Non-destructive sex-specific monitoring of early embryonic growth rate in light brown broiler eggs using light transmission and its correlation with hatching time and chick weight.

1. Monitoring early embryonic growth rate (EGR) has significant economic and animal welfare benefits. This study focuses on monitoring sex-specific early EGR using light transmission, and correlating this with hatching time and chick weight. For broiler eggs in particular, spectral masking of the light brown eggshells needed to be addressed. This was done using longitudinal visible transmission spectroscopy combined with eggshell colour image analysis. 2. Prior to incubation, colour images of eggs were captured followed by daily measurements of transmission spectra of eggs from days one to nine of incubation. The sex of the eggs was subsequently verified 2 d after hatching. 3. To accurately and sensitively determine sex differences in EGR using light transmission, while minimising interference from eggshell colour and thickness, the ratio of longitudinal transmissions was determined to be most effective at 575 and 610 nm. 3. Embryonic growth was detectable from d 3 (72 h) of incubation, 24 h earlier than previously reported lateral transmission measurements. However, at this time, low blood levels meant that no significant sex-differences (P > 0.05) for the mean T575/T610 ratio were detectable. This may have been due, in part, to spectral masking from the light brown eggshells. At d 7, female embryos had a significantly lower (P < 0.05) mean T575/T610 ratio than males. 4. Although the T575/T610 ratio had low correlations with hatching time and hatch-weight of chicks, this could be a good starting point for further non-destructive investigations for such predictions. 5. In conclusion, the methodology had the sensitivity to differentiate sex-specific early EGR in broiler eggs, even with pigmented eggshells, and has the potential to advance precision hatchery management and poultry research.

Nondestructive sex-specific monitoring of early embryonic development rate in white layer chicken eggs using visible light transmission.

1. Sex-specific variations in early embryonic development rates may pre-empt later variations in embryonic development through to pipping and hatching. Given that erythropoiesis (blood production) can be equated with early embryonic growth rate, it was hypothesised that blood pigment haemoglobin can act as a specific spectral fingerprint for changes in growth rate. Moreover, by measuring longitudinal, rather than lateral, spectral transmission through the egg, a more consistent spectrum with a higher signal-to-noise ratio could be captured.2. Longitudinal visible transmission (T575/T598 ratio), which is sensitive to haemoglobin, was used to monitor sex-specific early embryonic development rate in white layer chicken eggs from d 0 to 8 of incubation. The sex of these eggs was subsequently confirmed two days after hatching.3. Embryonic development was detectable from d 3 (72 h) of incubation, 36 h earlier than previously reported lateral spectral measurements, supporting the greater sensitivity of longitudinal measurements.4. At d 3, the mean T575/T598 ratio for male embryos was significantly lower (P < 0.001) (i.e. higher absorbance of haemoglobin) than for female embryos, which was thought to be due to sex-differences in early embryogenesis. On the other hand, female embryos had a significantly lower (P < 0.05) mean T575/T598 ratio than male embryos at d 7 of incubation, presumably due to the combined effects of oestrogen synthesis receptors and enzymes on erythropoiesis in female embryos at this time.5. In conclusion, the proposed methodology has the sensitivity to differentiate sex-specific embryonic development rates during early incubation and the potentiality to advance precision incubation management and poultry research.

A novel D-phenylalanine-derivative hypoglycemic agent A-4166 increases cytosolic free Ca2+ in rat pancreatic beta-cells by stimulating Ca2+ influx.

It has recently been shown that N-[(trans-4-isopropylcyclohexyl)-carbonyl]D-phenylalanine (A-4166), a new nonsulfonylurea oral hypoglycemic agent, reduces blood glucose levels in nondiabetic and diabetic animals in a quicker and shorter lasting manner than sulfonylureas, and that the hypoglycemic effect of A-4166 is due to the stimulation of insulin release. However, the mechanism by which A-4166 stimulates insulin release is still unknown. In the present study, we investigated the effect of A-4166 on the cytosolic free Ca2+ concentration ([Ca2+]i) in pancreatic beta-cells from normal rats by dual wavelength fura-2 microfluorometry. In the presence of 2.8 mM glucose, A-4166 produced a rapid increase in [Ca2+]i in a concentration-dependent manner over the range of 3-30 microM. The increase in [Ca2+]i was transient, oscillatory, or sustained. A-4166 did not evoke any decrease in [Ca2+]i, whereas a high concentration of glucose (16.7 mM), a metabolized secretagogue, produced an initial decrease and a subsequent increase in [Ca2+]i. In the presence of 16.7 mM glucose, low concentrations (0.03-1 microM) of A-4166 produced an increase in [Ca2+]i in some of the beta-cells tested. The [Ca2+]i response to A-4166 was completely and reversibly inhibited under Ca(2+)-free conditions as well as by nitrendipine, a blocker of the L-type Ca2+ channel. Nitrendipine also inhibited insulin release from perfused rat pancreases stimulated by A-4166. Diazoxide, an opener of the ATP-sensitive K+ channel, blocked the [Ca2+]i response to A-4166. Sulfonylureas such as tolbutamide and glibenclamide increased [Ca2+]i in a manner similar to A-4166. These results indicate that at basal glucose concentrations, A-4166 increases [Ca2+]i in rat pancreatic beta-cells by stimulating Ca2+ influx through L-type Ca2+ channels, and that this effect is markedly augmented at elevated glucose concentrations. It appears that the increase in [Ca2+]i is related to the stimulation of insulin release by A-4166. Inhibition of ATP-sensitive potassium channels, but not stimulation of beta-cell metabolism, may be involved in the increase in [Ca2+]i by A-4166.

The ability of a new hypoglycaemic agent, A-4166, compared to sulphonylureas, to increase cytosolic Ca2+ in pancreatic beta-cells under metabolic inhibition.

1. N-(trans-4-isopropylcyclohexanecarbonyl)-D-phenylalanine (A-4166) is a new non-sulphonylurea oral hypoglycaemic agent which stimulates insulin release by increasing cytosolic Ca2+ concentration ([Ca2+]i) in beta-cells. 2. We studied comparative effects of A-4166 and sulphonylureas on [Ca2+]i, measured by dual-wavelength fura-2 microfluorometry, in single rat pancreatic beta-cells under normal conditions and conditions where glucose metabolism was inhibited. 3. A glucokinase inhibitor, mannoheptulose (10 mM), a mitochondrial respiratory inhibitor, KCN (100 microM), and uncouplers, dinitrophenol (DNP, 50 microM) and carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP, 0.3 microM), were used to abolish glucose-induced increases in [Ca2+]i in a reversible manner. 4. Under control conditions, A-4166 was one order more potent than tolbutamide in increasing [Ca2+]i, and maximal responses were evoked by 30 microM A-4166 and 300 microM tolbutamide. These equipotent concentrations were employed for the comparative study where glucose metabolism was inhibited. 5. In the presence of mannoheptulose, [Ca2+]i responses to tolbutamide, but not those to A-4166, were attenuated in a reversible manner. 6. KCN, DNP and FCCP inhibited [Ca2+]i responses to tolbutamide to a much greater extent than those to A-4166. Responses to tolbutamide even at 3.3 times the equipotent concentration (1000 microM) were also markedly attenuated by these inhibitors. Responses evoked by another sulphonylurea, gliclazide, were inhibited by DNP to a larger extent than A-4166-induced responses. 7. The results indicate that A-4166 acts more effectively than sulphonylureas to increase [Ca2+]i in beta-cells during metabolic inhibition.

Hypoglycaemic and insulinotropic effects of a novel oral antidiabetic agent, (-)-N-(trans-4-isopropylcyclohexanecarbonyl)-D-phenylalanine (A-4166).

1. (-)-N-(trans-4-isopropylcyclohexanecarbonyl)-D-phenylalanine (A-4166), a novel oral hypoglycaemic agent is a non-sulphonylurea insulin secretagogue. 2. We investigated the insulin-releasing action and hypoglycaemic effect of A-4166 compared to sulphonylureas in vitro and in vivo. 3. A-4166 stimulated insulin secretion from rat freshly isolated pancreatic islets at concentrations from 3 x 10(-6) M to 3 x 10(-4) M in the presence of 2.8 mM glucose. There was no obvious difference in glucose dependency between the insulinotropic effect of A-4166 and that of glibenclamide, and no additive or synergistic effect was observed between these two drugs. 4. A-4166 displaced [3H]-glibenclamide bound to intact HIT-T15 cells in a concentration-dependent manner. The Ki value was 4.34 +/- 0.04 x 10(7) M, and the displacement potency of A-4166 was between that of glibenclamide and tolbutamide, being similar to that of gliclazide. 5. Inf fasted beagle dogs, A-4166 showed a dose-dependent hypoglycaemic effect after oral administration over the range 1 to 10 mg kg-1. The hypoglycaemic action of A-4166 showed an earlier onset and a shorter duration than that of sulphonylureas. 6. Simultaneous measurement of plasma insulin levels revealed that the hypoglycaemic effect of A-4166 was caused by a rapid-onset and brief burst of insulin secretion. 7. The pharmacokinetic profile of A-4166 was consistent with the changes of the blood glucose and plasma insulin levels. 8. Although the in vitro insulin-releasing effect of A-4166 was similar to that of sulphonylureas, its hypoglycaemic effect was more rapid and shorter-lasting, associated with rapid absorption and clearance. Thus, A-4166 may be useful in suppressing postprandial hyperglycaemia in patients with non-insulin-dependent diabetes mellitus.

Somatostatin and insulin secretion due to common mechanisms by a new hypoglycemic agent, A-4166, in perfused rat pancreas.

N-[(trans-4-isopropylcyclohexyl)-carbonyl]-D-phenylalanine (A-4166) is a nonsulfonylurea hypoglycemic agent that decreases blood glucose levels in nondiabetic and diabetic animals. In the present study, we attempted to determine the effect of A-4166 on hormone secretion from the in vitro-perfused rat pancreas and to examine the underlying secretory mechanisms. In the presence of basal glucose (3 mmol/L), A-4166 markedly stimulated insulin and somatostatin release in a concentration-dependent manner over 0.03 to 3 mmol/L. A sulfonylurea, tolbutamide, also stimulated insulin and somatostatin release. A-4166 and tolbutamide elevated the level of glucagon release; however, the change lacked a clear concentration-dependent property. A-4166 at 0.3 mmol/L and tolbutamide at 3 mmol/L exhibited maximal stimulation of insulin release to a similar extent, indicating that A-4166 is one log-order more potent than and as effective as tolbutamide. By contrast, A-4166 stimulated somatostatin release to a threefold greater extent than tolbutamide. A-4166 evoked an increase in the cytosolic free-Ca2+ concentration ([Ca2+]i) in rat pancreatic beta cells. [Ca2+]i and insulin secretory responses to A-4166 were inhibited by nitrendipine (NTD), a blocker of the L-type Ca2+ channel, and by diazoxide (DAZ), an opener of the adenosine triphosphate (ATP)-sensitive K+ channel. Furthermore, A-4166-stimulated somatostatin release was also inhibited by NTD and by DAZ. The results indicate that A-4166 and tolbutamide stimulate the release of insulin and somatostatin, and that A-4166 is much more effective than tolbutamide in releasing somatostatin, a hormone that attenuates hyperglycemia under certain circumstances. It is concluded that A-4166-induced insulin release is mediated by an increase in [Ca2+]i in beta cells. An inhibition of ATP-sensitive K+ channels and a consequent activation of L-type Ca2+ channels appear to play a key role not only in insulin secretion from beta cells, but also in somatostatin secretion from delta cells in response to A-4166.

[Transnasal microsurgery using a micro-pressure-suction-irrigation system for pituitary adenomas].

Micro-pressure-suction-irrigation system (MPSIS), introduced by Luedecke et al, is an instrumentation for the direct transnasal pituitary procedure. We improved this system for use in Japan. The irrigation system can effectively clean the operating field by one-hand manipulation and dissect tumor tissue by its rapid flow. The pressure of suction and irrigation can be adjusted respectively by a device in the handpiece. The MPSIS is applicable to different stages of intervention because it is equipped with separate tips of various diameters, lengths and angles. This system is especially useful in combination with a micromirror or an endoscopy for direct inspection of the eccentric tumor sites such as the cavernous sinus, the upper part of the planum sphenoidale, or the posterior suprasellar regions. The use of the MPSIS helps to avoid injury to normal tissue structures, and prevents tiny soft microadenoma from being lost during preparation. We have proved the suitability and usefulness of the MPSIS in 23 surgical interventions for transnasal microsurgery of pituitary adenomas.

[A combination of low-dose carboplatin (CBDCA) and radiation therapy in head and neck cancer patients--response and hematologenic toxicity].

A combination of carboplatin (CBDCA) and radiation therapy was performed in patients with head and neck cancer. The intravenous administration of CBDCA at a weekly dose of 100 mg/body was combined with external irradiation at a dose of 1.8 Gy/day x 5/week during the same therapy period. We evaluated the effects of this method not only on survival and neoadjuvant chemotherapy in patients with advanced cancer but also on local control and prevention of distant metastases in patients with early cancer. The subjects consisted of 31 patients with head and neck cancer who visited the Department of Otolaryngology, Showa University Hospital, Kanto Rosai Hospital and Yokohama Rosai Hospital between March 1993 and March 1995. Squamous cell carcinoma was found in all but one patient who had adenocarcinoma in the parotid gland. The patients were 27 males and 4 females ranging between 40 and 81 years of age (mean, 59 years). Twenty-six patients had previously untreated tumor, while 5 patients had recurrent tumor. The clinical stage was Stage I in 6, Stage II in 10, Stage III in 5 and Stage IV in 10 patients. The original site of cancer was the larynx in 9, hypopharynx in 6, nasopharynx in 6 and elsewhere in 10 patients. The combined therapy was repeated for 2-9 courses (mean, about 5 courses). The total dose of CBDCA was 500 mg on average with a maximum of 900 mg. The total effective rate of the combined therapy was 93.3% with 12 cases of complete response (CR) and 16 cases of partial response (PR). The effective rate in patients with previously untreated tumor was 92% because one patient showed a minor response (MR) and one patient showed no change (NC). The effective rate in patients with recurrent tumor was 100%. Concerning the clinical stage, all patients with Stage I-III disease showed CR or PR, while MR was found in 1, NC in 1 and progressive disease (PD) in 1 of 10 patients with Stage IV disease, resulting in an effective rate of 71.4%. There were no CRs in Stage IV patients. Therapy was terminated due to side effects in 2 patients. Excellent safety was confirmed by laboratory data.

[Clinical study of noise-induced deafness. Part 12: Results of tests on cases of tinnitus and evaluation].

For 80 cases of noise-induced deafness, various tests on tinnitus were performed to obtain results as follows. 1) When tinnitus was analyzed by the tinnitus questionnaire, many had bilateral tinnitus all the time, while few noted changes in the tone and loudness of sound. 2) As for the tinnitus frequency obtained by the pitch match test, cases with 4 kHz or more were many, accounting for 81 percent. The average tinnitus frequency was 5500 Hz. The rate of agreement with the frequency for the maximum hearing loss was 55 percent. In cases where the tinnitus frequency was not in agreement with the frequency for the maximum hearing loss, the tinnitus frequency was noted in the lower range of tone than the frequency for the maximum hearing loss. 3) The loudness of tinnitus is measured by the loudness balance test was not more than 5 dB in 86 percent of the cases. 4) As for evaluation on the subjective expression, expressions such as "zee", "keen", "meen" were many. On the pitch-match test, most of tinnitus was of the pure tone but there might be some composed of noise judging from onomatopoeic words. As for evaluation, the subjective expression was considered close to the tinnitus frequency when emphasis is placed on 2 points of evaluation. 5) As for results of the masking test, convergence type and distance type according to the Feldmann's classification were found in many cases. 6) When the tinnitogram was classified into 5 types, the high-pitched tone obliquely dip type and the dip type accounted for 80 percent. The masking inability was noted in 9 percent. The agreement between the lowest frequency for masking in the masked cases and the tinnitus frequency in the pitch-match test was found in 82%, if similar cases are included. This shows that the tinnitus frequency can be found from the masking test as well. 7) A look at the masking level at each frequency indicated that the band noise at 125 Hz might be a sound that can be masked regardless of the tinnitus frequency. 8) The rate of RI appearance was low at 36 percent, indicating that the white noise 80 dB failed to mask tinnitus.

A unique repetitive DNA sequence in the Myxococcus xanthus genome.

We found a novel type of repetitive DNA sequence in the Myxococcus xanthus genome. The first repetitive sequence is located in the spacer region between the ops and tps genes. We cloned five other repetitive sequences using the first repetitive sequence as a probe and determined their nucleotide sequences. Comparison of these sequences revealed that the repetitive sequences consist of a 87-bp core sequence and that some clones share additional homology on their flanking regions.

Quantitative evaluation of capacity-limited hepatobiliary transport based on hepatocellular diffusion model by MULTI(FEM).

The dose-dependency of hepatic uptake and hepatobiliary transport of a drug was evaluated by means of a nonlinear least square program incorporating the finite element method, MULTI(FEM). A perfusion experiment using isolated rat livers following a pulse input (i.e., under nonsteady-state conditions) was performed at three dose levels of cefpiramide as a model drug. The hepatic extraction ratio (E(H)) of cefpiramide decreased with an increase in dose, which demonstrates that the hepatic uptake is capacity-limited. The outflow time-profiles from the liver were represented by a two-compartment dispersion model with central Michaelis-Menten elimination, and the maximal elimination rate per central compartment volume (Vmax) and the Michaelis constant (Km) were estimated to be 1420 microg/ml/min and 235 microg/ml, respectively. The biliary mean transit time (t(bile)) increased slightly with an increase in dose. The hepatocellular diffusion model under non-steady-state conditions considering nonlinear transport across the bile canalicular membrane was adopted to evaluate dose-dependency in the biliary excretion of cefpiramide. The maximal penetration velocity across the bile canalicular membrane per liver (V=(bcm)max) and the affinity constant of penetration across the bile canalicular membrane (k(bcm)m = K(bcm)m A(H)L) were estimated to be 40.1 microg/min and 123 microg, respectively. Considering that the volume of a rat liver (A(H)L) is approximately 10 ml, the Michaelis constant of penetration (K(bcm)m), which is an apparent parameter, was estimated to be approximately 12.3 microg/ml. In conclusion, MULTI(FEM) is useful for evaluation of capacity-limited local disposition.

Effect of a new hypoglycemic agent, A-4166 [(-)-N-(trans-4-isopropylcyclohexanecarbonyl)-D-phenylalanine], on postprandial blood glucose excursion: comparison with voglibose and glibenclamide.

(-)-N-(trans-4-Isopropylcyclohexanecarbonyl)-D-phenylalanine (A-4166) is a new nonsulfonylurea hypoglycemic agent that lowers blood glucose by stimulating insulin release. In the present study, we examined the effects of A-4166, voglibose (an alpha-glucosidase inhibitor), and glibenclamide (a sulfonylurea) on the postprandial glycemic increase in rats with or without diabetes mellitus. Oral administration of A-4166 (25-100 mg/kg) dose-dependently decreased blood glucose with a rapid onset and short duration in normal rats. On the other hand, glibenclamide (1-4 mg/kg) showed a slower onset of its hypoglycemic action, and voglibose (0.2 mg/kg) had no effect. In the case of postprandial glucose excursion, the carbohydrate-induced increase in blood glucose was reduced by oral administration of either A-4166 or voglibose without causing sustained hypoglycemia in both normal and neonatal streptozotocin-induced diabetic rats. However, the efficacy of voglibose varied with the type of carbohydrate load. Glibenclamide produced a prolonged decrease in blood glucose without any appreciable effect on the initial glucose excursion. After sucrose loading, plasma insulin levels during the initial 1 h were significantly higher in A-4166-treated rats than in control rats, while voglibose completely inhibited the insulin response to sucrose. In glibenclamide-treated rats, an augmented insulin response was not seen. In conclusion, unlike other hypoglycemic agents, A-4166 suppresses postprandial glucose excursions by stimulating the early phase of insulin secretion.

Physical and genetic analyses of IncI2 plasmid R721: evidence for the presence of shufflon.

A physical map of the 75.1-kb IncI2 plasmid R721 was constructed by using 15 restriction enzymes, and the regions of several genetic determinants including the origins of replication and of conjugal DNA transfer were located on the physical map. It was found that R721 bears a DNA region which undergoes DNA rearrangement similar to the shufflon of R64.

FruA, a putative transcription factor essential for the development of Myxococcus xanthus.

A new developmental gene, fruA, of Myxococcus xanthus was cloned using a one-step cloning vector, TnV. DNA sequencing of the wild-type allele of the fruA gene indicated that the fruA gene encodes a protein of 229 amino acid residues with a calculated molecular weight of 24672. The deduced amino acid sequence of FruA protein showed similarity to those of many bacterial regulatory proteins carrying a DNA-binding helix-turn-helix motif. The transcription-initiation site of the fruA gene was determined by a primer-extension experiment. Development of M. xanthus cells with a disrupted fruA gene stopped at the stage of mound formation. Although cells were able to aggregate to form mounds, myxospores were not formed. By Northern and Western blot analysis, it was found that the fruA expression was not detected during vegetative growth but initiated at around 6 h and reached the highest level at 12 h after the onset of development. Expression of the fruA gene was dependent on the expression of asg, bsg, csg, dsg, and esg genes, indicating that a series of intercellular signalling is necessary for the expression of the fruA gene. The effects of the fruA mutation on beta-galactosidase expression of various developmentally regulated genes fused with the lacZ gene were analysed; three developmental lacZ fusions (omega 4469, omega 4273 and omega 4500) were either poorly induced or not induced at all, while three other lacZ fusions (omega 4408, omega 4521 and omega 4455) expressed at the early stage of development were normally induced but were unable to be repressed at a later stage of development as in the wild-type strain. Interestingly, in the fruA mutant, tps (the gene for protein S) was not activated. From these results together with analysis of the amino acid sequence of FruA, we propose that FruA is a putative transcription factor required for the development of M.xanthus.