction of urinary sediment tended to be higher in the CGM group (P<0.10), although urinary pH was similar in the two groups. These results suggest that CGM is comparable with FM in respect to nutritional value and the urine acidifying effect, but FM may be preferable to CGM for the prevention of constipation and struvite urolithiasis in cats.

Animal Feed↗

PubMed2001

Effect of supplementation of dry cat food with D,L-methionine and ammonium chloride on struvite activity product and sediment in urine.

Feeding dry foods supplemented with urine acidifier (D,L-methionine (Met) or ammonium chloride) decreased urinary pH and struvite activity product in clinically normal cats. As a result, the number of struvite crystals in urine was greatly reduced. Supplementation with 3% Met but not 1% Met caused decrease in the urinary concentration of sediment, which resulted from a reduction in the HCl-soluble fraction. The concentration of HCl-insoluble sediment was not affected by supplementation with the urine acidifier.

Ammonium Chloride↗

PubMed2001

Expression and localization of activin receptors during endochondral bone development.

The expression and localization of activins (dimeric protein of inhibin beta subunit) and activin receptors in skeletal tissue were examined. RT-PCR revealed that cultured chondrocytes expressed mRNAs of inhibin/activin betaA and four activin receptors (two type I (ActRI and ActRIB) and two type II (ActRII and ActRIIB)). Immunohistochemical analyses showed that activin betaA, ActRI and ActRII were localized in proliferating chondrocytes and osteoblasts in tibiae of neonatal rats, and in implants of demineralized bone matrix, a well-established model of ectopic bone formation. The immunoreactivities of osteoblasts were decreased with aging in the tibiae and with progressing endochondral bone development in the implants. The strong expression of ActRI was also detected in hypertrophic chondrocytes both in the tibial growth plate and in the implants, whereas immunoreactive ActRII was lower in hypertrophic chondrocytes. Western blot analysis also showed that immunoreactive ActRI, migrating at 52 kDa, was detected only in the implants on days 9 and 11, the period of conversion from cartilage to bone. In view of the sharing of type II receptors between activins and bone morphogenetic proteins (BMPs), our findings suggest that activin/BMP activity involves in bone modeling, especially during active chondro- and osteogenesis and during the conversion from cartilage to bone.

Activin Receptors↗

PubMed2001

Adverse effects of excess lysine in calves.

Two main trials and three preliminary experiments were conducted in order to examine adverse effects of excess lysine in 140- to 150-kg Holstein bull calves. The animals had been trained to maintain reflex closure of the reticular groove after weaning and were fed a corn and soybean meal diet. In Trial 1 (n = 30), administration via the reticular groove of 0 to 64 g/d of lysine as L-lysine monohydrochloride resulted in a linear decrease in DMI and N utilization efficiency, with notably lower values at 64 g/d, although ADG and gain/feed ratio were not affected. Plasma arginine and ornithine did not decrease but rather increased over that range. Free lysine but not free arginine was detected in urine. In addition, free ornithine was excreted into urine only when 64 g/d was administered. Unexpectedly, severe but transient diarrhea occurred when 64 g/d of lysine were administered. Preliminary experiments revealed that a single administration of more than 32 g of lysine as L-lysine monohydrochloride could result in diarrhea, and the diarrhea was proven to be due to the lysine itself and not to the HCl portion. In Trial 2 (n = 15), a single administration of 40 or 60 g of lysine as L-lysine monohydrochloride resulted in increased fecal excretion of free lysine and ornithine, especially the latter, although free arginine was not detected in feces. These results suggested that diarrhea could occur almost concurrently with an imbalance in calves when 64 g/d of lysine was administered. However, lysine did not antagonize arginine at that level or at lower levels. The remarkable increase in fecal ornithine may be somehow related to the development of diarrhea from excess lysine.

Animals↗

PubMed2000

Activin A stimulates type IV collagenase (matrix metalloproteinase-2) production in mouse peritoneal macrophages.

The role of activin, a dimer of inhibin beta subunit, in mouse peritoneal macrophages was evaluated. Activin activity in the cultured macrophages was augmented in response to activation by LPS. In Western blot analysis, immunoreactive activin A was detected in the culture medium only when the macrophages were stimulated by LPS. Although mRNA expression of betaA subunit was detected, that of alpha and betaB subunit was not found in macrophages by reverse RT-PCR. The activin betaA mRNA level was increased in macrophages by LPS, suggesting that the activin production augmented by LPS is regulated at the mRNA level of the betaA gene. The mRNAs of four activin receptors (ActRI, ActRIB, ActRII, and ActRIIB) were also detected in the peritoneal macrophages, and the mRNA levels, except for ActRIB, were decreased during the LPS treatment. Exogenous activin A stimulated the mRNA expression and gelatinolytic activity of matrix metalloproteinase-2 (MMP-2) in macrophages in both the presence and the absence of LPS. In contrast, activin did not affect the production of MMP-9 in macrophages. These results suggested that 1) mouse peritoneal macrophages produced activin A; 2) expression of activin A was enhanced with activation of the macrophages; 3) the macrophages also expressed activin receptors; and 4) exogenous activin A stimulated MMP-2 expression and activity, implicating activin A as an positive regulator of MMP-2 expression. Considering that MMP-2 constitutes the rate-limiting proteinase governing the degradation of basement membrane collagens, activin A may be involved in migration and infiltration of macrophages through the basement membrane in an inflammatory state.

Activin Receptors↗

PubMed2000

Recombinant expression and purification of smad proteins.

Smads transduce intracellular signals initiated by members of the transforming growth factor beta (TGF beta) family, including activins, TGF betas, and bone morphogenetic proteins. Recently, various models concerning the mechanism of Smad action have been proposed; however, these models are basically qualitative. Quantitative verification of the validity of the models requires significant amounts of purified Smad proteins, but purification of full-length Smad protein has not been straightforward even using recombinant protein expression systems. Here, we report purification of Smad proteins expressed in E. coli as glutathione S-transferase-fused proteins. By glutathione-Sepharose affinity purification, ATP treatment, DEAE-Sepharose and hydroxylapatite columns, expressed Smads were purified to near homogeneity as judged by SDS-PAGE; protein recovery was ca. 1 mg/l culture for Smad2 and 100 microg/l culture for Smad4. The purified Smad proteins had three known in vitro activities: Smad2 phosphorylation by TGF beta receptor complexes immunoprecipitated from COS7 cells, Smad4 binding to Smad-binding DNA element, and Smad2 interaction with calmodulin. The data suggest that purified proteins could be useful for biochemical analyses to evaluate the current models quantitatively.

Animals↗

PubMed2000

Identification and characterization of constitutively active Smad2 mutants: evaluation of formation of Smad complex and subcellular distribution.

Smads mediate activin, transforming growth factor beta (TGFbeta), and bone morphogenetic protein signaling from receptors to nuclei. According to the current model, activated activin/TGFbeta receptors phosphorylate the carboxyl-terminal serines of Smad2 and Smad3 (SSMS-COOH); phosphorylated Smad2/3 oligomerizes with Smad4, translocates to the nucleus, and modulates transcription of defined genes. To test key features of this model in detail, we explored the construction of constitutively active Smad2 mutants. To mimic phosphorylated Smad2, we made two Smad2 mutants with acidic amino acid substitutions of carboxyl-terminal serines: Smad2-2E (Ser465, 467Glu) and Smad2-3E (Ser464, 465, 467Glu). The mutants enhanced basal transcriptional activity in a mink lung epithelial cell line, L17. In a Smad4-deficient cell line, SW480.7, Smad2-2E did not affect basal signaling; however, cotransfection with full-length Smad4, but not transfection of Smad4 alone, resulted in enhanced basal transcriptional activity, suggesting that the constitutively active Smad2 mutant also requires Smad4 for function. In vitro protein interaction analysis revealed that Smad2-2E bound more tightly to Smad4 than did wild-type Smad2; dissociation constants were 270 +/- 66 nM for wild-type Smad2:Smad4 complexes and 79 +/- 18 nM for Smad2-2E:Smad4 complexes. Determination of the subcellular localization of Smad2 revealed that a greater percentage of Smad2-2E was localized in the nucleus than wild-type Smad2. These results suggest that Smad2 phosphorylation results in both tighter binding to Smad4 and increased nuclear concentration; those changes may be responsible for transcriptional activation by Smad2.

Animals↗

PubMed2000

mRNA expression of type I and type II receptors for activin, transforming growth factor-beta, and bone morphogenetic protein in the murine erythroleukemic cell line, F5-5.fl.

OBJECTIVE: Intracellular signaling of activin and transforming growth factor-beta (TGF-beta) is thought to be mediated by the same molecules (Smad2/3 and Smad4). Although differentiation of murine erythroleukemia F5-5.fl cells is induced by activin, it is not induced by TGF-beta, suggesting that at some point TGF-beta signaling is defective. The aim of this study was to investigate the unresponsiveness of F5-5.fl cells to TGF-beta. DESIGN: mRNA expression of ligands, receptors, and signal mediators for the TGF-beta family was examined in F5-5.fl cells using RT-PCR. RESULTS: Activin induced erythrodifferentiation of F5-5.fl cells in a dose-dependent manner. Neither TGF-beta1 nor bone morphogenetic protein (BMP)-4 affected the differentiation of F5-5.fl cells in the presence or absence of activin. Although mRNAs of TGF-betas (TGF-beta1, TGF-beta2 and TGF-beta3) were detected, those of inhibin/activin (alpha-, betaA- and betaB-subunits) and BMPs (BMP-2, BMP-4 and BMP-7) could not be detected in the cells, suggesting that neither activins nor BMPs are produced in F5-5.fl cells. The expression of both type I (ALK-4/ActRIB) and type II (ActRII) receptors for activin was detected in F5-5.fl cells. In contrast, while the expression of type I receptor for TGF-beta (ALK-5/TbetaRI) was detected, that of type II receptor (TbetaRII) was not. The mRNA of all Smads examined was detected in F5-5.fl cells. CONCLUSIONS: A defect in the type II receptor might cause unresponsiveness to TGF-beta in F5-5.fl cells. An erythrodifferentiation assay using F5-5.fl cells would be useful for measuring net activin activity because it would not be necessary to consider endogenous activins and BMPs.

Activin Receptors, Type I↗

PubMed2000

Methionine imbalance and toxicity in calves.

The occurrence of methionine imbalance and toxicity was examined using 70- and 100-kg Holstein bull calves. The animals had been trained to maintain reflex closure of the reticular groove after weaning at 5 wk of age, and Trials 1 (n = 30) and 2 (n = 24) were conducted on animals at 7 and 12 wk of age, respectively. Calves received a corn-soybean meal diet in Trial 1 and a corn-corn gluten meal diet in Trial 2. In Trial 1, postruminal administration of 6 g of DL-methionine/d increased ADG, feed intake, gain/feed, and N retention compared with a control group receiving N-free supplement. However, the administration of 12 g of DL-methionine/d did not improve these variables, whereas both 18 and 24 g/d resulted in BW loss and decreased gain/feed and N utilization efficiency. In Trial 2, postruminal administration of 16 g/d of L-lysine from L-lysine monohydrochloride increased ADG, gain/feed, and N utilization efficiency compared with a control group receiving a N-free supplement. The administration of 8 g of DL-methionine/d in addition to L-lysine did not exert an adverse effect on these variables. However, the additional supplementation of 16 and 24 g of DLmethionine/d negated the improvement, whereas 32 g/d resulted in BW loss and decreased gain/feed and N utilization efficiency. These results showed that a methionine imbalance and toxicity occurred in calves with even a modest excess of DL-methionine, and 70-kg calves were more susceptible to methionine toxicity than 100-kg calves. Plasma concentrations of branched-chain amino acids and phenylalanine linearly decreased with increasing amounts of additional DL-methionine from 0 to 32 g/d in Trial 2. However, such a decrease occurred mainly within the range from 0 to 12 g/d in Trial 1. This decrease was suggested to occur in relation to methionine metabolism via the transsulfuration pathway.

Amino Acids↗

PubMed2000

Short communication: estimation of plasma osmolality from plasma electrolytes, glucose, and urea in calves.

Plasma osmolality estimated from plasma concentrations of Na+, Cl-, K+, glucose, and urea was compared with measured osmolality in preweaned Holstein calves. When calves (n = 5) were fed only milk replacer after fasting for 24 h, measured osmolality fluctuated almost in parallel with estimated osmolality during the 8-h period after feeding, although estimated values were about 90% of measured values. When calves (n = 5) were fed only calf starter after fasting for more than 16 h, measured osmolality did not parallel the estimated osmolality during the 8-h period after feeding. Some factors depressed measured osmolality in the first 2 h.

Animal Feed↗

PubMed1999

Immunological localization and ontogenetic development of inhibin alpha subunit in rat brain.

This study examined the immunolocalization and ontogeny of the inhibin-specific alpha subunit in the brain of male rats. Immunohistochemistry using antiserum directed against the mature region of porcine inhibin alpha (1-19, Tyr20) revealed positive reactions in process-bearing cells resembling astroglia in several regions, especially in the dorsal region of the third ventricle, medial and ventral arcuate nucleus, hippocampal dentate gyrus, and layers 1-3 of the cerebral cortex. Generally, inhibin alpha-positive cells in the limbic cortex had larger cell bodies and longer processes than those in the hypothalamus. These inhibin alpha-positive cells were verified to be positive for glial fibrillary acidic protein (GFAP), a differentiated astroglial marker, by double immunolabelling. The expression of inhibin alpha mRNA was higher in the brains of neonatal rats than in those of adult rats, as revealed by reverse transcription-competitive polymerase chain reaction, although the similar changes of immunoreactive inhibin alpha subunit in the brain was not observed. Orchiectomy did not affect expression of inhibin alpha mRNA in the hypothalamic area. This study suggests that inhibin-related peptide is produced by differentiated astrocytes, especially in the hypothalamic arcuate nucleus, the hippocampal dentate gyrus, and the cerebral cortex, and that the expression of inhibin alpha is regulated during brain development.

Animals↗

PubMed1999

Borna disease virus infection in domestic cats: evaluation by RNA and antibody detection.

Borna disease virus (BDV) infection has been suggested to cause spontaneous neurological disease in cats referred to as staggering disease. However the evaluation of BDV infection in neurologically asymptomatic cats remained unclear. In the present study, BDV infected, asymptomatic cats in Tokyo were surveyed both by the presence of plasma antibodies against BDV-p24 and -p40 and by RNA detection in peripheral blood mononuclear cells. Seven of 32 domestic cats (21.9%) were serologically or genetically judged to be BDV-infected. Six cats were positive for anti-BDV antibody and two cats were positive for BDV RNA. Within the 2 RNA-positive cats, only one was positive for anti-BDV antibodies. Furthermore, the findings of anti-BDV-p40 and anti-BDV-p24 antibody-positive cats did not completely overlap. These results suggest that there are neurologically asymptomatic domestic cats infected with BDV present in the Tokyo area.

Animals↗

PubMed1999

Adverse effects of excess DL-methionine in calves with different body weights.

Adverse effects of excess methionine were examined using 12 Holstein bull calves trained to maintain reflex closure of the reticular groove even after weaning at 5 wk of age. Two nitrogen balance experiments were conducted for 2 wk each from 6 wk (Stage 1; BW = 62 kg) and 12 wk of age (Stage 2; BW = 103 kg) by dividing the calves into three groups at each stage. Calves were fed a corn-soybean meal diet at 62 g/kg of metabolic BW at both stages. At Stage 1, feed efficiency (gain:feed intake) and nitrogen retention did not differ between the group supplemented with .333 g of DL-methionine and .111 g of L-lysine monohydrochloride/kg BW per day and the group supplemented with isonitrogenous diammonium citrate, although the level of DL-methionine was considered to be enough to induce toxicity. Conversely, administration of isonitrogenous casein increased nitrogen retention. At Stage 2, administration of the same levels of methionine and lysine resulted in reduced feed intake, depressed nitrogen retention, and BW loss. Conversely, administration of the isonitrogenous casein did not increase nitrogen retention compared with the supplement of isonitrogenous diammonium citrate. Administration of excess methionine and lysine increased plasma methionine concentrations up to 230 (Stage 1) or 190 micromol/dL (Stage 2). Plasma lysine concentrations were less than 24 micromol/dL at every stage. Administration of the amino acid mixture decreased plasma concentrations of branched-chain amino acids and phenylalanine more obviously at Stage 2 than at Stage 1. These results indicated that abomasal administration of .333 g of DL-methionine/kg BW per day induced methionine toxicity at Stage 2 but methionine imbalance at Stage 1.

Animals↗

PubMed1999

Absence of limiting amino acids in calves fed a corn and soybean meal diet past three months of age.

We conducted three nitrogen balance trials using Holstein bull calves older than 16 wk (Trial 1; n = 8), 13 wk (Trial 2; n = 6), and 15 wk of age (Trial 3; n = 9) in a 4 x 4 (Trial 1) or 3 x 3 Latin square design (Trials 2 and 3) to identify limiting amino acids for a corn and soybean meal diet. All calves were trained to maintain reflex closure of the reticular groove after weaning at 5 wk of age. The basal diet was fed daily at 20 or 27 g/kg BW (Trial 1) and at 20 g/kg BW (Trials 2 and 3). The lower feeding level resulted in reduced urinary excretion of purine derivatives, suggesting reduced synthesis of ruminal microbial protein (Trial 1). In Trials 1 and 2, administration of DL-methionine plus L-lysine monohydrochloride through the reticular groove did not increase N retention compared with the supplement of isonitrogenous L-glutamine at either level of intake. In Trial 3, administration of either casein or isonitrogenous monosodium glutamate increased N retention to a similar extent above that observed with a N-free supplement. Results suggested that no specific amino acids were limiting for the corn-soybean meal diet. Administration of methionine plus lysine resulted in a remarkable increase in plasma methionine (Trials 1 and 2), especially at the lower intake level (Trial 1), and a decrease in plasma branched-chain amino acids at either intake level. Glutamine supplementation did not increase plasma branched-chain amino acids compared with the supplementation of diammonium citrate (Trial 2).

Animal Feed↗

PubMed1999

Water balance and fecal moisture content in suckling calves as influenced by free access to dry feed.

Holstein bull calves were used to examine the effect of dry feed on water balance and fecal moisture content during the suckling period. In Experiment 1 (n = 20 calves), free access to concentrate and timothy hay decreased urine volume and increased apparent water retention, fecal water excretion, and fecal moisture content by 2 wk, although daily amounts of milk replacer also affected water balance when DMI from dry feed was low. In Experiment 2 (n = 20 calves), free access to concentrate and hay from wk 1 increased reabsorption of water from renal tubules during wk 2, resulting in reduced urine volume and increased plasma volume. In Experiment 3 (n = 10 calves), supplementation of 500 g/d of milk replacer plus free access to concentrate and hay from wk 1 increased plasma antidiuretic hormone by 2 wk compared with the concentration in calves receiving 200 g/d of milk replacer alone. Plasma antidiuretic hormone concentrations were highly correlated with plasma concentrations of acetate and ketone bodies but not with glucose and urea. In Experiment 4 (n = 16 calves), apparent water retention and fecal moisture content during wk 2 were increased by free access to concentrate from wk 1 but were not affected by rice straw as an inert bulk source.

Acetic Acid↗

PubMed1999

Factors affecting water balance and fecal moisture content in suckling calves given dry feed.

Holstein bull calves were used to examine factors affecting water balance and fecal moisture content in suckling calves given dry feed for 2 wk from 1 wk of age. In Experiment 1 (n = 16), the shift of water balance (decreased urine volume, and increased water retention and fecal water excretion) and elevation of fecal moisture content were greatest when calf starter and Sudangrass hay were fed in addition to liquid milk replacer, compared with calves receiving only milk replacer. Intermediate changes occurred when calves were fed milk replacer and calf starter or milk replacer, calf starter, and rice straw. Water retention was correlated positively with digestible DMI and negatively with urine volume. Fecal water excretion was highly correlated with fecal DM excretion. In Experiment 2 (n = 18), water balance and fecal moisture content during wk 2 were affected by free access to calf starter and hay from wk 1. Urine volume of calves fed dry feed and milk replacer was lower than that of calves fed only milk replacer; however, when water was available in addition to dry feed, urine volume was similar to that of calves fed only milk replacer. Fecal water excretion was highly correlated with water retention rather than with fecal DM excretion, suggesting a close relationship to extracellular fluid volume. Ruminal fermentation would be an important factor affecting both water balance and fecal moisture content in suckling calves given dry feed.

Animal Feed↗

PubMed1998

Utilization of nitrogen and macro-minerals in response to nutritional status in clinically normal adult cats.

Five male cats were used to examine utilization of nitrogen and macro-minerals (calcium, phosphorus and magnesium) in response to food restriction and subsequent repletion. For the first week, each cat was daily given 135 g of dry cat food (baseline period), followed by a restriction period for 1 week; during this period, daily food was individually restricted to 40% of the amount consumed by each cat during the baseline period. Food provision was then returned to the daily 135 g for the final week (recovery period). Fecal weight changed in association with changes in daily food intake, but urine volume changed less with the periods. Fecal and urinary excretion of nitrogen rapidly decreased during the restriction period, but the decreases were smaller than the decrease in nitrogen intake, leading to net nitrogen loss. On the other hand, the food restriction had relatively smaller effects on retention of macro-minerals, and calcium retention was not significantly affected by daily food provision, although the plasma concentration of magnesium was increased during the restriction period and tended to return during the recovery period. Nitrogen retention was increased by the removal of food restriction, but did not exceed the original level of nitrogen retention during the baseline period. These findings suggested that restriction of diet had a serious effect on nitrogen balance, and the impaired protein nutrition might not be easily recovered by subsequent nutritional repletion.

Animals↗

PubMed1998

Limiting amino acids for a corn and soybean meal diet in weaned calves less than three months of age.

Holstein bull calves (n = 51) weaned at 6 wk of age were used in four N balance trials for 4 wk (Trials 1 to 3) or for 2 wk (Trial 4) from 8 wk of age to identify limiting amino acids for a corn and soybean meal diet. The calves were trained to maintain reflex closure of the reticular groove throughout the trials. In Trial 1, administration of .111 g of DL-methionine plus .333 g of L-lysine monohydrochloride/kg BW through the reticular groove increased N retention compared with the control that received isonitrogenous L-glutamine, but administration of .333 g L-lysine monohydrochloride/kg BW alone did not increase N retention in Trial 2. In Trial 3, administration of .111 g of DL-methionine/kg BW improved N balance compared with the control, although the improvement was not detected when DL-methionine was restricted to .022 g/kg BW. In Trial 4, administration of the mixture of .111 g of DL-methionine, .333 g of L-lysine monohydrochloride, and .055 g of L-tryptophan/kg BW increased N retention in calves compared with calves that received an isonitrogenous mixture of .111 g of DL-methionine and .274 g of L-glutamine/kg BW, or .111 g of DL-methionine, .055 g of L-tryptophan, and .234 g of L-glutamine/kg BW. The present results suggest that methionine was the first-limiting and that lysine was probably the second-limiting amino acid for the corn and soybean meal diet in weaned calves less than 11 wk of age, although tryptophan may be either co-limiting with lysine or third-limiting.

Amino Acids↗