Short communication: Menaquinone-4 (vitamin K2) induces proliferation responses in bovine peripheral blood mononuclear cells.

The effects of vitamin K (VK) on immune cells in ruminants are yet to be fully investigated. The objective of this study was to examine the effects of VK on peripheral blood mononuclear cells (PBMC) in Holstein dairy cows. A cell proliferation assay was performed to evaluate the effect of menaquinone-4 (MK-4, the biologically active form of VK) on immune response of PBMC. The proliferation of PBMC stimulated by MK-4 was significantly higher than that of nonstimulated controls. The expression of T cell-related genes in PBMC, stimulated with MK-4, was assessed by quantitative PCR. No significant changes were observed in the mRNA expression levels of both CD4 and CD8 as helper T cell and cytotoxic T cell markers, respectively. The present study demonstrated that MK-4 positively influenced cow PBMC proliferation and suggested the possibility of bovine-specific immune cell activation. The present study lays a foundation for understanding the physiological role of VK in cattle.

The Clinical Outcome of Anatomical Reattachment of the TFCC to the Ulnar Fovea using an ECU Half-Slip and an Interference Screw.

We developed an original reattachment technique using a half-slip of the extensor carpi ulnaris (ECU) tendon with a very small titanium interference screw for chronic foveal avulsion of the TFCC. The clinical outcome of 66 wrists with foveal detachment of the TFCC treated by this procedure was examined.A distally based ECU half-slip was harvested, inserted into the TFCC, sutured to the remnant of the TFCC, and pulled out through a 2.5-mm bone tunnel at the centre of the fovea. The ECU half-slip was subsequently anchored to the ulnar fovea with a small titanium interference screw. We evaluated 66 wrists of 65 patients with a minimum follow-up of 1 year. Ulnar variance was neutral in 47 wrists, negative in 5 and positive in 14 wrists. Causes of injury were falls in 34 patients, traffic accidents in 12, sports activities in 9, labour in 2 and unknown in 8 patients. In the positive variance wrists, ulnar shortening was performed before the reattachment. The clinical outcome was evaluated using our original DRUJ evaluating system.Preoperatively, severe wrist pain was reported in 50 wrists and moderate pain in 16 wrists. Severe no-endpoint DRUJ instability was noted in 65 wrists, while 1 wrist demonstrated moderate DRUJ instability. Only 2 wrists had supination loss by 20 degrees. At the final follow-up, no pain was felt in 55 wrists, mild pain in 3 wrists, and 8 patients had moderate pain. One wrist exhibited a 30-degree loss of supination. The DRUJ was stable in 55 wrists, mildly unstable in 3, moderately unstable in 4 and severely unstable in 4 wrists. There were 50 excellent, 9 good, 3 fair and 4 poor results.The technique of anatomical reattachment of the TFCC to the ulnar fovea using an ECU half-slip tendon is effective for chronic foveal avulsion of the TFCC with severe DRUJ instability.

Ultraviolet photochemical reaction of [Fe(III)(C2O4)3](3-) in aqueous solutions studied by femtosecond time-resolved X-ray absorption spectroscopy using an X-ray free electron laser.

Time-resolved X-ray absorption spectroscopy was performed for aqueous ammonium iron(III) oxalate trihydrate solutions using an X-ray free electron laser and a synchronized ultraviolet laser. The spectral and time resolutions of the experiment were 1.3 eV and 200 fs, respectively. A femtosecond 268 nm pulse was employed to excite [Fe(III)(C2O4)3](3-) in solution from the high-spin ground electronic state to ligand-to-metal charge transfer state(s), and the subsequent dynamics were studied by observing the time-evolution of the X-ray absorption spectrum near the Fe K-edge. Upon 268 nm photoexcitation, the Fe K-edge underwent a red-shift by more than 4 eV within 140 fs; however, the magnitude of the redshift subsequently diminished within 3 ps. The Fe K-edge of the photoproduct remained lower in energy than that of [Fe(III)(C2O4)3](3-). The observed red-shift of the Fe K-edge and the spectral feature of the product indicate that Fe(III) is upon excitation immediately photoreduced to Fe(II), followed by ligand dissociation from Fe(II). Based on a comparison of the X-ray absorption spectra with density functional theory calculations, we propose that the dissociation proceeds in two steps, forming first [(CO2 (•))Fe(II)(C2O4)2](3-) and subsequently [Fe(II)(C2O4)2](2-).

A high-protein diet induces dissociation between plasma concentrations of growth hormone and ghrelin in wethers.

High-carbohydrate or high-fat diets have been demonstrated to change ghrelin concentrations in plasma; however, there remains a need to clarify the effects of dietary protein on the interaction between circulating GH and ghrelin concentrations in the ruminant. In this study, we investigated the postprandial changes in plasma concentrations of GH and ghrelin and their interactions when wethers were fed either a high-protein (HP; 40% CP) or a low-protein (LP; 10% CP) diet for 2 wk. The wethers were divided into 2 groups and fed once a day for 2 wk in a randomized crossover design. Each diet contained the same level of ME. Blood was collected from the animals at specific times over 24 h to measure hormones and metabolites. Feeding once a day caused a prompt reduction in the GH and ghrelin concentrations regardless of the type of diet that the wethers consumed. The preprandial concentrations (P = 0.04), area under the curve (AUC; P = 0.04), and incremental AUC (iAUC; P = 0.06) for ghrelin in HP-fed wethers were or tended to be greater than those in LP-fed wethers although concentrations for GH were the same for both diets (P = 0.23). In addition, the time it took for the postprandial ghrelin concentrations to recover to the preprandial concentrations was greater in HP-fed wethers than in LP-fed wethers although this was not true for GH concentrations. Similarly, as for ghrelin, postprandial increase (P < 0.001) and AUC (P = 0.03) for insulin concentration was greater in the HP-fed wethers than in the LP-fed wethers. From these findings, we concluded that dietary proteins (or some other derived metabolites) may dissociate the interaction between plasma concentrations of GH and ghrelin in wethers.

Effects of dietary uridine 5'-monophosphate on immune responses in newborn calves.

When compared with normal milk, bovine colostrum contains a large amount of uridine 5'-monophosphate (UMP) and its derivatives. In the present study, we carried out 2 experiments to determine the effects of dietary UMP (2 g/d) on the immune status of newborn calves. In Exp. 1, newborn Holstein bull calves were fed milk replacer alone (control group) or milk replacer supplemented with UMP (UMP group) from d 4 to 10 after birth. The increase in interferon-gamma concentration by peripheral blood mononuclear cells (PBMC) on d 24 tended to be greater in the UMP group than in the control group (P = 0.06). The IgA concentration of the ileal mucosa was greater in the UMP group than in the control group (P < 0.05), although there was no difference between groups in the jejunal mucosa. In Exp. 2, newborn Holstein bull calves were fed milk replacer alone (control group) or milk replacer supplemented with UMP (UMP group) from d 4 to 56 after birth. The proliferation of PBMC was greater in the UMP group than in the control group on d 14, 28, and 42 (P < 0.01). The increase in interferon-gamma concentration by PBMC was greater in the UMP group than in the control group on d 28 and 42 (P < 0.05). From these results, we concluded that dietary UMP affected the immune responses of newborn calves.

Changes in hepatic key enzymes of dairy calves in early weaning production systems.

The objective of the present study was to describe plasma hormonal and metabolite profile and mRNA expression levels and activities of the enzymes pyruvate carboxylase (PC), phosphoenolpyruvate carboxykinase (PEPCK), and acetyl-coenzyme A (CoA) carboxylase in the liver of male Holstein calves before (1 and 3 wk of age) and after (8, 13, and 19 wk of age) weaning at 6 wk of age. The mean plasma concentration of acetate and beta-hydroxybutyrate increased, and that of plasma lactate and nonesterified fatty acids decreased with week, particularly after weaning. Plasma glucose concentration was lowest at 8 wk of age. The mean plasma concentration of insulin and glucagon did not change with time, and that of cortisol was greatest at 1 wk of age. In the liver, enzyme activity of PC was greatest at 1 wk of age and decreased with time. There was a significant relationship between the activity and the mRNA level for PC. Activity of PEPCK also decreased with week. Acetyl-CoA carboxylase activity tended to decrease with week, and activity at 13 wk of age was lower than that at other times. Expression of PC mRNA, but not that of PEPCK and acetyl-CoA carboxylase alpha, decreased with week. We conclude that the hepatic gluconeogenic enzymes and acetyl-CoA carboxylase activities tend to decrease with age, reflecting changes in plasma metabolites in early weaning production systems.

Changes of activity and mRNA expression of urea cycle enzymes in the liver of developing Holstein calves.

Urea is an important reutilizable nitrogen source for the ruminant and is mainly synthesized through the urea cycle in the liver. The cycle is undertaken by 5 enzymes: carbamoyl phosphate synthetase (CPS), ornithine transcarbamoylase (OTC), arginino-succinate synthetase (AS), argininosuccinate lyase (AL), and arginase. The purpose of this study was to investigate changes in the activity of the enzymes and mRNA expression, given that previous observations have indicated an increase in plasma urea concentrations with age in Holstein calves. First, plasma concentrations of metabolites and hormones were determined in calves at 1, 3, 8, 13, and 19 wk of age (n = 4, weaned at 6 wk of age). The plasma concentration of urea drastically increased after weaning (P < 0.001). The plasma concentration of glucose was lowest at 8 wk. The plasma concentration of IGF-I gradually increased with age, although those of NEFA, glucagon, and cortisol decreased (P < 0.001). Concentrations of triglyceride, alpha-amino nitrogen, growth hormone, and insulin did not change significantly with age of the calf. Next, using the liver tissues taken from calves at 2, 13, and 19 wk of age (n = 4 to 6 at each time point, weaned at 6 wk of age), we measured the activity and mRNA expression of the enzymes by biochemical methods and quantitative reverse transcription-PCR, respectively. The activities of CPS (P < 0.001), OTC (P = 0.001), and AS (P = 0.015) increased with age, whereas AL (P = 0.003) decreased. Although mRNA expression was decreased with age for AL (P = 0.002) and arginase (P = 0.007), no significant change was observed for CPS, OTC, or AS mRNA expression. We conclude that the increased urea production in the liver may be explained not only by an increase in the activities of the urea cycle enzymes, but also by increased ammonia production by rumen fermentation and gluconeogenesis from amino acids around weaning time.

Different responses in postprandial plasma ghrelin and GH levels induced by concentrate or timothy hay feeding in wethers.

Ghrelin and growth hormone (GH) play a key role in regulating energy balance, metabolic hormone secretion and food intake. Ghrelin and GH responses to dietary compositions have not yet been fully clarified, although there may be significant relationships between dietary compositions and ghrelin and GH responses. In the present study, therefore, we assessed whether dietary compositions influence postprandial plasma ghrelin and GH levels in wethers. Four wethers were respectively fed concentrate (C) or timothy hay (R) for 14 days. The levels of total digestive nutrients (TDN) and crude protein (CP) were adjusted to be at the same level. The basal ghrelin in both groups was rapidly and significantly decreased after feeding. Although the decline of ghrelin levels in C was greater and shorter than that in R, no significant difference was observed in the area under the curve (AUC) or in the incremental area. The plasma GH levels were also rapidly and significantly decreased after feeding in both groups and a significant difference was observed between the two groups for AUC of GH. Interestingly, the circadian changes in the plasma ghrelin levels were close to those in the GH levels in C, but this was not the case in R. These data suggest that dietary compositions influence postprandial plasma ghrelin and GH levels, and that these differences may be caused by several factors, including nutrients and ruminal fermentation.

Interaction of GH polymorphism with body weight and endocrine functions in Japanese black calves.

We assessed the interaction of GH gene polymorphisms (AA, AB and BB genotypes) with body weight and measures of endocrine function in Japanese black calves at 10 months of age. The average body weight for the BB genotype (281+/-5 kg) was significantly lower (P=0.0017, ANOVA) than those for the AA (324+/-9 kg) and AB (317+/-7 kg) genotypes. Plasma concentrations of insulin and IGF-I were greater for the AA genotype than for the AB genotype, and AB and BB genotypes, respectively. There were significant differences in the triglyceride and cholesterol concentrations among the GH genotypes. The area under the basal GH concentration was significantly greater (P=0.0314) for the AA genotype than for the two other genotypes. The incremental area over the basal GH concentrations in response to intravenous GHRH injection (0.4 microg/kg BW) was significantly smaller (P=0.0005) for the BB genotype than for the two other genotypes. In addition, linear regression analysis between GH incremental area induced by GHRH and body weight demonstrated that there was a positive linear correlation (r=0.6496, P<0.002) for incremental areas less than 600 ng min/ml, but a negative correlation (r=0.6473, P<0.05) for incremental areas over 600 ng min/ml. These findings indicate that the GH genotypes of the animals could be associated with difference in the GH response in Japanese black cattle at 10 months of age. We also observed a relationship between genotype and animal performances, but other studies on more animals in different conditions must be realized to make a definite conclusion.

Characteristics of developmental changes in the kinetics of glucose and urea in Japanese Black calves: comparison with Holstein calves.

The current study was conducted to clarify the characteristics of glucose and urea kinetics in male Japanese Black calves, using a glucose and urea dilution method with stable isotopes, at preweaning (3 wk of age) and postweaning (13 and 26 wk of age) stages, in comparison with the kinetics of glucose and urea in male Holstein calves. Six Japanese Black and 6 Holstein calves were arranged in a 2 (breed) x 3 (stage) factorial block design. These 12 suckling calves were fed only whole milk, offered concentrate and or chardgrass hay after 3 wk of age, and weaned at 6 wk of age. Under steady-state conditions, glucose challenges (1.0 mg/kg of BW; [U-13C]d-glucose) and urea challenges (both 2.2 mg/kg of BW; [13C]urea and [15N2]urea) were performed at the 3 stages examined. There were no significant differences in plasma glucose concentrations between Japanese Black and Holstein calves at any stage, but the glucose concentrations at 3 wk of age were greater (P <0.05) than those at 13 wk of age in both breeds. The glucose pool size in Japanese Black calves was smaller (P <0.05) than that in Holstein calves at all stages. Within each breed, there were no significant differences between the glucose pool sizes at 3 and 13 wk of age, but the glucose pool size was larger (P <0.05) at 26 wk of age. Moreover, Japanese Black calves had greater glucose irreversible loss rates (P <0.01) than Holstein calves at 3 wk of age, and the glucose irreversible loss rates were less (P <0.05) on and after 13 wk of age in both breeds. Japanese Black calves had greater plasma urea N concentrations (P <0.05) than Holstein calves at all stages, and in both breeds, the urea N concentrations increased (P <0.05) with age. The urea pool size was smaller (P <0.01) in Japanese Black calves than in Holstein calves at all stages, and in both breeds, the urea pool size increased (P <0.05) with age. In comparison with Holstein calves, Japanese Black calves had greater urea irreversible loss rates (P <0.05) on and after 13 wk of age and greater urea recycling rates (P <0.05) at 26 wk of age. In addition, in both breeds, urea irreversible loss rates and urea recycling rates increased (P <0.05) with age. We conclude that Japanese Black calves have partially different glucose and urea kinetics from Holstein calves and that the kinetics of these metabolites in both Japanese Black and Holstein calves are strongly influenced by weaning.

Identification of differentially expressed transcripts in bovine rumen and abomasum using a differential display method.

The rumen has several important physiological functions: absorption, transport, metabolic activity, and protection. To clarify the molecular basis underlying the physiological function of the rumen, reticulum, omasum, and abomasum, we used mRNA differential display to isolate and identify differentially expressed genes in these tissues. We isolated 18 transcripts that coexpressed in the rumen, reticulum, and omasum. Five genes, ribosomal protein 19 (RPS19), basic helix-loop-helix domain containing class B2 (BHLHB2), NADH dehydrogenase flavoprotein 2 (NDUFV2), exosome component 9 (EXOSC9), and ribosomal protein 23 (RPS23), were highly expressed in the rumen of adult Holstein and Japanese Black cattle. Significant differences of expression were observed in the abomasum compared with the rumen, reticulum, and omasum. To investigate the expression pattern of these genes during the neonatal growth stage, the relative levels of gene expression were analyzed in the rumen and abomasum of 3-wk-, 13-wk-, and 18- to 20-mo-old Holstein cattle. The expression level of RPS19 did not change with age in the rumen and abomasum. The levels of BHLHB2, NDUFV2, and EXOSC9 mRNA in the abomasum decreased (P < 0.05) after weaning and declined (P < 0.05) further in adults; in contrast, expression in the rumen was not altered. Interestingly, the levels of RPS23 mRNA in the rumen increased (P < 0.05) after weaning and further increased in the adult; however, the level of expression of this gene decreased (P < 0.05) in the abomasum with weaning and age. This indicates that the 4 tissues, especially the rumen and abomasum, have different developmental pathways after birth and subsequent onset of rumination.

Isolation and characterization of sex chromosome rearrangements generating male muscle dystrophy and female abnormal oogenesis in the silkworm, Bombyx mori.

In deletion-mapping of W-specific RAPD (W-RAPD) markers and putative female determinant gene (Fem), we used X-ray irradiation to break the translocation-carrying W chromosome (W( Ze )). We succeeded in obtaining a fragment of the W( Ze ) chromosome designated as Ze (W), having 3 of 12 W-RAPD markers (W-Bonsai, W-Yukemuri-S, W-Yukemuri-L). Inheritance of the Ze (W) fragment by males indicates that it does not include the Fem gene. On the basis of these results, we determined the relative positions of W-Yukemuri-S and W-Yukemuri-L, and we narrowed down the region where Fem gene is located. In addition to the Ze (W) fragment, the Z chromosome was also broken into a large fragment (Z(1)) having the +( sch ) (1-21.5) and a small fragment (Z(2)) having the +( od ) (1-49.6). Moreover, a new chromosomal fragment (Ze (W)Z(2)) was generated by a fusion event between the Ze (W) and the Z(2) fragments. We analyzed the genetic behavior of the Z(1) fragment and the Ze (W)Z(2) fragment during male (Z/Z(1) Ze (W)Z(2)) and female (Z(1) Ze (W)Z(2)/W) meiosis using phenotypic markers. It was observed that the Z(1) fragment and the Z or the W chromosomes separate without fail. On the other hand, non-disjunction between the Ze (W)Z(2) fragment and the Z chromosome and also between the Ze (W)Z(2) fragment and the W chromosome occurred. Furthermore, the females (2A: Z/Ze (W)Z(2)/W) and males (2A: Z/Z(1)) resulting from non-disjunction between the Ze (W)Z(2) fragment and the W chromosome had phenotypic defects: namely, females exhibited abnormal oogenesis and males were flapless due to abnormal indirect flight muscle structure. These results suggest that Z(2) region of the Z chromosome contains dose-sensitive gene(s), which are involved in oogenesis and indirect flight muscle development.

Postprandial changes in plasma concentrations of growth hormone and ghrelin around weaning in the goat.

We measured and compared plasma levels of GH and ghrelin in response to feeding in 4-week-old (milk replacer-fed) and 13-week-old (alfalfa hay cube-fed) goats, in order to elucidate whether or not the postprandial regulation of these hormone levels changes around weaning. Furthermore, we examined the effects of suckling from the dam or intravenous glucose administration on both hormone and insulin levels in kids. In 4-week-old goats, feeding of a milk replacer diet significantly increased plasma GH levels without changing level of ghrelin. In contrast, in 13-week-old goats, feeding of hay cubes did not change the levels of either ghrelin or GH. Suckling of milk directly from the dams significantly increased the levels of GH and insulin, but not ghrelin, in kids. Finally, intravenous injection of glucose (0.625 mmol/kg BW) did not cause any significant increase in the levels of GH or ghrelin, despite a significant increase in the levels of insulin and glucose. From these results, we conclude that the regulatory system of the somatotropic axis is altered by weaning or weaning-associated processes, and that ghrelin levels may not be involved in this alteration in young goats.

Developmental changes in the kinetics of glucose and urea in Holstein calves.

Because weaning is the point when the nutrient composition of feed changes for the neonatal ruminant, the present experiment was conducted to assess the developmental changes in the kinetics of glucose and urea over this period, using stable isotopes of glucose and urea, at 4, 13, and 24 wk in calves. Plasma concentrations of nonesterified fatty acids, amino-N, urea-N, and insulin-like growth factor-I increased, but that of growth hormone decreased with age. The plasma glucose concentration increased at 13 wk of age and thereafter decreased at 24 wk of age. The glucose irreversible loss and recycling rates were significantly higher at 4 wk of age than at 13 and 24 wk of age. On the other hand, the irreversible loss and recycling rates of urea, as well as the urea pool size, were higher at 24 wk of age than at 4 and 13 wk. It is concluded that weaning at 6 wk is the pivotal time for the alteration of glucose kinetics. However, the aging process, but not weaning, is important for changes in the kinetics of urea in calves.

Molecular cytogenetic characterization and chromosomal distribution of the satellite DNA in the genome of Oxya hyla intricata (Orthoptera: Catantopidae).

The genomic DNA of the grasshopper (Oxya hyla intricata) was subjected to electrophoresis after digestion with HaeIII, and the result showed two bands of highly repetitive DNA, approximately 200 and 400 bp in length. The 200-bp HaeIII-digested fragment was cloned and characterized by sequencing and fluorescence in situ hybridization (FISH). The results showed the presence of two distinct satellite DNA (stDNA) families: one consisting of a 169-bp repeated element having an A+T content of 60.9% and the other consisting of a 204-bp repeated element having an A+T content of 53.9%. No significant homology between the two stDNA families was observed. FISH showed that the chromosomal locations of these families are different from each other. The 169-bp element was located in the C-band-positive regions of the short arms of most of the chromosomes, whereas the 204-bp element was located in the centromeric regions of three chromosome pairs. These results imply that the origins of these two DNA families are different. The results of zoo-blot hybridization to the genomic DNA from four Oxya species, O. hyla intricata, O. japonica japonica, O. chinensis formosana, and O. yezoensis, suggest that the two stDNA families found in the present study are species-specific for O. hyla intricata.

Responses induced by arginine-vasopressin injection in the plasma concentrations of adrenocorticotropic hormone, cortisol, growth hormone and metabolites around weaning time in goats.

In order to assess the biological significance of weaning and water deprivation on the control of plasma concentrations of adrenocorticotropic hormone (ACTH), cortisol, growth hormone (GH) and metabolites in response to stimulation with arginine-vasopressin (AVP) and corticotropin-releasing hormone (CRH), we carried out three experiments in which male goats before and after weaning were intravenously injected with AVP or CRH alone, or in combination with each other. In experiment 1, 17-week-old (post-weaning) goats were intravenously injected with AVP or CRH alone at the doses of 0.1, 0.3 and 1.0 nmol/kg body weight (BW). The AVP injection significantly and dose dependently increased plasma levels of ACTH, cortisol, GH and metabolites, whereas the injection with CRH did not cause significant increases in the levels of these parameters. In experiment 2, 4-week-old (pre-weaning) and 13-week-old (post-weaning) goats were injected with either AVP or CRH alone, followed by a combined injection of both secretagogues at a dose of 0.3 nmol/kg BW. Although the basal levels of the hormones and metabolites, with the exception of glucose, were greater in the 4-week-old goats, the hormone responses induced by stimulation with AVP were weaker than those induced in 13-week-old goats. Additionally, there were no responses in any hormone patterns to CRH stimulation in 4-week-old goats. In experiment 3, 13-week-old goats were injected with CRH alone followed by injection with AVP for two consecutive days of water deprivation. The animals were subjected to withdrawal of up to 20% of the total blood volume and water deprivation for up to 28 h. However, no significant differences in plasma ACTH, cortisol or GH levels were observed between days 1 and 2. Based on these results, we concluded that: (1) AVP is a more potent stimulant than CRH in terms of its ability to induce increases in plasma levels of ACTH, cortisol and GH; (2) the role of AVP as a secretagogue of hypothalamus-pituitary-adrenal hormones is strengthened, whereas the ineffective role of CRH remains unaltered, by weaning; (3) acute stress such as massive withdrawal of blood volume and subjection to water deprivation may not be sufficient burdens to alter stress-related hormone levels in young goats.

Deep lamellar keratoplasty by deep parenchyma detachment from the corneal limbs.

AIM: To improve the deep lamellar keratoplasty technique. METHOD: For the easy and reliable perfomance of deep lamellar keratoplasty (DLKP), detachment of Descemet's membrane through the corneal limber flap was improved. To expose Descemet's membrane, the parenchyma was detached by hydrodelamination through a sclerocorneal flap made in the corneal limbs. The parenchyma was removed after the pseudochamber between it and Descemet's membrane was maintained with viscoelastic material. The corneal graft was placed with a running suture. 22 eyes were treated. RESULTS: Complete exposure of Descemet's membrane was obtained in 20 of the 22 eyes (91%). The membrane was perforated in five of the 22 eyes (23%) during surgery, and two of the 22 eyes (9%) were converted to penetrating keratoplasty. These two eyes developed keratoconus after acute corneal hydrops. CONCLUSION: Compared with the conventional procedure, this new method provides easy, reliable exposure of Descemet's membrane.

Karyotypic evolution and organization of the highly repetitive DNA sequences in the Japanese shrew-moles, Dymecodon pilirostris and Urotrichus talpoides.

The karyological relationship and organization of highly repetitive DNA sequences in Japanese shrew-moles were studied by zoo-blot hybridization and fluorescence in situ hybridization (FISH). When the genomic DNA of the eastern race of Urotrichus talpoides was digested with PstI, three fragments of highly repetitive DNA sequences, approximately 0.7, 0.9, and 1.4 kb in length, were observed as distinct bands. The results of FISH in the eastern race of U. talpoides using these three fragments separately as probes showed that the 0.7-kb PstI fragment was distributed in the centromeric regions of most chromosomes, and that the 0.9- and 1.4-kb fragments were predominantly located in the C-heterochromatin region of chromosome 13p. Although the western race of U. talpoides also had three PstI fragments, 0.9- and 1.4-kb PstI fragments were more ambiguous than those of the eastern race. The PstI- digested genomic DNA in Dymecodonpilirostris produced only a faint 0.9-kb band, and its signal patterns obtained by zoo-blot hybridization were clearly different from those of U. talpoides. The 0.7-kb fragment of U. talpoides hybridized strongly with the 0.9-kb fragment of D. pilirostris. In a FISH analysis, the 0.9-kb fragment of D. pilirostris hybridized with highly repetitive DNA in the centromeric regions of most chromosomes from both D. pilirostris and U. talpoides. Zoo-blot hybridization and FISH analyses suggest that the 0.9- and 1.4-kb PstI fragments were generated specifically in the genome of U. talpoides after the common ancestor differentiated into two extant shrew-mole species. A difference in the length of the centromeric elements between U. talpoides and D. pilirostris might be observed due to certain modifications of the repeating unit.

Effects of 5'-uridylic acid feeding on postprandial plasma concentrations of GH, insulin and metabolites in young calves.

Postprandial changes in plasma concentrations of GH, insulin, IGF-I, leptin and metabolites were compared between young Holstein bull calves fed with milk alone (control group) and with milk+5'-uridylic acid (UMP) (UMP group). UMP (2 g/day) was given with milk at 0830 h and 1530 h for 11 days from the 4th to the 14th day after birth. The perirenal fat weight was significantly lower in the UMP group than in the control group, but there was no significant difference in the weights of the liver, spleen and heart between the groups. Basal GH concentrations in the UMP group were slightly higher, but the postprandial increase in plasma insulin level and the area under the curve for insulin in the UMP group were significantly lower than those in the control group. There was no significant difference in IGF-I levels between the groups. In addition, the postprandial glucose concentrations were lower in the UMP group as reflected by the insulin level, and nonesterified fatty acid concentrations were not different. In the muscle (M. longissimus thoracis) sampled at 14 days of age, the triacylglycerol (TAG) content was significantly greater but glycogen content was significantly lower in the UMP group than in the control group. From these results, we have concluded that feeding 5'-UMP at 2 g/day for 11 days significantly alters TAG accumulation in the body and plasma concentrations of GH and insulin in young bull calves.

Suppressing actions of butyrate on growth hormone (GH) secretion induced by GH-releasing hormone in rat anterior pituitary cells.

We assessed the inhibitory effects of butyrate on the growth hormone (GH) secretion in order to investigate the cellular mechanisms in rat somatotrophs. Isolated anterior pituitary cells were cultured in DMEM for several hours, either in the presence (1, 3, or 10mM) or absence of butyrate, and then stimulated with 10(-7)M GHRH for 30 min, in the presence of butyrate at the concentrations used for the previous culture. The increase in GHRH-induced GH release was significantly reduced in a time-dependent and concentration-dependent manner in the cells previously cultured with butyrate. GH content (the sum of GH released into the medium induced by GHRH stimulation and the GH remaining in the cells after stimulation) was reduced by the culture of cells in the presence of butyrate, which was also inversely dependent on the concentrations used for the culture. Simultaneous addition of an L-type Ca(2+) channel blocker, nifedipine (10 pM), to the medium during 10(-9)M GHRH stimulation significantly reduced the stimulated GH release, which was further significantly decreased by a simultaneous addition of 10 mM butyrate. Butyrate blunted the GHRH (10(-9)M)-induced increase in cellular cyclic AMP and calcium ion concentrations, the activity of protein kinases (A and C), and GHmRNA expression. The expression of mRNA for GPR 41 and 43, known as receptors for short-chain fatty acids, was confirmed in the anterior pituitary cells. These findings suggest that butyrate inhibits GHRH-induced GH release as well as GH production, and the cellular inhibitory actions of butyrate occur in diverse cellular signaling pathways of rat somatotrophs.