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The blood luteinizing hormone (LH) surge in cows is well studied. However, little is known about urinary LH in cows. This study examined urinary LH concentrations after administration of gonadotropin-releasing hormone (GnRH) in six Japanese black cows to induce LH secretion from the pituitary gland into the bloodstream. Abrupt rises in plasma and urinary LH were observed after GnRH administration. Plasma and urinary LH peaked at 2 and 5 hr, respectively. A positive correlation was observed between plasma LH concentrations and urinary LH amounts. Ovulation was confirmed in the cows after 48 hr of GnRH administration. These data strongly suggest that urinary LH is derived from plasma LH, which triggers ovulation in cows.

Background Liver‐type fatty acid‐binding protein (L‐FABP) is a biomarker for early detection of renal disease in humans. Liver‐type fatty acid‐binding protein is cytotoxic oxidation products secreted from proximal tubules under ischemia and oxidative stress. Objective To examine renal expression and quantify urinary excretion of L‐FABP in catswith renal disease. Animals One hundred and thirty‐four client‐owned cats including 34 cats with serum creatinine (sCre) values >1.6 mg/dL and 10 other cats that died in clinics. Methods Tissue expressions of L‐FABP were examined by reverse transcription polymerase chain reaction and Western blotting. Urinary L‐FABP (uL‐FABP) and serum L‐FABP (sL‐FABP) levels were determined by enzyme‐linked immunosorbent assay. Anti‐liver‐type fatty acid‐binding protein antibody immunostained renal sections. Results Feline kidneys express L‐FABP. Strong L‐FABP signals were observed in the lumens of proximal tubular cells in 5 cats with high uL‐FABP excretion, but not in 5 cats with low uL‐FABP excretion. In 9 normal cats, uL‐FABP index was 10.0 μg/g uCre) were detected in 7 of 100 cats with low sCre (1.6 mg/dL). There was a weak correlation between L‐FABP index and sCre, serum symmetric dimethylarginine (SDMA), or blood urea nitrogen (BUN), and these correlation coefficients were increased by analyzing only data of cats with sCre >1.6 mg/dL. There was a weak correlation between u L‐FABP index and sL‐FABP in all tested cats, but not in cats with high sCre. Conclusions and Clinical Importance This study demonstrates correlations between L‐FABP and current renal biomarkers for chronic kidney disease in cats, such as sCre and SDMA. Liver‐type fatty acid‐binding protein may be a potential biomarker to predict early pathophysiological events in feline kidneys.

Fruit malformation is a major constrain in fruit production world-wide resulting in substantial economic losses. The farmers for decades noticed that the chilling temperature prior to the blooming often caused malformed fruits. However, the molecular mechanism underlying this phenomenon is unclear. Here we examined the fruit development in response to cold stress in tomato, and demonstrated that short-term cold stress increased the callose accumulation in both shoot apical and floral meristems, resulting in the symplastic isolation and altered intercellular movement of WUS. In contrast to the rapidly restored SlWUS transcription during the recovery from cold stress, the callose removal was delayed due to obstructed plasmodesmata. The delayed reinstatement of cell-to-cell transport of SlWUS prevented the activation of SlCLV3 and TAG1, causing the interrupted feedback-inhibition of SlWUS expression, leading to the expanded stem cell population and malformed fruits. We further showed that the callose dynamics in response to short-term cold stress presumably exploits the mechanism of bud dormancy during the seasonal growth, involving two antagonistic hormones ABA and GA. Our results provide a novel insight into the cold stress regulated malformation of fruit.

Several manufacturers recommend to feed mixture comprising equal amounts of oral rehydration salt (ORS) solution and milk for diarrheic calves after milk withdrawal. Such a feeding method is expected to supply more nutrients and energy compared to feeding only the ORS solution. However, little is known about the effects of feeding milk diluted with ORS solution on calves' digestive process. This study examined the abomasal contents, volumes, and emptying rates in calves fed whole milk, milk diluted by 50% with ORS solution (50% ORS-milk), and ORS solution. Ultrasonography identified curds in the milk-fed calves, but not in the 50% ORS-milk-fed or the ORS-fed calves. The abomasal fluid of the 50% ORS-milk-fed calves contained not only β-lactoglobulin but also α-casein (CN), β-CN, and κ-CN, which were used for curd formation and undetectable in the milk-fed calves. Abomasal pH was relatively higher in the 50% ORS-milk-fed than that in the milk-fed calves. Abomasal emptying rates were significantly faster in the ORS-fed than in the 50% ORS-milk-fed and the milk-fed calves. These data indicate that the formation of abomasal curd is inhibited in the 50% ORS-milk-fed calves due to the resultant high abomasal pH and low κ-CN concentration. The 50% ORS-milk may not provide rehydration as quickly as the ORS solution. In conclusion, we do not recommend feeding 50% ORS-milk to calves.

Liver-type fatty acid–binding protein (L-FABP) is a biomarker for the early detection of renal diseases in humans. It is secreted along with cytotoxic oxidation products from proximal tubular epithelial cells under conditions of ischemia and/or oxidative stress. This study examined urinary L-FABP excretion under renal ischemia in feline acute kidney injury (AKI) model. L-FABP excretion increased immediately after renal ischemia/reperfusion, despite the absence of obvious structural damage to the kidneys, in the two AKI model cats studied. L-FABP was detected in the renal tubular lumen immediately after renal ischemia/reperfusion in the two cats, but not in a sham surgery cat. These results suggested that high L-FABP excretion is a pathophysiological response associated with antioxidant defense in proximal tubules with renal ischemia and/or oxidative stress in a feline model.

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Cats anoint themselves with plant metabolites that confer mosquito repellence., Domestic cats and other felids rub their faces and heads against catnip (Nepeta cataria) and silver vine (Actinidia polygama) and roll on the ground as a characteristic response. While this response is well known, its biological function and underlying mechanism remain undetermined. Here, we uncover the neurophysiological mechanism and functional outcome of this feline response. We found that the iridoid nepetalactol is the major component of silver vine that elicits this potent response in cats and other felids. Nepetalactol increased plasma β-endorphin levels in cats, while pharmacological inhibition of μ-opioid receptors suppressed the classic rubbing response. Rubbing behavior transfers nepetalactol onto the faces and heads of respondents where it repels the mosquito, Aedes albopictus. Thus, self-anointing behavior helps to protect cats against mosquito bites. The characteristic response of cats to nepetalactol via the μ-opioid system provides an important example of chemical pest defense using plant metabolites in nonhuman mammals.

Di-N-acetylchitobiase (Ctbs) degrades β-1,4 glycoside bonds of the chitobiose core of free asparagine-linked glycan. This study examined whether Ctbs degrades chitin-oligosaccharides to GlcNAc in mammals. We analyzed Ctbs mRNA and protein expression in mouse tissues and characterized enzymatic activity using recombinant mouse Ctbs expressed in Escherichia coli. Ctbs mRNA and protein were expressed in various tissues of mouse, including the stomach. Optimal conditions for recombinant Ctbs were pH 3.0 and 45°C, and the recombinant enzyme was retained more than 94% activity after incubation at pH 3.0–7.0 and below 37°C. The recombinant Ctbs hydrolyzed (GlcNAc)3 and (GlcNAc)6 at pH 3.0 and produced GlcNAc. The K m of Ctbs was lowest with (GlcNAc)3 as a substrate. k cat/K m was fourfold as high with (GlcNAc)3 and (GlcNAc)4 as substrates than with (GlcNAc)2. These results suggest that Ctbs digests chitin-oligosaccharides or (GlcNAc)2 of reducing-end residues of oligosaccharides and produces GlcNAc in mouse tissues. Di-N-acetylchitobiase (Ctbs) is most active at pH 3.0 and retained more than 94% activity after incubation at pH 3.0–7.0 and below 37°C. The preferred substrate of recombinant Ctbs is (GlcNAc)3.

Scent emitted from anal sac secretions provides important signals for most Carnivora. Their secretions emit a variety of volatile compounds, some of which function as chemical signals with information about the scent owners. The domestic cat has a pair of anal sac glands to secrete a pungent liquid. Their anal sac secretions may give information about sex, reproductive state, and recognition of individuals. However, little is known about the volatile compounds emitted from anal sac secretions and their biological functions in cats. In this study, we examined the volatile chemical profiles of anal sac secretions in cats and their olfactory ability to discriminate intraspecific anal sac secretions. Analysis with gas chromatography–mass spectrometry showed that the major volatile compounds were short-chain free fatty acids, whose contents varied among individuals, as well as other carnivores. There was no sex difference in the volatile profiles. In temporal analyses of individual anal sac secretions performed 2 months apart, the profiles were highly conserved within individuals. Habituation–dishabituation tests showed that cats can distinguish individual differences in the odor of anal sac secretions. These results suggest that cats utilize short-chain free fatty acids emitted from anal sac secretions to obtain scent information for individual recognition rather than species or sex recognition.

Calves are fed milk and milk replacer for their growth until approximately 2 mo after birth. During this period, their abomasa produce curd and whey from milk. It has been thought that curd formation is important for digestion and absorption of milk nutrients and immune substances in calves. However, no study has been done observing abomasal contents in neonatal calves after first ingestion of first milking colostrum. Here we report curd formation in neonatal calves and its physiological function with a focus on immunoglobulin absorption. We first examined curd formation by ultrasonography in 3 neonatal calves after first ingestion of first-milking colostrum. Between 0.5 and 8 h after colostrum ingestion, a curd was visualized as a large echogenic image with a clear outline, which was surrounded by an anechoic image corresponding to whey. We next compared serum IgG and IgA concentrations in 10 calves fed the pooled colostrum and 7 calves fed the whey solution that did not coagulate into curds. Serum from 1 calf in the pooled colostrum sample set was excluded due to incomplete curd formation in that the whole colostrum did not coagulate into a large mass of curd and a portion of the colostrum remained as its residue caseins detectable from the abomasal fluid. Serum IgG and IgA concentrations were significantly higher in the 9 calves fed the colostrum than the 7 calves fed the whey solution. One calf exhibiting incomplete curd formation showed low levels of serum IgG and IgA after ingestion, similar to the calves fed the whey solution. These results indicate that curd formation is associated with IgG and IgA absorption in neonatal calves after first ingestion of colostrum.

Neonatal calves show a remarkable increase in serum IgG levels after first ingestion of colostrum. They can absorb high-molecular IgG from colostrum in the small intestine by nonspecific receptor-independent fluid pinocytosis within 24 h after birth. However, little is known about the temporal changes in serum small-molecule metabolites, such as carbohydrates and AA, in neonatal calves after first colostrum ingestion. In this study, we examined temporal changes in serum metabolites of neonatal calves after first ingestion of colostrum by comprehensive 2-dimensional gas chromatography mass spectrometry (GC×GC-MS). Forty serum samples obtained from 5 calves at 8 time points between 0 and 12 h after first colostrum ingestion were analyzed in triplicate by GC×GC-MS. Multivariate analyses of 120 GC×GC-MS results revealed significant variations in the serum metabolites, primary individual differences among the calves, and secondary temporal changes within each individual calf. Several serum metabolites increased temporally after ingestion in each calf, but only a limited number of compounds were increased universally in all 5 calves. Eight compounds, including oligosaccharides such as lactose, were associated with temporal changes in IgG. Some essential AA that must be supplied from the diet increased temporally after ingestion, but differed from the temporal pattern of the oligosaccharides and IgG. These results suggest that the colostral contents may be absorbed by complex mechanisms that include intestinal pinocytosis for IgG and oligosaccharides, along with others such as specific transporters in the intestinal epithelial cells for AA in calves.

Bovine digital dermatitis (BDD) is the most prevalent infectious cause of lameness in cattle. Because Treponema infection is a major etiology of BDD, the most common treatment of BDD is an antibiotic. Nonetheless, dairy cows require a withdrawal period after antibiotic treatment before their milk can be marketed. To address the problem, in this study, we tested whether 3 nonantibiotic agents (used separately)-allyl isothiocyanate (AITC), sodium alginate, and calcium hydroxide-alleviate BDD lesions in dairy cows. The AITC treatment improved the BDD lesions, whereas the sodium alginate and calcium hydroxide treatments did not. Therapeutic efficacy of AITC was similar to that of lincomycin, a topical antibiotic prescribed for BDD. These results suggest that AITC is a promising nonantibiotic agent for BDD treatment in dairy cows.

Pheromones are crucial for eliciting social and sexual behaviors in diverse animal species. The vomeronasal receptor type-1 (V1R) genes, encoding members of a pheromone receptor family, are highly variable in number and repertoire among mammals due to extensive gene gain and loss. Here, we report a novel pheromone receptor gene belonging to the V1R family, named ancient V1R (ancV1R), which is shared among most Osteichthyes (bony vertebrates) from the basal lineage of ray-finned fishes to mammals. Phylogenetic and syntenic analyses of ancV1R using 115 vertebrate genomes revealed that it represents an orthologous gene conserved for400 My of vertebrate evolution. Interestingly, the loss of ancV1R in some tetrapods is coincident with the degeneration of the vomeronasal organ in higher primates, cetaceans, and some reptiles including birds and crocodilians. In addition, ancV1R is expressed in most mature vomeronasal sensory neurons in contrast with canonical V1Rs, which are sparsely expressed in a manner that is consistent with the "one neuron-one receptor" rule. Our results imply that a previously undescribed V1R gene inherited from an ancient Silurian ancestor may have played an important functional role in the evolution of vertebrate vomeronasal organ.

Pheromones are crucial for eliciting social and sexual behaviors in diverse animal species. The vomeronasal receptor type-1 (V1R) genes, encoding members of a pheromone receptor family, are highly variable in number and repertoire among mammals due to extensive gene gain and loss. Here, we report a novel pheromone receptor gene belonging to the V1R family, named ancient V1R (ancV1R), which is shared among most Osteichthyes (bony vertebrates) from the basal lineage of ray-finned fishes to mammals. Phylogenetic and syntenic analyses of ancV1R using 115 vertebrate genomes revealed that it represents an orthologous gene conserved for >400 My of vertebrate evolution. Interestingly, the loss of ancV1R in some tetrapods is coincident with the degeneration of the vomeronasal organ in higher primates, cetaceans, and some reptiles including birds and crocodilians. In addition, ancV1R is expressed in most mature vomeronasal sensory neurons in contrast with canonical V1Rs, which are sparsely expressed in a manner that is consistent with the "one neuron-one receptor" rule. Our results imply that a previously undescribed V1R gene inherited from an ancient Silurian ancestor may have played an important functional role in the evolution of vertebrate vomeronasal organ.

An enzymatic method for 6-oxohexanoic acid production was developed using 6-aminohexanoic acid and an ω-amino group-oxidizing enzyme (ω-AOX) from Phialemonium sp. AIU 274. 6-Oxohexanoic acid was produced from 6-aminohexanoic acid with 100% yield by incubation with 0.3 U of the ω-AOX and 20 U of catalase at 30 °C for 30 h in 0.1 M potassium phosphate buffer (pH 7.0).

Removal of the major urinary protein, cauxin, a carboxylesterase, from cat urine is essential for distinguishing between physiological and abnormal proteinuria by a urine dipstick. We have previously developed a material for removing cauxin by using lens culinaris agglutinin (LCA) lectin which targets the N-linked oligosaccharides present in cauxin. To improve the affinity and specificity toward cauxin, we immobilized 1,1,1-trifluoro-3-(2-sulfanylethylsulfanyl) propane-2-one, an inhibitor of esterases, to a polymer chain grafted on to a porous hollow-fiber membrane by applying radiation-induced graft polymerization. Normal male urine was forced to permeate through the pores rimmed by the ligand-immobilized polymer chain. Cauxin could not be detected in the effluent from the membrane. The residence time of the urine across a membrane thickness of 1 mm was set at 7 s. The respective dynamic and equilibrium binding capacities of the membrane for cauxin were 2 and 3 mg/g. The developed cauxin-affinity membrane material was more effective for diagnosing cat kidney diseases than the LCA lectin tip.

The article Olfactory discrimination of anal sac secretions in the domestic cat and the chemical profiles of the volatile compounds, written by Tamako Miyazaki, Takashi Nishimura, Tetsuro Yamashita and Masao Miyazaki was originally published electronically on the publisher’s internet portal (currently SpringerLink) on 16 November, 2017 without open access.

Sendai virus (SeV) is an enveloped virus with a non-segmented negative-strand RNA genome. SeV envelope fusion (F) glycoproteins play crucial roles in the viral life cycle in processes such as viral binding, assembly, and budding. In this study, we developed a viable recombinant SeV designated F-EGFP SeV/ΔF, in which the F protein was replaced by an F protein fused to EGFP at the carboxyl terminus. Living infected cells of the recombinant virus were directly visualized by green fluorescence. The addition of EGFP to the F protein maintained the activities of the F protein in terms of intracellular transport to the plasma membrane via the ER and the Golgi apparatus and fusion activity in the infected cells. These results suggest that this fluorescent SeV is a useful tool for studying the viral binding, assembly, and budding mechanisms of F proteins and the SeV life cycle in living infected cells.

Objective—To evaluate urine cauxin immunoreactivity in geriatric cats with variable plasma creatinine concentrations and proteinuria and to assess urinary cauxin-to-creatinine concentration ratio (UC/C) as a predictor of developing azotemia. Animals—188 client-owned geriatric (≥ 9 years of age) cats. Procedures—A direct immunoassay was developed and validated for the quantification of urinary cauxin relative to a standard curve generated from a urine sample with high cauxin immunoreactivity. Relationships among UC/C, plasma creatinine concentration, and proteinuria were assessed. Nonazotemic cats were recruited and followed for 12 months. Urinary cauxin-to-creatinine concentration ratio was evaluated as a predictor of development of azotemia in these cats. Results—No relationship was evident between UC/C and plasma creatinine concentration. A weak positive correlation was identified between UC/C and urine protein-to-creatinine concentration ratio (r = 0.212). At entry to the longitudinal study, those cats that later developed azotemia had a UC/C that was significantly higher than in those remaining nonazotemic after 12 months. Conclusions and Clinical Relevance—The UC/C did not vary with severity of azotemia but appeared contributory to the feline urinary proteome. High UC/C values were predictive of the geriatric cats in our study developing azotemia. However, it seems unlikely that UC/C will provide additional information about the measurement of urine protein-to-creatinine concentration ratio as a biomarker for the development of azotemia in cats.

Seventy-two patients with dizziness or vertigo having C4-7 cervical disk disease had their systolic blood pressure (BP) measured before and after the head-up test with tonometry. We demonstrated that significant depression of the systolic BP was seen in the patient group compared with the normal group during the head-up test. There were 11 patients in whom vertigo or dizziness was induced by each head-up test, and all of them showed a BP depression of 19 mmHg or more. However, 26 patients had a BP depression of only 9 mmHg or less, which was within normal limits, and none showed vertigo or dizziness. It was considered whether the main cause of vertigo or dizziness induced by the head-up test among the patients with cervical lesion was BP depression. The other causes of vertigo or dizziness among the patients who showed BP depression of 18 mmHg or less could possibly be blood flow insufficiency in the brain stem, acoustic nerve ischemia, Powers syndrome, carotid artery stenosis, platelet hyper-aggregation or psychosomatic factors.

Cauxin is a carboxylesterase-like glycoprotein excreted as a major component of cat urine. Cauxin contains four putative N-glycosylation sites. We characterized the structure of an N-linked oligosaccharide of cauxin using nano liquid chromatography (LC)-electrospray ionization (ESI) and matrix-assisted laser desorption/ionization quadrupole ion trap time-of-flight mass spectrometry (MALDI-QIT-TOF MS) and MS/MS, and high-performance liquid chromatography (HPLC) with an octadecylsilica (ODS) column. The structure of the N-linked oligosaccharide of cauxin attached to (83)Asn was a bisecting complex type, Galbeta1-4GlcNAcbeta1-2Manalpha1-3(Galbeta1-4GlcNAcbeta1-2Manalpha1-6)(GlcNAcbeta1-4)Manbeta1-4GlcNAcbeta1-4(Fucalpha1-6)GlcNAc.

Des2 (degenerative spermatocyte 2) is a bifunctional enzyme that produces phytoceramide and ceramide from dihydroceramide. The molecular mechanism involved in C-4-hydroxylation has not been studied in detail. In the present paper, we report that C-4-hydroxylation requires an electron-transfer system that includes cytochrome b 5 and that the hydroxylase activity is reconstituted in an in vitro assay with purified recombinant Des2. FLAG-tagged mouse Des2 was expressed in insect Sf9 cells and was purified by solubilization with digitonin and anti-FLAG antibody affinity column chromatography. The activity of dihydroceramide:sphinganine C-4-hydroxylase was reconstituted with the purified FLAG–Des2, m b 5 (the membrane form of cytochrome b 5 ) and bovine erythrocyte membrane. The apparent K m and V max of Des2 for the substrate N -octanoylsphinganine were 35 μM and 40 nmol·h −1 ·mg of protein −1 respectively. The K m of the hydroxylase for m b 5 was 0.8 μM. Interestingly, m b 5 was not replaced with the soluble form of cytochrome b 5 , which lacks the C-terminal membrane-spanning domain. The erythrocyte membrane was separated into Triton X-100-soluble and -insoluble fractions, and the detergent-soluble fraction was replaced by the soluble or membrane form of b 5 R (NADH-cytochrome b 5 reductase). The Triton-X-100-insoluble fraction contained trypsin-resistant factors. The Des2 protein is found in the endoplasmic reticulum and is assumed to have three membrane-spanning domains. The findings of the present study indicate that the hydroxylation requires complex formation between Des2 and m b 5 via their membrane-spanning domains and electron transfer from NADH to the substrate via the reduction of m b 5 by b 5 R.

Mass spectrometric analysis of a prion protein (PrP)-containing complex isolated from ram cauda epididymal fluid revealed a protein that showed homology to a carboxylesterase-like protein previously identified in cat urine (cauxin). Using anti-cauxin antibodies, immunoreactive bands were detected in corpus and cauda epididymal fluid from all mammals tested (ram, boar, mouse, and cat). In the ram, the protein was also present in seminal fluid but not found to be associated with sperm. The bands reacting with the anti-cauxin antibody coincided with those having esterase activity in a zymographic assay and its levels paralleled the esterase activity of native epididymal fluids. A partial nucleotide sequence of 1143 bp, corresponding to 380 amino acids, was obtained by RT-PCR amplification from total RNA from the corpus epididymis (zone 6). The deduced protein sequence shows a high degree of homology (up to 90%) with the different cauxin proteins found in databases but only up to 60% with other known carboxylesterases. By PCR, strong mRNA expression was found in the corpus and cauda epididymis, while the testis, kidney, and caput epididymis had low expression. No mRNA was detected in the lung, heart, or liver. These data demonstrate that an epididymal form of the cauxin enzyme is secreted into mammalian epididymal fluid. In the ram, it is associated with a high molecular-weight PrP-associated complex and may be responsible for the majority of the esterase activity in the cauda epididymal fluid of this species.

Urinary excretion of N-acetyl-beta-D-glucosaminidase (NAG) was examined in healthy dogs and dogs with urinary diseases, and its clinical usefulness as an indicator of urinary diseases was discussed. Twenty-eight healthy dogs and 20 dogs with urinary diseases were used. Urinary NAG activity was measured using p-nitrophenyl N-acetyl-beta-D-glucosaminide as substrate, and expressed as units per gram of urinary creatinine (NAG index). Urinary NAG index in urine of healthy dogs was 3.2+/-2.4 U/g, and NAG index in the dogs with chronic renal failure or lower urinary tract infection accompanied by pyelonephritis was higher than that in healthy dogs. However, the dogs with lower urinary tract infection without pyelonephritis showed normal values of NAG index. Some dogs with diabetic mellitus showed elevated values of NAG index when control of blood sugar was not successful. Increase of NAG index was observed in some dogs with pyometra before increases of BUN and serum creatinine concentration. Therefore, NAG index in urine seems to be a good indicator for urinary diseases in dogs.

Phosphate-buffered sucrose (PBSc) solution is effective for short-term hypothermic preservation of tissue during feline kidney transplantation. A high-molecular-weight polyethylene glycol (35,000 Da, PEG35) reportedly enhanced the protective effects against cold-induced tubular injuries in animal kidney transplantation models. We investigated the ability of PBSc solution containing PEG35 to preserve cultured feline kidney cells using in vitro WST-8 cell proliferation assays. PEG35 significantly improved cell viability during 24 hr of cold preservation. PBSc containing 20 g/l PEG35 achieved an effect almost equal to that of University of Wisconsin (UW) solution, the gold standard preservation solution used in human clinical kidney transplantation, for up to 24 hr of preservation. Our results suggest that PBSc containing PEG35 provides an excellent medium for graft cold storage during feline kidney transplantation.

食品製造工程上の汚れ (主に残留蛋白質) を検出する洗浄度判定キットを用い, 各種食品の検出感度, 牛乳濃度と大腸菌数の増殖経時変化を調べた.フキトリマスターを用いると牛乳ではおよそ80mg/m2, 脱脂粉乳では2mg/m2, 固形チキンコンソメでは20mg/m2, 卵白においては4.2mg/m2の汚れ量の検出が可能であった.これは目視観察できる汚れ量のおよそ1/3から1/400であった.分光光度計を併用すればさらに検出感度は向上することがわかった.一方, 大腸菌はわずかな栄養源の存在下でも増殖する可能性が示唆された.すなわち菌が増殖するレベルまで汚れの残存 (清浄度) を管理するためには, 目視観察による判定では管理不十分と考えられた.以上の成績から, 製造工程の自主衛生管理のための洗浄度モニタリグの極めて有効な手法の一つとして市販の洗浄度判定キット (フキトリマスター: コニカ) の使用を提案する.

Protein C, a vitamin K-dependent protein, is a blood coagulation inhibitor. Its deficiency causes systemic thrombosis. A 31-year-old woman developed cerebral infarction followed by late psychomotor seizures, and thrombosis in the inferior mesenteric vein and bilateral crural veins. Her parents were first cousins. Her mother died of cerebral thrombosis in her 30's. Her elder brother died of suspected purpura fulminans immediately after birth. Her protein C activity and protein C antigen level decreased markedly and were less than 5% of those of normal controls and 0.3 microgram/ml, respectively. Her father, a paternal aunt and a maternal uncle also showed a low protein C activity and protein C antigen level. This patient seems to have congenital protein C deficiency which produced thrombosis in the leg veins and the mesenteric vein, probably cerebral infarction.

Mammalian carboxylesterase (CES or Ces) genes encode enzymes that participate in xenobiotic, drug, and lipid metabolism in the body and are members of at least five gene families. Tandem duplications have added more genes for some families, particularly for mouse and rat genomes, which has caused confusion in naming rodent Ces genes. This article describes a new nomenclature system for human, mouse, and rat carboxylesterase genes that identifies homolog gene families and allocates a unique name for each gene. The guidelines of human, mouse, and rat gene nomenclature committees were followed and “CES” (human) and “Ces” (mouse and rat) root symbols were used followed by the family number (e.g., human CES1). Where multiple genes were identified for a family or where a clash occurred with an existing gene name, a letter was added (e.g., human CES4A; mouse and rat Ces1a) that reflected gene relatedness among rodent species (e.g., mouse and rat Ces1a). Pseudogenes were named by adding “P” and a number to the human gene name (e.g., human CES1P1) or by using a new letter followed by ps for mouse and rat Ces pseudogenes (e.g., Ces2d-ps). Gene transcript isoforms were named by adding the GenBank accession ID to the gene symbol (e.g., human CES1_AB119995 or mouse Ces1e_BC019208). This nomenclature improves our understanding of human, mouse, and rat CES/Ces gene families and facilitates research into the structure, function, and evolution of these gene families. It also serves as a model for naming CES genes from other mammalian species.

The distribution and fine structure of [Met] -Enkephalin-Arg6-Gly7-Leu8-like immunoreactive (MEAGL-LI) neurons in the rat cerebellum were examined by light and electron microscopic immunohistochemistry. Immunoreactive neurons which were designated to be Golgi cells were found in the granular layer. These MEAGL-LI Golgi cells were distributed heterogeneously in the cerebellar cortex; the densest distribution was seen in the ventral and anterior vermis, moderate distribution in the pedunculofloccular lobe (PFL) and less in the dorsal and posterior parts of the cerebellar hemispheres. Immunoreactive Golgi cells showed well-developed organelles, such as rough-surfaced endoplasmic reticulum (r-ER) and Golgi complex as well as immunoreactive substances throughout the cytoplasm. On the surfaces of immunoreactive neurons non-immunoreactive axons made axo-somatic synapses. MEAGL-LI neuronal elements were distributed as components of the cerebellar glomeruli in the granular layer. Some of them were presynaptic axons which made axo-dendritic synapses with non-immunoreactive dendrites, and others were postsynaptic dendrites with which non-immunoreactive axons made axo-dendritic synapses. MEAGL-LI neuronal elements were also seen to make intimate contact with Purkinje cells in the Purkinje cell layer.

手術予定患者の不安を取り除くため,入院時より抗不安剤トフィソパムを約1週間投与し,その臨床的意義について考察した.抗不安剤の有用性は,21名の対象を任意にトフィソパム投与群とコントロール群の2群に分類し,入院時と手術当日の2回の心理テストState-Trait-Anxiety-Inventory (STAI)の特性不安と状態不安の変動より判定した.コントロール群において特性不安は変化していないが,状態不安は有意差は認めないものの上昇傾向を示しSTAIテストは術前の不安の判定に有用であるといえる.トフィソパム投与群では特性不安は変化しないが,状態不安は有意に低下した.トフィソパムは手術予定患者の不安に対して有用であり,副作用も少なく安全な薬剤であるといえる.

Direct coupling of high-performance thin-layer chromatography (HPTLC) to matrix-assisted laser desorption/ionization quadrupole ion trap time-of-flight mass spectrometry (MS) was shown to be a reliable and reproducible method to obtain structural information and fundamental properties of glycosphingolipids (GSLs).We report a protocol for the preparation of neutral GSL extracts frommouse tissues and demonstrate the applicability of HPTLC/MS to these preparations. The protocol consists of lipid extraction and ion exchange chromatography followed by a mild alkaline treatment and a reversed-phase cartridge extraction. Possible structures for each GSL are proposed based on HPTLC/MS analyses. This fast and simple method can be used to screen neutral GSL extracts obtained from tissues and cells without isolation and purification into individual GSLs.

We studied the effect of nicardipine on the canine cardiovascular system, especially on total blood volume and vascular compliance. Under light halothane anesthesia, nicardipine decreased total blood volume significantly (from 80.0 +/- 8.4 ml/kg in the control state to 75.3 +/- 8.0 ml/kg under nicardipine administration, p less than 0.01), while it increased central circulating blood volume (from 17.1 +/- 5.9 ml/kg to 25.5 +/- 8.2 ml/kg, p less than 0.01), increased cardiac output and central venous pressure, and decreased mean arterial pressure (from 134.3 +/- 16.2 mmHg to 93.9 +/- 17.1 mmHg, p less than 0.01) and total peripheral resistance. Vascular compliance derived from fluid infusion experiments showed a significant decrease (from 8.9 +/- 3.8 ml/mmHg/kg to 5.5 +/- 8.0 ml/mmHg/kg, p less than 0.01). In addition to the vasodilatory action of nicardipine on arteries, these findings also suggest that 1) nicardipine causes a fluid shift from the vascular to the interstitial fluid space as a result of increased capillary pressure, 2) it increases preload through blood redistribution from the peripheral to the central circulation, and 3) it decreases compliance of the vessels, perhaps due to an indirect splanchnic venoconstriction.

Gsl5 regulates the expression of a glycolipid and glycoproteins that contain the Le(X) epitope in the mouse kidney through tissue-specific transcriptional regulation of the core 2 beta-1,6-N-acetylglucosaminyltransferase (core 2 GnT) gene. The core 2 GnT gene has six exons and produces three alternatively spliced transcripts. Gsl5 regulates only the expression of the kidney-type mRNA, which is transcribed from the most 5'-upstream exon. By introducing a 159-kb bacterial artificial chromosome (BAC) clone that carries the mouse core 2 GnT gene and its 5'-upstream region into DBA/2 mice that carry a defective Gsl5 allele, we were able to rescue the deficient phenotype. The BAC clone was subsequently engineered to replace the core 2 GnT gene with the sequence of enhanced green fluorescent protein (EGFP) as a reporter by an inducible homologous recombination system in Escherichia coli. The transgenic mice derived from the modified BAC clone expressed EGFP in the kidney, which suggests that the candidate Gsl5 is in the 5'-upstream region of the core 2 GnT gene. Sequence analysis of the 5'-upstream regions of the BAC clone and DBA/2 genomic DNA revealed a candidate sequence for Gsl5 at about 5.5 kb upstream of exon 1. This sequence consisted of eight repeats of two GT-rich units in the wild-type mice, whereas it consisted of only one pair of GT-rich units with a minor modification in the DBA/2 mice. Transgenic mice produced with the EGFP reporter gene construct that included this candidate sequence expressed EGFP exclusively in the proximal straight tubular cells of the kidney. These results indicated that this unique repeat is indeed the Gsl5, and it is a cis-regulatory element responsible for proximal straight tubule cell-specific transcriptional regulation.

Mass spectrometric analysis of a prion protein (PrP)-containing complex isolated from ram cauda epididymal fluid revealed a protein that showed homology to a carboxylesterase-like protein previously identified in cat urine (cauxin). Using anti-cauxin antibodies, immunoreactive bands were detected in corpus and cauda epididymal fluid from all mammals tested (ram, boar, mouse, and cat). In the ram, the protein was also present in seminal fluid but not found to be associated with sperm. The bands reacting with the anti-cauxin antibody coincided with those having esterase activity in a zymographic assay and its levels paralleled the esterase activity of native epididymal fluids. A partial nucleotide sequence of 1143 bp, corresponding to 380 amino acids, was obtained by RT-PCR amplification from total RNA from the corpus epididymis (zone 6). The deduced protein sequence shows a high degree of homology (up to 90%) with the different cauxin proteins found in databases but only up to 60% with other known carboxylesterases. By PCR, strong mRNA expression was found in the corpus and cauda epididymis, while the testis, kidney, and caput epididymis had low expression. No mRNA was detected in the lung, heart, or liver. These data demonstrate that an epididymal form of the cauxin enzyme is secreted into mammalian epididymal fluid. In the ram, it is associated with a high molecular-weight PrP-associated complex and may be responsible for the majority of the esterase activity in the cauda epididymal fluid of this species.