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Lungfish are the fish related most closely to tetrapods. The olfactory organ of lungfish contains two distinct sensory epithelia: the lamellar olfactory epithelium (OE) and the recess epithelium (RecE). Based on their ultrastructural and histological characteristics, the lamellar OE and the RecE are considered to correspond respectively to the teleost OE and a primitive vomeronasal organ (VNO). In tetrapods, the OE and VNO have been shown to express different families of olfactory receptors; for example, in mammals, the OE expresses odorant receptors and trace amine-associated receptors, while the VNO expresses type 1 (V1Rs) and type 2 (V2Rs) vomeronasal receptors. In the present study, we examined the expression of V1Rs in the olfactory organs of two African lungfish, Protopterus annectens and Protopterus amphibius. RNA sequencing and phylogenetic analyses identified 29 V1R genes in P. annectens and 50 V1R genes in P. amphibius. Most V1Rs identified in these lungfish were classified as the tetrapod-type V1Rs initially found in tetrapods and distinct from fish-type V1Rs. In teleost, which all lack a VNO, all olfactory receptors are expressed in the OE, while in Xenopus V1Rs are expressed exclusively in the OE, and not in the VNO. In situ hybridization analysis indicated that lungfish V1Rs were expressed mainly in the lamellar OE and rarely in the RecE. These results imply that V1R expression in lungfish represents an intermediate step toward the complete segregation of V1R expression between the OE and VNO, reflecting the phylogenetic position of lungfish between teleosts and amphibians.

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.

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.

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.

Differential diagnosis between Parkinson's disease (PD) and atypical parkinsonism, such as multiple system atrophy (MSA), can be difficult, especially in the early stages of the disease. Deep learning using neural networks (NNs) makes possible the prediction of the diagnosis using various types of biomarkers, unlike conventional linear statistics. We aimed to differentiate the Parkinson's variant of MSA (MSA-P) from PD both in the early stages by clinical utilization of NN analyses before the hot cross-bun and putaminal rim imaging features of MSA appeared. Analysis by NN involved the data of voxel-based morphometry (VBM) that indicate morphological changes and magnetic resonance spectroscopy (MRS) that indicate qualitative changes. VBM analysis showed that compared with PD patients, MSA-P patients showed atrophy in the superior cerebellar peduncle, middle cerebellar peduncle, cerebellar hemisphere, pons, midbrain, and putamen, but not in the globus pallidus. Proton MRS on the globus pallidus in the diseased hemisphere, lacking atrophy as observed with VBM, revealed decreased neurons and gliosis in both groups. Clinical differentiation of MSA-P from PD using NN analysis, involved measuring the prediction potential using the area under the receiver operator characteristic (ROC) curves (AUC). Using both VBM and MRS data, NNs contributed adequately to the clinical diagnosis.

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.

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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.

Scents emitted from excretions provide important information about the owner. Volatile compounds with higher levels in a species and/or sex, or that vary among individuals could be odor cues for species and/or sex, or individual recognition. However, such compounds have been identified in only a few vertebrate species. In domestic cats (Felis silvestris catus), it is known that unburied cat feces are territorial markers asserting the border of their home range, but little was known which fecal compounds are scent cues for species, sex, and individual recognition in cats. In the present study, we demonstrated the chemical basis for species, sex, and individual recognition using feces of cats. For males, major contents were fatty acids and 3-mercapto-3-methyl-1-butanol (MMB), a derivative of the unusual amino acid, felinine. MMB emission levels from feces had sex-based differences (male > female) and dynamic temporal changes during aging. Cats distinguished fecal odors with and without MMB, and different fatty acid compositions among individuals. No cat-specific compound, such as MMB, was detectable from their anal odor emitting fatty acids. We concluded that fecal MMB is a male sex recognition pheromone in cats and also provides a temporal trace of the owner. After sensing MMB, they may distinguish individual differences of conspecific feces with variable subsets of fatty acids. In contrast to scent marks, since cats can obtain species information from visual cues before sniffing conspecific anal odors, they may use their efforts to distinguish individual differences of anal odors during sniffing.

We isolated Burkholderia sp. AIU M5M02, which accumulates poly(3-hydroxybutyrate) [P(3HB)] homopolymer in a nitrogen-limiting mineral salt medium containing mannitol as a sole carbon source, from a marine environment. When the isolated strain was cultured in a nitrogen-limiting mineral salt medium (pH 5.0) containing 10% (v/v) mannitol as a sole carbon source for 4 days, P(3HB) content and P(3HB) productivity reached 40 weight percent and 1.5 g/l, respectively. Thus, we demonstrated that Burkholderia sp. AIU M5M02 can be utilized for P(3HB) production from mannitol, which is a major sugar in seaweed. Furthermore, the isolate utilized not only mannitol but also gluconic acid, glycerol, starch, and sugars such as fructose, glucose, maltose, sucrose, and xylose to produce P(3HB). Based on these results, we expect to be able to apply Burkholderia sp. AIU M5M02 to the production of P(3HB) from other raw materials with low environmental loads.

Domestic cat urine contains large concentrations of the unusual amino acid, felinine (2-amino-7-hydroxy-5,5-dimethyl-4-thiaheptanoic acid). A felinine derivative, 3-mercapt-3-methylbutanol is a potential scent signal for letting other animals know that the scent owners are cats. 3-Methylbutanol-glutathione (MBG) is an upstream precursor of the biosynthetic pathway of felinine that may be produced in hepatocytes by conjugation of glutathione with isopentenyl pyrophosphate, an intermediate for cholesterol biosynthesis. However, little evidence exists to support the biosynthesis of MBG in the liver. This study examined the distribution of metabolites of the felinine biosynthetic pathway in multiple tissues, body fluids, and excretions of cats. MBG, the felinine precursor, 3-methylbutanol-cysteinylglycine (MBCG), felinine, and felinine N-acetyl derivative were quantified by liquid chromatography-electron spray ionization-tandem mass spectrometry. All compounds were detected in cat serum. In males, MBG and MBCG contents were significantly higher than felinine and N-acetylfelinine contents. MBG was detected in multiple tissues, including the salivary gland, heart, liver, spleen, gut, kidney, bladder, adipose tissue, and muscle. Sex differences in MBG levels were observed in the liver and other tissues. Felinine and N-acetylfelinine were also detected in those tissues. Furthermore, we detected all compounds in cat bile and fecal samples, indicating that felinine is excreted into the feces via bile from the liver. We conclude that MBG is synthesized in several tissues in a sex-dependent manner. These findings improve our understanding of felinine metabolism and function in cats.

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.

Foods and drinks emit many volatile compounds, some of which are key odorants producing characteristic odor of each food and drink. All of volatile compounds do not contribute to the formation of odor. Thus, it is very important to identify key odorants from each food and drink for developments of flavor and fragrance. The omission method is useful to identify key odorants. In the method, a standard sample consisted of all volatile compounds and test samples, which are omitted one or some volatiles, are reconstructed by mixing authentic chemicals. Then, odor qualities are compared between the standard and the test samples by sniffing; if odor qualities are different between two samples, the omitted compounds are key odorants. However, it is not easy to prepare omission samples, because we have to identify chemical structures of compounds consisting of target odors, obtain authentic compounds, and reconstruct target odors that may be consisted of several compounds. To overcome these problems, we developed a high-throughput GC-MS system to prepare the omission samples automatically. The developed system is not necessary to identify chemical structures of candidate compounds, and to prepare authentic compounds for the reconstruction of target odors. The system innovatively improves analytical methods for identification of key odorants from foods and drinks.

Domestic cats (Felis silvestris catus) are solitary and territorial, and mark their territories by spraying urine, which emits a strong odor produced by volatile organic compounds (VOCs). Previous studies have focused on identifications of specific VOCs, such as 3-mercpto-3-methyl-1-butanol, a cat-specific VOCs. However, little is known about how whole volatile profiles of their sprayed urine change over time or how the profiles differ among individuals. This study investigated temporal changes and individual differences of volatile profiles produced by whole VOCs in cat urine, and the ability of cats to discriminate between these scent differences. Volatile profiles of fresh and aged cat urine were analyzed by using two-dimensional gas chromatography–mass spectrometry with a VOC preconcentrator comprehensively. Volatile profiles produced by hundreds of VOCs emitted from cat urine were influenced primarily by the age of the urine, and secondarily by individuality. During habituation–dishabituation tests, subjects discriminated between fresh and 24 h-old samples of same individuals, and between odor of different individuals from 0 h-, 3 h-old, and 24 h-old samples. These results strongly suggest that cats can recognize conspecific individuals via olfaction. Since most VOCs varied among individuals but were not stable over time, their urine may contain unknown VOCs that vary among individuals, are stable over time, and act as individual recognition signals.

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.

The histidine-containing imidazole dipeptide carnosine and its methylated analogs anserine and balenine are present at high concentrations in vertebrate tissues. Although the physiological functions of the imidazole dipeptides have not been elucidated yet, it has been suggested that they play significant biological roles in animals. Despite increasing interest, few studies have challenged the quantifications of carnosine, anserine, and balenine by a single HPLC run because they have similar retention times. In this study, we developed a method to quantify these imidazole dipeptides in meat samples using an LC-ESI-MS/MS triple-quadrupole mass spectrometer. We improved the liquid chromatographic separation of the imidazole dipeptides by applying a mix-mode column, which provides both normal phase and ion exchange separations, and developed multiple reaction-monitoring of the transitions for quantification of m/z 227 → 110 for carnosine, m/z 241 → 126 for anserine, m/z 241 → 124 for balenine, and m/z 269 → 110 for L-histidyl-L-leucine (internal standard). The established method met all pre-defined validation criteria. Intra- and inter-day accuracy and precision were ±10.0% and ≤14.8%, respectively. The ranges of quantifications were 14.7 ng/mL to 1.5 mg/mL for carnosine, 15.6 ng/mL to 1.6 mg/mL for anserine, and 15.6 ng/mL to 1.6 mg/mL for balenine. In conclusion, the validated method was successfully applied to the quantification of imidazole dipeptides in biological samples without derivatization.

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).

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.

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.

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.

The aim of this study was to ultrasonographically evaluate curd formation in 29 pre-ruminant calves that were fed a clotting milk replacer. Abomasal curd was absent in 8/29 calves at 2 h after feeding. In these eight calves, abomasal contents were observed as an anechoic image with small echogenic spots (five calves), or as an echogenic image with an unclear outline (three calves), but there was no echogenic image with a clear outline corresponding to curd that was visualised in the other 21 calves. The curd was not observed until 7 h after feeding in the eight calves. Our analysis also indicated that the absence of curd formation in the pre-ruminant calves did not have a significant impact on the appearance, appetite and vigour of pre-ruminant calves or on their blood parameters, including serum triglyceride, blood urea nitrogen and glucose concentrations. The study provided the first evidence that the abomasum of some calves does not form curd despite ingestion of a clotting milk replacer.

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.

Recombinant adeno-associated virus (rAAV) vectors provide excellent gene delivery into the kidney in several mammals. This study evaluated gene delivery into the cat kidney using an rAAV vector. First, infection and reporter gene expression using rAAV vector encoding the enhanced green fluorescent protein gene (rAAV-EGFP) was examined in vitro in epithelial crandell reese feline kidney (CRFK) cells. At 12 h after transduction, green fluorescence was detected in cells. Next, the rAAV-EGFP construct was injected into the kidneys of two anesthetized cats via the skin, similar to a renal biopsy. On 3 and 12 days after injection, green fluorescence was detected in renal tubules localized near the injected site, but not in glomeruli, blood vessels, or interstitial cells. Finally, the rAAV-EGFP construct was transduced into kidney sections cultured ex vivo. EGFP was expressed in renal tubules between the outer cortex and inner medulla regions. These results demonstrate that rAAV vectors effectively mediate gene delivery into cat renal tubules, and may prove usefulness in gene therapy for cats with renal diseases.

Megalin, a receptor-like transporter glycoprotein, is expressed on kidney proximal tubular cells and reabsorbs small-molecular-weight proteins from the glomerular filtrate. Here, we report that mouse megalins differently modified with core 2 beta6GlcNAc transferase had different kinetic properties to a fluorescence-labeled ligand, retinol-binding protein (RBP). BALB/c mice, a wild-type strain in terms of the expression of kidney-specific core 2 beta6GlcNAc transferase, express megalin carrying the core 2 extended Le(x) epitope, while DBA/2 mice, a mutant-strain of the core 2 beta6GlcNAc transferase, express megalin lacking the epitope. We purified these two types of megalin using lentil lectin chromatography and measured the ligand-binding activities of the megalins using Cy5-labeled RBP by applying gel permeation chromatography (GPC) and fluorescence correlation spectroscopy (FCS). The analysis by GPC indicated that the apparent V(max) of the interaction between Cy5-labeled RBP and the megalins of BALB/c and DBA/2 mice was 60 microM and 30 microM, respectively, and the apparent K(m) was 11 microM and 17 microM, respectively. Scatchard analysis demonstrated the presence of two binding sites. Linear regression analysis resulted in a two-binding-site model characterized by a high-affinity site (K(dBALB)=12.0 microM; K(dDBA)=20.9 microM) and a low-affinity site (K(dBALB)=36.2 microM; K(dDBA)=58.8 microM). FCS analysis exhibited quite different K(m) and V(max) values from those obtained by GPC, but similar K(m) values for the two types of megalin, and a lower V(max) value for DBA/2 megalin than BALB/c megalin. These results suggest that the core 2 GlcNAc extended glycan chains on megalin can change the ligand-binding affinity and capacity.

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.

Cauxin, a member of mammalian carboxylesterases (EC 3.1.1.1), is excreted as a major urinary protein in the domestic cat. Urinary cauxin is derived from the kidney proximal straight tubules. Here, we report changes in the renal expression and urinary excretion of cauxin in cats with tubulointerstitial nephritis (TIN). Immunohistochemistry using anti-cauxin antibody showed fewer cauxin-positive tubules in 15 TIN cases than in normal animals. In areas with tubulointerstitial damage, fibroblasts and inflammatory cells replaced renal tubules, and cauxin-positive tubules consequently disappeared. Urine was analysed in six of the 15 cases. In the two cases with mild tubulointerstitial changes, urinary cauxin was detected using SDS-PAGE with Coomassie staining. In the four cases with severe tubulointerstitial changes, urinary cauxin was below the detection limit using Western blotting. These results indicate that the renal expression and urinary excretion of cauxin decrease with the progression of TIN in cats.