Circadian rhythms in heart rate, motility, and body temperature of wild-type C57 and eNOS knock-out mice under light-dark, free-run, and after time zone transition

The nitric oxide (NO) system is involved in the regulation of the cardiovascular system in controlling central and peripheral vascular tone and cardiac functions. It was the aim of this study to investigate in wild-type C57BL/6 and endothelial nitric oxide synthase (eNOS) knock-out mice (eNOS-/-) the contribution of NO on the circadian rhythms in heart rate (HR), motility (motor activity [MA]), and body temperature (BT) under various environmental conditions. Experiments were performed in 12:12 h of a light:dark cycle (LD), under free-run in total darkness (DD), and after a phase delay shift of the LD cycle by -6 h (i.e., under simulation of a westward time zone transition). All parameters were monitored by radiotelemetry in freely moving mice. In LD, no significant differences in the rhythms of HR and MA were observed between the two strains of mice. BT, however, was significantly lower during the light phase in eNOS-/- mice, resulting in a significantly greater amplitude. The period of the free-running rhythm in DD was slightly shorter for all variables, though not significant. In general, rhythmicity was greater in eNOS-/- than in C57 mice both in LD and DD. After a delay shift of the LD cycle, HR and BT were resynchronized to the new LD schedule within 5-6 days, and resynchronization of MA occurred within 2-3 days. The results in telemetrically instrumented mice show that complete knock-out of the endothelial NO system—though expressed in the suprachiasmatic nuclei and in peripheral tissues—did not affect the circadian organization of heart rate and motility. The circadian regulation of the body temperature was slightly affected in eNOS-/- mice.     

Lock and roll: single-molecule genotyping in situ using padlock probes and rolling-circle amplification

In this review I will describe the development of a technique that enables genotyping of individual DNA molecules in the context of morphologically preserved fixed cells, from the fundamental concept published in 1994 to the present status. The review describes enzyme-assisted histochemistry approaches to achieve highly specific molecular identification reactions coupled to efficient signal amplification. The primary molecular identification is accomplished through circularization of oligonucleotide probes, called padlock probes. The circularization reaction is catalyzed by a DNA ligase, which provides robust distinction between single-nucleotide variants under standard reaction conditions. To generate a detectable signal from individual circularized probe molecules, a DNA polymerase is added that replicates probe circles, generating a long tandem-repeated DNA product, easily visualized using a standard epi-fluorescence microscope. Individual signals are recorded as bright dots, providing digital information about the abundance of specific sequences and opportunities for simultaneous detection of several targets using spectral multiplexing. The importance of strictly target-dependent signal amplification will be discussed.Robert Feulgen Prize 2006 Winner lecture presented at the 48th Symposium of the Society for Histochemistry in Stresa, Lake Maggiore, Italy, 7–10 September 2006.     

Morphological, yield, cytological and molecular characterization of a bread wheat X tritordeum F1 hybrid

The morphological, yield, cytological and molecular characteristics of bread wheat X tritordeum F₁ hybrids (2n =6x = 42; AABBDH^ch) and their parents were analysed. Morphologically, these hybrids resembled the wheat parent. They were slightly bigger than both parents, had more spikelets per spike, and tillered more profusely. The hybrids are self-fertile but a reduction of average values of yield parameters was observed. For the cytological approach we used a double-target fluorescencein situ hybridization performed with total genomic DNA fromHordeum chilense L. and the ribosomal sequence pTa71. This technique allowed us to confirm the hybrid nature and to analyse chromosome pairing in this material. Our results showed that the expected complete homologous pairing (14 bivalents plus 14 univalents) was only observed in 9.59% of the pollen mother cells (PMCs) analysed. Some PMCs presented autosyndetic pairing of Hch and A, B or D chromosomes. The average number of univalents was higher in the wheat genome (6.8) than in the Hch genome (5.4). The maximum number of univalents per PMC was 20. We only observed wheat multivalents (one per PMC) but the frequency of trivalents (0.08) was higher than that of quadrivalents (0.058). We amplified 50 RAPD bands polymorphic between the F₁ hybrid and one of its parents, and 31 ISSR polymorphic bands. Both sets of markers proved to be reliable for DNA fingerprinting. The complementary use of morphological and yield analysis, molecular cytogenetic techniques and molecular markers allowed a more accurate evaluation and characterization of the hybrids analysed here.     

Genetic Relationships Between Brazilian Species of Molossidae and Phyllostomidae (Chiroptera, Mammalia)

A comparative analysis of G-banded karyotypes was performed for seven species of Chiroptera, representing two families (Phyllostomidae and Molossidae). Despite the differences in diploid and fundamental numbers, extensive homologies between six karyotypes were identified: A . planirostris, P. lineatus, S. lilium, G. soricina, P. hastatus (Phyllostomidae) and M. rufus (Molossidae). Robertsonian rearrangements and pericentric inversions account for the differences between the karyotypes of phyllostomid and molossid species. The homologies and rearrangements observed reinforce the monophiletic origin of phyllostomids and the inclusion of species in different subfamilies. In situ hybridization with genomic DNA revealed considerable conservation of the karyotypes, including C. perspicillata, that did not show G-band homologies with the other species analyzed. For the first time, chromosomal evidence is presented of a common origin for Phyllostomidae and Molossidae.     

Alterations in the composition of the supramucosal defense barrier in relation to disease severity of ulcerative colitis.

Mucin glycoproteins and trefoil peptides play an important role in protection and repair of the gastrointestinal epithelium. This study investigates alterations in mucin and trefoil peptide gene expression and product localization in ulcerative colitis (UC). Product localization and message expression of mucin MUC1 to 6 and trefoil peptide TFF1 to 3 genes was analyzed in rectosigmoid tissue from a cohort of patients with active UC and compared with that of normal colorectal mucosa. MUC1 expression was upregulated in severe UC at the site of rupture of crypt abscesses. Reduction in MUC2 expression occurred in UC adjacent to ulceration. No alteration in MUC3 or MUC4 gene expression was detectable in UC compared with normal colorectal mucosa. No ectopic expression of MUC5AC, MUC5B, or MUC6 was identified in UC. Ectopic TFF1 expression was identified in tissues eliciting histological features of severe disease. Decreased TFF3 localization was demonstrated in UC tissues, but no TFF2 expression was detected in any colorectal specimens. Subtle alterations in composition of the supramucosal defense barrier exist in UC and vary in relation to clinical severity of disease. There is upregulation in mucin MUC1 at crypt abscesses and neo-expression of TFF1 trefoil peptide in severe disease.     

人14p＋标记染色体的分子细胞遗传学研究

一例23岁女性患者因近五、六年来出现胡须、四肢多毛及偶有月经不规则而就诊。细胞遗传学检查发现一个短臂明显增大的亚中着丝粒的14号标记染色体14p ·p 区域GTG显带呈浅染,C-带暗染,都呈均匀的染色区。硝酸银染色在p 远侧端显现一个Ag-NOR,其大小与正常近端着丝粒染色体的无明显差异。应用~3H标记的7.3 kb长的rRNA基因探针进行染色体原位杂交,自显影银颗粒沿整个p 区域分布,p 上的银颗粒数是正常近端着丝粒染色体短臂上银颗粒平均数的5倍。这些结果排除了Y或其他染色体参加的重排形成p 的可能性,并表明Ag-NOR的大小或NOR的数目并不一定与rRNA基因的数量成正比。研究Dp 或Gp 类型的染色体变异,对了解人二倍体细胞内rRNA基因表达的调控有重要意义。     

Identification of diverse molecular forms of GnRH in reptile brain

Gonadotropin-releasing hormone (GnRH) molecular forms in the brains of three reptiles, Alligator mississippiensis (alligator), Calcides ocellatus tiligugu (skink) and Podarcis s. sicula (lizard) were characterized by high performance liquid chromatography (HPLC) and radioimmunoassay with region-specific antisera, and by assessment of luteinizing (LH)-releasing activity in chicken dispersed pituitary cells. In alligator brain two GnRHs had identical properties to the two known forms of chicken hypothalamic GnRH (Gln⁸-GnRH and His⁵,Trp⁷,Tyr⁸-GnRH) in their elution on two reverse phase HPLC systems, cross-reaction with region-specific GnRH antisera, and ability to release LH. In skink brain, one immunoreactive and bioactive GnRH form, which eluted in the same position as His⁵,Trp⁷,Tyr⁸-GnRH on reverse phase HPLC, was identified. Three bioactive and immunoreactive GnRHs were detected in lizard brain. One form had similar properties to salmon brain GnRH (Trp⁷,Leu⁸-GnRH). The other two GnRH-like peptides are novel forms. One of these forms eluted in the same position as Gln⁸-GnRH on HPLC but had different immunological properties, while the third form was a rather hydrophobic species which appeared to be modified in the middle region of the molecule.     

人类新基因WDR70的克隆及初步研究

运用基于基因组数据库的特定基因同源新基因的克隆策略得到一个人类新基因WDR70 ,该基因编码一个包含 12个WD4 0结构域的蛋白。WDR70的cDNA序列长 2 2 6 6bp ,预测编码蛋白含 6 30个氨基酸 ,理论分子量为 70× 10 3 u ,染色体定位为 17p13.1。以小鼠胚胎为模型进行整体原位杂交 ,结果显示WDR70基因在 8.5d小鼠胚胎中没有表达 ,而在 9.5d和 10 .5d的小鼠胚胎的脑部有特异表达。由此推断该基因对胚胎期脑部的发育有重要的影响。     

Lyt phenotype analysis of the effector cells responsible for evoking lymphocyte-induced angiogenesis (LIA)

Lymphocyte-induced angiogenesis (LIA) is a vascular response observed when allogeneic or semiallogeneic immunocompetent lymphocytes are inoculated intradermally into immunosuppressed or irradiated host mice. The reported experiments were carried out to characterize the effector cell population(s) responsible for causing LIA. Lyt 1.2, Lyt 2.2, and monoclonal Thy 1.2 antisera were used for negative selection with complement (C′) to investigate the ability of selected subsets of lymphocytes to evoke angiogenesis. Treatment of C57BL/6 spleen cells with either anti-Lyt 1.2 or anti-Thy 1.2 and C′ resulted in an almost complete abrogation of the LIA reaction. In contrast, depletion of Lyt 2+ cells, under conditions which fully abrogated their ability to generate cell-mediated cytotoxicity in allogeneic mixed leukocyte cultures, resulted only in a partial (45%) reduction in the induced vascular response. Synergistic interaction between cell preparations treated separately with either anti-Lyt 1.2 or anti-Lyt 2.2 serum was not observed. We conclude that (i) Lyt 1 + 2?T lymphocytes can induce a significant LIA reaction; (ii) lymphocytes resistant to negative selection with anti-Lyt-1.2 serum are incapable of inducing such a reaction; and (iii) Lyt 1 + 2+ cells directly or indirectly play an additional role in generating a maximal LIA response.     

Métabolisme de l’Acide 2-Aminoéthylphosphonique (Ciliatine) chez le Poulet

For Podospora anserina, several studies of cellulolytic enzymes have been established, but characteristics of amylolytic enzymes are not well understood. When P. anserina grew in starch as carbon source, it accumulated glucose, nigerose, and maltose in the culture supernatant. At the same time, the fungus secreted α-glucosidase (PAG). PAG was purified from the culture supernatant, and was found to convert soluble starch to nigerose and maltose. The recombinant enzyme with C-terminal His-tag (rPAG) was produced with Pichia pastoris. Most rPAG produced under standard conditions lost its affinity for nickel-chelating resin, but the affinity was improved by the use of a buffered medium (pH 8.0) supplemented with casamino acid and a reduction of the cultivation time. rPAG suffered limited proteolysis at the same site as the original PAG. A site-directed mutagenesis study indicated that proteolysis had no effect on enzyme characteristics. A kinetic study indicated that the PAG possessed significant transglycosylation activity.