Immunoelectron microscopic studies on centromere-kinetochore complexes detached from chromosomes

The centromere-kinetochore complexes of Chinese hamster ovary (CHO) cells were detached and separated from the condensed chromatin by treatment with hydroxyurea and caffeine. By labelling the complex for immunoelectron microscopy (immuno-EM) with a mixture of antibodies against centromere proteins (anti-CENP-A,-B, -C) in some cells, we could demonstrate complete detachment of the complexes. No remnants were left at the bulk of condensed chromatin in these cells. In some mitotic cells complex and chromatin were found side by side. It could be shown that the fine structure of the separated material of the complex differs significantly from that of the rest of chromatin. The complex consists of proteins and DNA. This leads us to suppose that the organization of chromatin in the centromere-kinetochore complex is different.     

Effect of temperature variation (22˚C- 44˚C) on halothane and caffeine contracture testing in normal humans

Background: Malignant hyperthermia (MH) susceptibility is diagnosed using halothane-caffeine contracture testing of a muscle sample maintained at 37˚C. However, there has not been a systematic study that examines the effect of different temperatures on the response of normal muscle to halothane and caffeine. We hypothesized that altering bath temperature would modify the contracture responses.
Methods: We obtained muscle samples from 20 patients undergoing surgical procedures of the lower extremities. The samples were dissected into 245 bundles and the bundles were exposed to halothane 3% or incremental caffeine, according to the North American MH group protocol. Several bundles from each patient were simultaneously studied at four different temperatures (22˚C, 30˚C, 37˚C and 44˚C). Each bundle was studied at only one temperature, the muscle samples of 3 patients were simultaneously studied at all four temperatures for halothane and caffeine.
Results: Maximum contracture to caffeine (32 mM) was highest at 37˚C; however, at lower caffeine concentrations (2–4 mM), there was no consistent effect of temperature on contracture response. Likewise, temperature did not alter contracture responses to halothane. The extremes of temperature (22˚C and 44˚C) were associated with lack of twitch in response to electrical stimulation. For the bundles exposed to halothane at 22"C, the absence of a twitch was associated with the presence of a contracture, although these were never above the diagnostic threshold.
Conclusions: We conclude that temperature has little effect on responses of normal muscle to halothane and caffeine.     

In vitro contracture test for diagnosis of malignant hyperthermia following the protocol of the European MH Group: Results of testing patients surviving fulminant MH and unrelated low-risk subjects

Background: Determination of sensitivity and specificity of the in vitro contracture test (IVCT) for malignant hyperthermia (MH) susceptibility using the European MH Group (EMHG) protocol has been performed in some laboratories but only on a small sample from the combined EMHG. Thus, the purpose of the present study was to determine combined EMHG sensitivity and specificity of the test. Methods: Results of IVCT of patients with previous fulminant MH and normal, low-risk subjects (controls) were collected from 22 centresof the EMHG. IVCT was performed according to the EMHG protocol. Patients were included inthe study if the clinical crisis had a score of at least 50 points with the Clinical Grading Scale. Low-risk subjects were included provided they did not belong to a family with known MH susceptibility, they had not developed any signs of MH at previous anaesthetics, and they did not suffer from any neuromuscular disease. For inclusion of both MH patientsand low-risk subjects, at least 1 muscle bundle in the IVCT should have twitches of 10 mN(1 g) or more. For evaluation of individual tests, only muscle bundles with twitch heights of 10 mN (1 g) or more were used. Results: A total of 1502 probands had undergone IVCT because of a previous anaesthesia with symptoms and signs suggestive of MH. Of these, 119 had clinical scores of 50 and above. From these 119 MH-suspected patients and from 202 low-risk subjects, IVCT data were collected. Subsequently, 14 MH-suspected patients were excluded from further analysis for thefollowing reasons: In 3 patients, the suspected MH episode could be fully explained by diseases other than MH; in 11 MHS patients, IVCT was incomplete (n=l), data were lost (n=3), or none of the muscle bundles fulfilled twitch criteria (n=7). Of the remaining 105 MH-suspected patients, 89 were MHS, 10 MHEh, 5 MHEc, and one MHN. Thus, we observed a diagnostic sensitivity of the IVCT of 99.0% if the MHE group is considered susceptible(95% confidence interval 94.8–100.0%). Of the 202 low-risk subjects, 3 were MHS, 5 MHEh, 5 MHEc, and 189 MHN. This gives a specificity of the IVCT of 93.6% (95% confidence interval 89.2–96.5%). Conclusion: The IVCT for diagnosis of MH susceptibility in Europe has a high sensitivity and a satisfactory specificity.     

Age, fiber type composition and in vitro contracture responses in human malignant hyperthermia

Muscle fiber typing and in vitro contracture tests were performed in 59 patients investigated for susceptibility to malignant hyperthermia (MH). Eighteen patients were found to be susceptible to MH. There was no difference in age or fiber type distribution between MH susceptible and non-susceptible patients. No correlation was found between age and fiber type distribution. Separate analyses for each diagnostic group revealed no relationship between age or fiber type distribution and response to halothane or caffeine. When all caffeine results were pooled, however, there was a significant effect of age on the caffeine specific concentration (the concentration eliciting a contracture of 1 g), but not on the caffeine threshold concentration (the minimal concentration eliciting an increase in tension). It is concluded that age and fiber type distribution have no influence on MH diagnosis, if the protocol of the European MH Group for evaluation of susceptibility to MH is followed.     

去痛片中四组分的HPLC测定

以十八烷基硅烷键合硅胶为固定相，采用ＨＰＬＣ法测定去痛片中氨基比林、非那西丁、咖啡因、苯巴比妥的含量。各组分的回收率分别为９９．８±０．５０％、９９．７±０．７６％、９９．３±０．５４％、９９．６±０．５８％。     

Pentoxifylline acts synergistically with A23187 to increase the penetration of zona-free hamster oocytes by cryopreserved human spermatozoa

The number of cryopreserved human spermatozoa which penetrated zona-free hamster oocytes afier stimulation with 2μmol A23187 per litre was increased by the hrther addition of 0.6 or 3.6 mmol pentoxifjrlline per litre. With spermatozoa prepared by washing by repeated centrifugation, the median numbers of sperm headd/egg were 1.9, 7.9 and 10.8 in the presence of 0, 0.6 or 3.6 mmol pentoxifylline per litre, respectively. A similar effect was observed with spermatozoa prepared on a Percoll gradient. As A23187 inhibited sperm motility, and this was exacerbated by pentoxifylline, the increased penetration rate of hamster oocytes cannot be explained by improved sperm motility. The number of spermatozoa stimulated to acrosome react by 2 μmol A23187 per litre was increased 3-fold by 3.6 mmol pentoxifylline per litre and 4-fold by 5 mmol caffeine per litre. These data suggest that CAMP may act synergistically with Ca²⁺ to stimulate the acrosome reaction. Pentoxifjrlline may improve the fertility of poor-quality human spermatozoa by enhancing their ability to respond to the Ca²⁺ signal produced by binding to the zona pellucida.     

Effect of ketoconazole and terbinafine on the pharmacokinetics of caffeine in healthy volunteers

The effects of single oral doses of ketoconazole 400 mg and terbinafine 500 mg on the hepatic microsomal system have been investigated in 8 healthy male volunteers. Microsomal activity caffeine was assessed by following the metabolism of 3 mg/kg bodyweight i.v. administered 1 h after the drug. The inhibitory effect of terbinafine was more pronounced than that of ketoconazole: clearance was decreased from 1.34 ml.kg-1.min-1 in controls to 1.06 and 1.21 ml.kg-1.min-1, respectively, and the corresponding half-life was increased from 5.8 h in controls to 7.6 and 6.7 h, respectively. The apparent volume of distribution remained unchanged. The serum levels of the antimycotics were within the therapeutic range in each subject. Although all three substances are metabolised by microsomes, the kinetic parameters (Cmax, half-life, elimination constant) of the antimycotics were poorly if at all correlated with the elimination of caffeine.