Selective changes of neurotransmitter receptors in middle-aged gerbil brain

Age-related alterations in major neurotransmitter receptors and voltage dependent calcium channels were analyzed by receptor autoradiography in the gerbil brain. [³H]Quinuclidinyl benzilate (QNB). [³H]cyclohexyladenosine (CHA), [³H]muscimol, [³H]MK-801, [³H]SCH 23390, [³H]naloxone, and [³H]PN200-110 were used to label muscarinic acetylcholine receptors, adenosine A₁ receptors, γ-aminobutyric acid_A (GABA_A) receptors, (NMDA) receptors, dopamine D₁ receptors, opioid receptors, and voltage dependent calcium channels, respectively. In middle-aged gerbils (16 months old), the hippocampus exhibited a significant elevation in [³H]QNB, [³H]MK-801, [³H]SCH 23390, [³H]naloxone, and [³H]PN200-110 binding, whereas [³H]CHA and [³H]muscimol binding showed a significant reduction in this area, compared with that of young animals (1 month). On the other hand, the cerebellum showed a significant alteration in [³H]QNB, [³H]CHA, and [³H]naloxone binding and the striatum also exhibited a significant alteration in [³H]SCH 23390 and [³H]CHA binding in middle-aged gerbils. The neocortex showed a significant elevation only in [³H]CHA binding in middle-aged animals. The nucleus accumbens and thalamus also showed a significant alteration only in [³H]muscimol binding. However, the hypothalamus and substantia nigra exhibited no significant alteration in these bindings in middle-aged gerbils. These results demonstrate the age-related alterations of various neurotransmitter receptors and voltage dependent calcium channels in most brain regions. Furthermore, they suggest that the hippocampus is most susceptible to aging processes and is altered at an early stage of senescence.     

Purification and Heterogeneous Localization of Sarcophaga Lectin Receptor on the Surface of Imaginal Discs of Sarcophaga peregrina (Flesh Fly)

Previously, we reported autocrine involvement of Sarcophaga lectin in the development of Sarcophaga imaginal discs (Kawaguchi et al. , Dev. Biol. 144 , 86–93 (1991)). In this study, we purified Sarcophaga lectin binding protein from the membrane fraction of cultured embryonic cells of Sarcophaga to near homogeneity and raised a monoclonal antibody against it. Histochemical analysis using the monoclonal antibody revealed that this binding protein is distributed heterogeneously on the surface of leg imaginal discs. This binding protein was especially clearly localized in the central region of the basal side of leg discs which forms the junction between the leg and body, suggesting the participation of Sarcophaga lectin in morphogenesis of the basal region of the developing leg.     

Promotion of Zygote Formation by Protein Kinase Inhibitors during the Sexual Development of Dictyostelium mucoroides

To analyze the possible involvement of protein kinases in the sexual development (macrocyst formation) of the cellular slime mold Dictyostelium mucoroides-7 (Dm7), the effects of several protein kinase inhibitors were examined. K252a, a potent inhibitor of protein kinase activities, promoted the sexual cell fusion, through enhancement of gamete formation. In contrast, staurosporine (structurally and functionally similar to K252a) inhibited markedly the progress of development including cell aggregation, thus resulting in the failure of cells to form mature macrocysts. The effective period of K252a was 5–7 hr after starvation, during which Dm7 cells could acquire fusion competence, and the inhibitory effect of cAMP on zygote formation was nullified by the co-application of K252a. Although KT5720 (a specific inhibitor of cAMP-dependent protein kinase) and W7 (a calmodulin inhibitor) had no effects on zygote formation when applied separately, their combined application enhanced zygote formation like K252a did. Neither calphostin C (a specific inhibitor of Ca²⁺-dependent protein kinase) nor herbimycin A (a specific inhibitor of tyrosine kinase) exerted a stimulative influence upon macrocyst formation. These results strongly suggest that the two signal transduction pathways mediated by cAMP-dependent protein kinase (PKA) and calmodulin are closely related to zygote formation, their blockage being favorable to zygote formation.     

Transient Gene Expression by SV40 Promoter Characterizes Sequential Differentiation of Embryohal Carcinoma F9 Cells into Primitive and Visceral Endoderm

Transient expression of the chloramphenicol acetyl-transferase (CAT) gene under the control of simian virus 40 (SV40), Moloney murine leukemia virus, human T cell leukemia virus, and cytomegalovirus promoters was stimulated by the differentiation of F9 stem cells into primitive endoderm, but repressed again by further differentiation into visceral endoderm. Deletion mutants of the SV40 enhancer showed that a similar set of motifs is critical for CAT expression at all stages of F9 differentiation, but differentiation dependency was observed even in their absence. The stability of transient gene expression under the control of the SV40 promoter was markedly dependent on F9 differentiation. Appreciable expression was detected even in undifferentiated F9 cells immediately after gene transfection, was maximal at 12 h and declined rapidly thereafter. On the other hand, expression in primitive endoderm increased until 72 h. The decline was accelerated again in visceral endoderm. This shift was somewhat specific to the virus promoter since CAT expression in undifferentiated F9 cells under the control of the elongation factor 1α promoter was more stable than for virus promoters tested. Thus, the change in stability of expression is important for differentiation-dependent virus promoter activity.     

Data on the CGG repeat at the fragile X site in the non-retarded Japanese population and family suggest the presence of a subgroup of normal alleles predisposing to mutate

The fragile X mutation is the result of amplification in the repeat number of p(CGG) _n in FMR-1; alleles with more than 52 repeats have been shown to be so unstable as to mutate in the repeat number in almost every transmission. To improve our understanding of mutations in normal alleles of FMR-1, the following studies were carried out in the Japanese population: a study on length variation in the repeat to determine the allele distribution of the repeat length in a non-retarded population, family studies to observe new mutations in normal allele, and haplotype analyses with microsatellite markers flanking the repeat to confirm estimated mutation rates and founder chromosomes in the fragile X syndrome. Analysis of the p(CGG) _n in 370 unrelated males detected 24 distinct alleles with repeats of 18–44. A comparison with previously reported data suggests the presence of racial/ethnic differences in the allele distribution. No premutation allele was found in 824 unrelated X chromosomes examined by the polymerase chain reaction and Southern blot analysis. Family studies detected one new mutation in a total of 303 meioses. However, the mutation rate was not in accordance with the expected or observed heterozygosities in the population or with linkage disequilibrium observed between the repeat numbers and the haplotypes of the markers flanking the CGG. The haplotype in the chromosome in which the new mutation was found was the same as that frequently found in the Japanese fragile X chromosomes, and the variance in the CGG repeat number was wider in chromosomes with the haplotypes frequently found in the fragile X chromosome than in those with the other haplotypes. These observations suggest that a subgroup is present in normal alleles and that this subgroup is more liable to mutate than others.     

基于Golden Gate技术的齐整小核菌转录单元高效组装系统

【目的】为齐整小核菌代谢工程研究建立高效的转录单元组装系统。【方法】通过应用Golden Gate技术,以mobius assembly为基础,分别设计并构建DNA元件标准化接口改造、单转录单元组装、应用质粒(多转录单元)组装等功能的载体,从而形成一套完整的多转录单元组装系统。【结果】构建了2个用于DNA元件标准化接口改造的Level 0载体,4个用于单转录单元组装的Level 1载体,4个用于应用质粒组装的Level 2载体和13个应用质粒组装的辅助质粒。然后应用此系统为齐整小核菌组装了若干经过标准化接口改造的DNA元件质粒、单转录单元质粒和硬葡聚糖相关基因的功能分析质粒。所构建的最终应用质粒可以同时适用于齐整小核菌的根癌农杆菌介导转化法、电穿孔转化法和原生质体转化法。【结论】此质粒系统具有强大的DNA设计、组装和容纳能力,为未来齐整小核菌代谢工程和功能基因组学研究提供了高效的质粒构建技术平台。     

Recovery of aSasa tsuboiana population after mass flowering and death

The recovery process of aSasa tsuboiana population after a mass flowering and death in 1977 was investigated by 15 years of observation in the Hira Mountains, Kinki district, western Japan. Seed production was high (6600–13 800 seeds m⁻² inSasa plots and 3900 seeds m⁻² in a forest plot) but emergent seedling density was low (14–21 seedlings m⁻²), probably because of seed predation byMicrotus montebelli occurring between seed shedding and the next spring. The seedling density had decreased further by the next year and theS. tsuboiana population recovered from only a limited number of seedlings. In spite of such a low initial density, theS. tsuboiana population was able to regenerate successfully and attained the previous full stand height in 7–16 years.Miscantbus sinensis invaded and delayed the recovery ofS. tsuboiana in one plot, butS. tsuboiana became dominant as it caught up with the height ofM. sinensis. Seedling growth patterns, such as frequent tillering, the onset of rhizome extension in the early stage of seedling growth and frequent culm production from rhizomes, played important roles in the successful regeneration ofS. tsuboiana.     

Simple and rapid quantitative assay of C-labelled urea in human serum using liquid chromatography-atmospheric pressure chemical ionization mass spectrometry

A simple and rapid quantitative method for ¹³C-labelled urea ([¹³C]urea) in human serum was developed by using high-performance liquid chromatography-atmospheric pressure chemical ionization mass spectrometry (HPLC-APCI-MS). This method is used to establish and normalize the [¹³C]urea breath test, which is considered as an effective diagnostic method for Helicobacter pylori infection. HPLC-APCI-MS, involving a simple pretreatment process such as diluting serum with water, was shown to be able to discriminate the extrinsic [¹³C]urea from intrinsic urea present at high concentration in serum. In addition, a ¹³C nuclear magnetic resonance spectroscopic quantitative method for [¹³C]urea in human urine is also described. The precision and accuracy of measured concentrations in these two methods were found to be within the acceptable limit. An application of these methods to investigate the pharmacokinetic profile of orally administered [¹³C]urea in human serum and urine is also presented.