Application of good laboratory practice (GLP) to culture collections of microbial and cell cultures

Although the principles and the necessity for good laboratory practice (GLP) guidelines to confirm the credibility, integrity, and quality of non-clinical laboratory studies have been known for more than a decade, culture collection activities are not subject to them. Because of recent advances in biotechnology, culture collections face increased demands not only for quality cultures but also current information. When applied in culture collections, GLP guidelines prove to be an excellent management tool as well as a cost-effective system of providing authentic and reliable microbial and cell cultures and associated data.     

Familial and iatrogenic cortisol receptor resistance.

Primary cortisol receptor resistance has been reported in 6 patients and 14 asymptomatic family members. We observed an additional 6 patients (2 males and 4 females). The male patients presented with hypertension. The female patients presented with acne, hirsutism and irregular menstruations. Dexamethasone therapy (1-1.5 mg/day) was of considerable clinical benefit. All 6 patients showed insufficient suppression of cortisol after 1 mg dexamethasone. The diurnal rhythm of ACTH and cortisol was intact, albeit at an elevated level. There was a normal increase of ACTH, cortisol, and GH to insulin-induced hypoglycemia, while cortisol production was (slightly) elevated. Adrenal androgen levels were increased in all patients. Glucocorticoid receptors measured in a whole cell dexamethasone binding assay in mononuclear leukocytes showed a lowered affinity in 1, and lowered numbers of receptors in 4 patients. In 1 patient no abnormalities were found. As a "bioassay" for glucocorticoid action dexamethasone suppressibility of mitogen-stimulated incorporation of [3H]thymidine in mononuclear leukocytes was measured. In this last patient dexamethasone suppressibility of [3H]thymidine incorporation was significantly lowered. Twelve months' treatment with 200 mg RU 486 per day in meningioma patients induced a similar biochemical picture as observed in primary cortisol receptor resistance. Partial cortisol receptor resistance might be less rare than previously thought. In the 6 patients presented at least 3 different forms can be recognized. Therapy with dexamethasone was successful in female patients with acne and hirsutism, as the secondary overproduction of adrenal androgens was effectively controlled. Chronic therapy with RU 486 causes a biochemical picture similar to primary cortisol receptor resistance.     

Production of cellulolytic enzymes from theXylaria andHypoxylon species of xylariaceae

Eighteen strains of xylariaceous fungi have been screened for higher activities of cellulolytic enzymes,Trichoderma reesei QM 9414 was also examined for comparison. Strains ofXylaria anisopleura andX. regalis had higher endocellulase (CMCase) and exocellulase (Avicelase) activities after 2 weeks' incubation.Hypoxylon stygium produced the highest activity of -glucosidase 3 days after inoculation. The optimum pH for these cellulolytic enzymes was approx. 5.0 and the optimum temperatures ranged from 37 to 50°C. A mixed culture process usingT. reesei QM 9414 andH. stygium was developed to obtain enhanced synthesis of cellulase. -Glucosidase activities in the mixed culture increased within 48h whenH. stygium was introduced after 24h.     

活性氧对巨噬细胞呼吸爆发影响及云芝多糖的保护作用

用化学发光法观察到叔丁基氢过氧化物对培养的小鼠腹腔巨噬细胞呼吸爆发有强烈的抑制作用。云芝多糖经腹腔注射后,能增强巨噬细胞呼吸爆发功能对叔丁基氢过氧化物损伤的抵抗力。云芝多糖处理的巨噬细胞谷胱甘肽过氧化物酶基础活力显著提高,在叔丁基氢过氧化物作用下,云芝多糖处理的巨噬细胞仍有较高的谷胱甘肽过氧化物酶活力。说明巨噬细胞的免疫功能与谷胱甘肽过氧化物酶活力有关,非特异性免疫多糖可提高细胞抗氧化能力,减轻活性氧损伤作用。     

Monoclonal antibodies reveal evolutionary conservation of alternative splicing of the alpha A-crystallin primary transcript

Because of their specificity and sensitivity, monoclonal antibodies are powerful tools in studies of protein structure and function. Therefore, we raised monoclonal antibodies against alpha A-crystallin and identified the antigenic determinant for two of these antibodies. Applying limited-digestion methods, we show that the region spanning residues 158-168 of alpha A-crystallin contains the epitope for the two monoclonal antibodies. These monoclonals were then used to study the occurrence in the lenses of different vertebrates of the elongated alpha Ains-crystallin chain, a product of alternative splicing. It appears that the mutational event resulting in the alternative splicing pattern of the alpha A-crystallin gene took place at least 70 million years ago. This alternative splicing phenomenon has been maintained in rodents and some other, unrelated mammals, but disappeared again in most mammalian lineages.     

Blue Light-Induced Phototropic Response and the Intracellular Photoreceptive Site in Adiantum Protonemata

Blue light-induced phototropism in Adiantum protonemata wasinvestigated with microbeam irradiation. Brief irradiation withblue light effectively induced a phototropic response when itwas applied to a half-side of the apical 200d µm regionof a protonema. The phototropic response was partly reversedby the subsequent far-red light irradiation but the full reversalof the response was not observed even when the fluence of far-redlight was increased. In the far-red reversible part of the response,blue/far-red photoreversibility was repeatedly observed. Thus,both phytochrome and a blue light-absorbing pigment (other thanphytochrome) seem to be involved in the blue light-induced phototropicresponse in Adiantum protonemata. Furthermore, detailed studiesof the far-red light effect on the fluence-response curve forblue lightinduced phototropism revealed that the concomitantmediation by the two receptors was limited to the response inthe relatively higher fluence range of blue light and that theblue light-absorbing pigment alone was responsible in the lowerfluence range. In the higher fluence range, the response mediatedby the blue light-absorbing pigment became saturated and thephytochrome response became evident, indicating a differencein the sensitivities of the two receptor pigments to blue light. When various regions of half-sides of protonemata were irradiatedwith a blue microbeam of 10 µm width, irradiation at theapical 5–25 µm region was most effective both forphytochrome- and blue light-absorbing pigment-mediated response,indicating that the site of blue light perception is almostidentical for each response. (Received July 14, 1986; Accepted September 26, 1986)     

Manganese reduction by a marine Bacillus species.

Mature dormant spores of marine Bacillus sp. strain SG1 catalyze the oxidation of Mn(II) to MnO2. We report that vegetative cells of the same strain reduced MnO2 under low-oxygen conditions. The rate of reduction was a function of cell concentration. The process had a pH optimum of 7.5 to 8.0 and was inhibited by HgCl2, by preheating of the cells at 80 degrees C for 5 min, by antimycin A, and by N-heptyl-hydroxy-quinoline-N-oxide. At a nonlimiting O2 concentration, little MnO2 reduction was observed. Under these conditions, the process could be induced by the addition of NaN3. Spectrophotometric analysis of the Bacillus cells indicated the presence of type b and c cytochromes. Both types can be oxidized in situ by addition of MnO2 to the cells.     

Specific binding of three neurotoxins with phospholipase A2 activity to synaptosomal membrane preparations from the guinea pig brain

Snake presynaptic toxins such as crotoxin, β-bungarotoxin and taipoxin block neuromuscular transmission through inhibiting the release of acetylcholine by their phospholipase A₂ activities. On the other hand, many other phospholipase A₂s show little neurotoxicity. It is likely that the difference lies in whether high affinity binding to nerve cell membranes exists or not. To test this idea, crotoxin, β-bungarotoxin and taipoxin were first radioactively labeled with Na(¹²⁵I) without loss of their neurotoxicity. Using the radioactive toxins we have found that each of the three showed specific binding to synaptosomal membranes from guinea pig brain. In contrast, we could not detect specific binding of a non-neurotoxic pancreatic phospholipase A₂. Crotoxin and taipoxin, but not β-bungarotoxin, also bound specifically to membrane preparation from other tissues. The binding of each toxin was not greatly affected by the other two toxins. The photoaffinity labeling technique has been used to obtain further information about the components which bind crotoxin. For this purpose, (¹²⁵I) crotoxin was derivatized with N-hydroxysuccinimidyl-4-azidobenzoate. Autoradiographic analysis of the membranes following photoirradiation in the presence of the modified crotoxin revealed that an 85K dalton component was preferentially covalently conjugated with the crotoxin analogue in a specific manner.