Circadian clock molecules CLOCK and CRYs modulate fibrinolytic activity by regulating the PAI-1 gene expression.

Disruptions of circadian rhythms are associated with the development of many disorders. However, whether a disruption of the circadian clock can cause anomalies of the hemostatic balance remains unknown. The present study examines coagulation and fibrinolytic activities in circadian clock mutants, a homozygous Clock mutant and Cry1/Cry2 double knockout (Cry1/2-deficient) mice. The euglobulin clot lysis time (ELT) showed circadian variations that peaked at 21:00 (early night) in wild-type mice, suggesting that fibrinolytic activity is lowest at this time. The ELT was continuously reduced in Clock mutants, while the ELT was significantly increased and did not differ between day and night (9:00 and 21:00) in Cry1/2-deficient mice. The prothrombin time (PT) and activated partial prothrombin time (APTT) were constant in all genotypes. To identify which factors cause the loss of ELT rhythm, we measured fibrinolytic parameters in Clock mutant and Cry1/2-deficient mice. The robust circadian fluctuation of plasma plasminogen activator inhibitor 1 (PAI-1) that peaked at early night was damped to trough levels in Clock mutant mice. On the other hand, PAI-1 levels in Cry1/2-deficient mice remained equivalent to the peak levels of those in wild-type mice at both 9:00 and 21:00. Circadian changes in plasma PAI-1 levels seemed to be regulated at the level of gene expression, because the plasma PAI-1 levels in Clock mutant and Cry1/2-deficient mice were closely correlated with the level of PAI-1 mRNA transcript in these mice. Plasma plasminogen and hepatic mRNA levels were not rhythmic in wild-type mice, and continuously higher in Clock mutant than in wild-type or Cry1/2-deficient mice. In contrast, the activity and mRNA levels of tissue type plasminogen activator (t-PA), plasma levels and mRNA levels of plasminogen, and plasma levels of alpha2 plasmin inhibitor (alpha2PI) in all genotypes were constant throughout the day. Coagulation parameters such as factor VII, factor X, prothrombin and fibrinogen remained constant throughout the day, and were not affected by clock gene mutations. These results suggest that circadian clock molecules play an important role in hemostatic balance by regulating the fibrinolytic systems.

Quantities and types of ceramides and their relationships to physical properties of the horn covering the claws of clinically normal cows and cows with subclinical laminitis.

Quantities and types of ceramides and their relationships to physical properties of the horn covering the claws of clinically normal cows and cows with subclinical laminitis were investigated. Total ceramide content of the horn covering the sole and wall from cows with subclinical laminitis was 872.2 +/- 146.6 microg/g and 528.6 +/- 61.3 microg/g, respectively, and was significantly (P < 0.01, 0.05) lower than that from clinically normal cows. The mean moisture content in the claws from cows with subclinical laminitis (43.5% +/- 4.3%) was significantly (P < 0.05) higher than that in the claws from clinically normal cows. The hardness of claws from cows with subclinical laminitis (35.2 +/- 3.5) was significantly (P < 0.05) less than that of claws from clinically normal cows. Significant correlations between ceramides and moisture content (P < 0.001) and between ceramide and hardness (P < 0.001) were found in clinically normal cows and cows with subclinical laminitis. Our results indicate that decreases in ceramide contents may be related to changes in physical properties of the horn covering the claw in cows with subclinical laminitis.

Effects of biotin supplementation on serum biotin levels and physical properties of samples of solar horn of Holstein cows.

Effects of dietary biotin supplementation on serum biotin levels and physical properties of sole horn of 40 Holstein cows were evaluated. The mean serum biotin level in biotin-supplemented cows after 10 mo of biotin supplementation (1163.2 +/- 76.2 pg/mL) was significantly higher (P = 0.007) than that in control cows (382.0 +/- 76.2 pg/mL). The sole horn of biotin-supplemented cows was significantly harder (P = 0.026) and had a significantly lower moisture content (P = 0.021) than that of control cows. No morphologic differences in horn tubules or intertubular horn were found between the biotin-supplemented and control cows. The total lipid content of sole horn was significantly higher (P = 0.030) in the biotin-supplemented cows than in the control cows. These results suggest that dietary biotin supplementation causes increases in serum biotin levels and changes in physical properties and fat content of sole horn.

[Successful treatment for tachyarrhythmia with ultra short acting beta-blocker during beating coronary artery bypass grafting; report of a case].

A 62-year-old man with supraventricular/ventricular tachycardia associated with myocardial infarction developed tachycardia during beating coronary artery bypass grafting (CABG). Intravenous administration of an ultra short acting beta-blocker, landiolol hydrochloride, controlled heart rate and improved tachyarrhythmia without significant change of blood pressure. Landiolol hydrochloride is effective and useful for the treatment of tachyarrhythmia during beating CABG.

Physiological changes in the concentrations of biotin in the serum and milk and in the physical properties of the claw horn in Holstein cows.

Physiological changes in the concentrations of biotin in the serum and milk and in the physical properties of the claw horn were examined in Holstein cows. A lower concentration of biotin in the serum and a higher concentration of biotin in milk were found during early and late lactation and during the dry period, and a significant (p<0.05) inverse correlation was found between serum and milk biotin concentrations. A high moisture content and a low level of hardness of the claw horn were found during mid-lactation. Our results indicate that change in the serum biotin concentration probably results from the loss of biotin in the milk of cows during each stage of lactation and also confirm that the moisture content and hardness of the claw horn undergo physiological changes.

[Analysis of factors related to the high incidence of tuberculosis in the city of Wakayama--analysis according to age groups, and sputum test results].

Over the last 20 years, the decrease in the incidence of Tuberculosis (TB) in Japan has slowed down. As of 1999, the incidence rate was 34.6 per 100,000 population in Japan, which was higher than that of the other developed countries, and the incidence rate in the city of Wakayama, one of the prefectural capital cities in Japan, during the same period was 42.9 per 100,000 population. We investigated the causes of this high incidence rate of TB in Wakayama City according to the analysis by age groups and sputum test results when patients are newly registered. Comparing our data during the period from 1.1.1998 to 12.31.1999 with data during the same period in the whole country and the rest of Wakayama Pref., the following results were obtained. Observing by age-groups, the incidence of TB in Wakayama City as well as in the rest of Wakayama Pref. and in the whole country was highest in the age-group above 70 years of age, though the rate of Wakayama City was significantly higher (146.2 per 100,000 population) than that in the rest of Wakayama Pref. (98.5 per 100,000 population) and that in the whole country (90.3 per 100,000 population). Furthermore, the incidence rate of cases diagnosed as TB without bacteriological proof in Wakayama City (57.1 per 100,000 population) was significantly higher than that of the whole country (33.7 per 100,000 population). Therefore, we concluded that one of the causes of high incidence of TB in Wakayama City was due to inappropriate method of diagnosing TB. More extensive use of sputum examination and strict evaluation of cases without bacteriological proof are desirable to increase the accuracy of TB diagnosis in Wakayama City.

Serum lipid peroxide and alpha-tocopherol concentrations and superoxide dismutase activity in captive bottle-nosed dolphins.

OBJECTIVE: To evaluate serum lipid peroxide (LPO) and alpha-tocopherol concentrations and superoxide dismutase (SOD) activity in captive bottle-nosed dolphins and to evaluate effects of storage on production of LPO in various marine fish. ANIMALS: 16 bottle-nosed dolphins. PROCEDURE: 8 dolphins (group A) were fed chub mackerel and herring (high fat) and arabesque greenling and banded blue-sprat (low fat); the other 8 dolphins (group B) were fed chub mackerel and Pacific saury (high fat) and shishamo smelt and Japanese horse mackerel (low fat). Each group had been on these respective diets for 3 years. Serum LPO and alpha-tocopherol concentrations, serum SOD activity, and superoxide production by neutrophils were measured. All types of marine fish were frozen at -20 C for 6 months, and concentrations of LPO were measured at various time points. RESULTS: Serum LPO concentrations in group-A dolphins were significantly higher than those in group B. Serum alpha-tocopherol concentrations and SOD activity in group A were significantly lower than those in group B. A significant negative correlation was found between serum LPO and alpha-tocopherol concentrations in all 16 dolphins. The LPO concentrations in mackerel and herring fed to group-A dolphins were higher than those of other fish. Concentrations of LPO in herring stored for 3 and 6 months at -20 C were higher than those in herring before freezing and in herring stored for 1 month. CONCLUSIONS AND CLINICAL RELEVANCE: Serum LPO and alpha-tocopherol concentrations in captive bottle-nosed dolphins may be strongly influenced by high amounts of polyunsaturated fatty acid and LPO found in marine fatty fishes. High concentrations of serum LPO, as found in group-A dolphins, were associated with decreased antioxidative states. Monitoring of serum LPO and alpha-tocopherol concentrations and serum SOD activity may be useful for the management of captive marine mammals.

High-performance liquid chromatographic-fluorimetric assay for cathepsin A (lysosomal protective protein) activity.

A rapid and sensitive assay for the determination of cathepsin A activity is reported. This method is based on fluorimetric detection of a dansylated peptide, 5-dimethylaminonaphthalene-1-sulfonyl-L-Phe, enzymatically formed from the substrate 5-dimethylaminonaphthalene-1-sulfonyl-L-Phe-L-Leu, after separation by high-performance liquid chromatography using a C18 reversed-phase column and isocratic elution. This method is sensitive enough to measure 5-dimethylaminonaphthalene-1-sulfonyl-L-Phe at concentrations as low as 300 fmol, yields highly reproducible results and requires less than 7.0 min per sample for separation and quantitation. The optimum pH for cathepsin A activity was 4.5-5.0. The Km and Vmax values were respectively 14.9 microM and 27.91 pmol/microg/h with the use of enzyme extract obtained from mouse kidney. Cathepsin A activity was strongly inhibited by Ag+, Hg2+, diisopropylfluorophosphate and p-chloromercuriphenylsulphonic acid. Among the organs examined in a mouse, the highest specific activity of the enzyme was found in kidney. The sensitivity and selectivity of this method will aid in efforts to examine the physiological role of this peptidase.

Complete nucleotide sequences of 93-kb and 3.3-kb plasmids of an enterohemorrhagic Escherichia coli O157:H7 derived from Sakai outbreak.

Enterohemorrhagic Escherichia coli (EHEC) O157:H7, derived from an outbreak in Sakai city, Japan in 1996, possesses two kinds of plasmids: a 93-kb plasmid termed pO157, found in clinical EHEC isolates world-wide and a 3.3-kb plasmid termed pOSAK1, prevalent in EHEC strains isolated in Japan. Complete nucleotide sequences of both plasmids have been determined, and the putative functions of the encoded proteins and the cis-acting DNA sequences have been analyzed. pO157 shares strikingly similar genes and DNA sequences with F-factor and the transmissible drug-resistant plasmid R100 for DNA replication, copy number control, plasmid segregation, conjugative functions and stable maintenance in the host, although it is defective in DNA transfer by conjugation due to the truncation and deletion of the required genes and DNA sequences. In addition, it encodes several proteins implicated in EHEC pathogenicity such as an EHEC hemolysin (HlyA), a catalase-peroxidase (KatP), a serine protease (EspP) and type II secretion system. pOSAK1 possesses a ColE1-like replication system, and the DNA sequence is extremely similar to that of a drug-resistant plasmid, NTP16, derived from Salmonella typhimurium except that it lacks drug resistance transposons.

Colchicine-induced elevation of tissue metallothionein contents is mediated by inflammation-independent serum factor.

Subcutaneous injection of colchicine caused dose-dependent and time-dependent induction of hepatic MT in mice. Other than colchicine, similar MT induction was observed in vincristine- or vinblastine-injected mice, but not in beta-lumicolchicine-injected mice. MT contents were also elevated in the kidney, spleen, lung and heart by colchicine injection. Isoforms of colchicine-induced MT in the liver were identified to be MT-I and II by immunoblot analysis. Unlike turpentine-induced MT synthesis, dexamethasone, an anti-inflammatory agent, could not block the MT-inducing activity of colchicine. Therefore, the MT-inducing activity of colchicine does not appear to be due to inflammation. Mouse serum, obtained at 4-24 h after colchicine treatment, stimulated MT induction in rat hepatoma H4IIEC3 cells. The MT-inducing activity in the serum from colchicine-treated mice was determined to be highest at 12 h after colchicine injection. The MT-inducing activity from sera of colchicine-treated mice was completely blocked by glucocorticoid antagonist, RU38486, similar to such activity in the serum from lipopolysaccharide-treated mice. The ability of sera to induce MT was abolished by heat treatment (56 degrees C, 30 min). The molecular weight of the MT-inducing factor estimated by gel filtration was approximately 20 000 Da. Thus, colchicine-induced stimulation of MT production is mediated by some humoral factor. The production of the MT-inducing factor was not blocked by dexamethasone. We conclude that the mediator is not an inflammatory cytokine or a glucocorticoid and suspect that the disruption of microtubule triggers production or release of such humoral mediator which stimulates MT induction.

[Tizanidine for preanesthetic medication].

The effect of tizanidine given as a premedication on perioperative hemodynamics (mean blood pressure, heart rate), sedation, hypnosis and midazolam requirements for induction were assessed in 68 patients scheduled for elective surgery under general anesthesia. Patients were assigned to three groups. Group 1 was premedicated 90 min prior to induction with tizanidine 4 mg po (n = 28); group 2 was premedicated with tizanidine 2 mg po (n = 12); group 3 received no premedication (n = 28). In group 1, increase of mean blood pressure on anesthesia induction was attenuated significantly and sedative and hypnotic effects were stronger significantly compared with other groups. We also found that the amounts of midazolam necessary for loss of consciousness were significantly less in patients who had received tizanidine 4 mg. In conclusion, tizanidine is a useful drug as preanesthetic medication for general anesthesia.

Mucha-Habermann disease and its febrile ulceronecrotic variant.

In 1916 Mucha and in 1925 Habermann reported an acute form of pityriasis lichenoides characterized by the abrupt onset of papulovesicular eruptions and gave the name, pityriasis lichenoides et varioliformis acuta (PLEVA) or Mucha-Habermann disease (MH). In 1966, Degos reported a rare febrile ulceronecrotic variant of MH. MH occurs mainly in young adults, while febrile ulceronecrotic Mucha-Habermann's disease (FUMHD) occurs more frequently in children. The etiology of MH remains obscure, but it may be the result of a hypersensitivity reaction to an infectious agent. Although clinical and histologic features of the disease in children are similar to those of adults, more diseases need to be differentiated in pediatric patients. In addition, a number of effective therapeutic options in adults with MH are unsuitable for use in pediatric patients, to whom beginning with oral antibiotics, usually erythromycin, is recommended. A summary of previously reported fifteen cases with FUMHD, including our case, is listed.

Small bowel obstruction caused by a medication bezoar: report of a case.

We report herein the rare case of a 26-year-old woman who developed a small-bowel obstruction caused by a medication "bezoar" or enterolith, following the long-term ingestion of magnesium oxide cathartics for constipation. Medication bezoars resulting from laxatives or cathartics have rarely been reported and we were only able to find two other such cases in the literature.

[The catecholamine concentrations of collected autologous blood during adrenalectomy for pheochromocytoma].

We studied the catecholamine concentrations in collected autologous blood of a patient undergoing adrenalectomy for pheochromocytoma. In the preoperative laboratory data, plasma concentrations (normal ranges) of epinephrine, norepinephrine and dopamine were 60180 pg.ml-1 (< 100), 11090 pg.ml-1 (100-450) and 104 pg.ml-1 (< 20), respectively. The catecholamine levels of collected blood were epinephrine 2490000 pg.ml-1, norepinephrine 352300 pg.ml-1 and dopamine 6100 pg.ml-1 before wash. Wash of collected blood with 1000 ml saline diluted the catecholamines to epinephrine 212000 pg.ml-1, norepinephrine 18700 pg.ml-1 and dopamine 4900 pg.ml-1. Platelet activation by contact with tissue collagen or thrombin results in the release of catecholamine concentrated in the dense body. The mechanical stimulation by suction, roller pump and centrifugation during blood collection may accelerate the catecholamine release from platelets. Thus, saline wash hardly reduces catecholamine concentrations of collected blood from a patient with pheochromocytoma. In this particular case, plasma catecholamines seem to exceed the potential capacity of platelets in amount. The dilution effect for epinephrine and norepinephrine probably reflects the washout of greater amount of plasma catecholamines. However, saline wash was unable to reduce catecholamines contained in the collected blood to a safe level, and hypertension following autotransfusion was predictable. We conclude that hemodynamic change should be monitored carefully during intraoperative autotransfusion in a case of pheochromocytoma.

[A method for the quantification of interleukin-4 in serum (sandwich ELISA) and IL-4 levels in patients with atopic dermatitis].

We have developed a sandwich enzyme-linked immunosorbent assay (ELISA) using chemiluminescent enzyme substrate AMPPD for the quantification of interleukin 4 (IL-4) in serum. IL-4 in serum could be measured in concentrations from 0.17 pg/ml. The IL-4 levels in patients with atopic dermatitis were significantly higher than those in normal controls.

[Optimal glucose dose in the preoperative fluid infusion].

We have measured blood concentrations of glucose, 3-hydroxybutyrate (3-OHBA), non-esterified fatty acid (NEFA) and lactate before and after glucose administration in 105 patients to determine the optimal glucose dose during the period of preoperative fasting state. The patients, scheduled for laparotomy in the afternoon, received fluid infusion containing an arbitrary glucose dose for 4 hours prior to surgery. The glucose dose showed a negative correlation with the ratio of 3-OHBA or NEFA prior to and after glucose administration (r = 0.40, r = 0.41 respectively, P < 0.05). There was no correlation between lactate ratio and glucose dose (r = 0.06). Glucose administration more than 0.4 g.kg-1 significantly suppressed 3-OHBA production (P < 0.05). Hyperglycemia (> 200 mg.dl-1) developed more often in the groups given 0.5 g.kg-1 or more glucose (P < 0.05), while significant hypoglycemia (< 60 mg.dl-1) occurred in the groups given less than 0.2 g.kg-1 of glucose (P < 0.05). This study demonstrates that preoperative infusion of glucose 0.4-0.5 g.kg-1 is useful to maintain the energy metabolism during the fasting state in patients scheduled for operation in the afternoon.

[Succinylcholine neuromuscular blockade in a case of pseudocholinesterase variant].

Since there is little or no pseudocholinesterase at the motor end plate, the neuromuscular blockade by succinylcholine is terminated by its diffusion away from the end-plate into extracellular fluid. Pseudocholinesterase (ChE), therefore, influences the duration of action of succinylcholine (SCC) by controlling the rate of hydrolysis before it reaches the end-plate. SCC neuromuscular blockade can be prolonged in the presence of an atypical form of ChE. A 37 year old male with the deficiency of ChE activity underwent pancreatico-duodenectomy under neuroleptanesthesia. The mode, duration and treatments of neuromuscular blockade following succinylcholine (SCC) were studied using train-of-four (TOF). SCC was administered 6 times (total 170 mg) during the operation. Prolonged apnea was noted after the first intravenous injection of SCC (60 mg, 1 mg.kg-1). It was found that spontaneous resolution of the neuromuscular block had four phases and there was a pronounced fade of the TOF response. After the second injection of SCC (30 mg), a remarkable fade of the TOF was detected and the character of the block was considered to be a desensitizing block (phase II block). The block was treated by intravenous infusion of fresh frozen plasma (FFP) (400 ml) which contained ChE, at the rate of 231 +/- 56 IU/l/37 degrees C begun shortly after the fifth injection of SCC (20mg). The duration of the block was remarkably shortened but the recovery curves were almost unchanged, indicating the diffusion of SCC away from the end-plate and/or that receptor-channel is presumably slow. The neuromuscular blockade was fully reversed by additional injection of FFP (400 ml) before extubation.(ABSTRACT TRUNCATED AT 250 WORDS)