H－ras在颊粘膜正常、良性、癌前及恶性变上皮中的表达：免疫组织化学定量研究

本文通过建立图象分析方法对免疫组织化学反应结果进行定量,检测观察Ｈ－ｒａｓ在口腔颊粘膜上皮在正常（Ｎ）、慢性炎症（ＩＦ）、癌旁上皮（ＥＡＣ）和鳞癌（ＳＣＣ）的变化过程中的表达并进行分析。结果显示Ｈ－ｒａｓ在ＳＣＣ组中,以中等分化的ＳＣＣ无论是Ｈ－ｒａｓ表达的量还是细胞阳性率都较高。此外,组织学观察显示,Ｈ－ｒａｓ在处于分化末期但尚未角化的正常上皮细胞中有较高的表达。本文结果显示了Ｈ－ｒａｓ的过表达与上皮细胞的会化程度密切相关。本研究还显示,所采用的阳性区域透光值、平均总透光值及阳性反应区域与阴性反应区域比值可靠并有相关性。这进一步说明了用免疫组化定量方法检测Ｈ－ｒａｓ癌基因表达的精确和可靠性。     

Isolation and characterization of plasma membrane-associated cortical granules from sea urchin eggs

Cortical granules, which are specialized secretory organelles found in ova of many organisms, have been isolated from the eggs of the sea urchins Arbacia punctulata and Strongylocentrtus pupuratus by a simple, rapid procedure. Electron micropscope examination of cortical granules prepared by this procedure reveals that they are tightly attached to large segments of the plasma membrane and its associated vitelline layer. Further evidence that he cortical granules were associated with these cell surface layers was obtained by (125)I-labeling techniques. The cortical granule preparations were found to be rich in proteoesterase, which was purified 32-fold over that detected in a crude homogenate. Similarly, the specific radioactivity of a (125)I-labeled, surface glycoprotein was increased 40-fold. These facts, coupled with electron microscope observations, indicate the isolation procedure yields a preparation in which both the cortical granules and the plasma membrane-vitelline layer are purified to the same extent. Gel electrophoresis of the membrane-associated cortical granule preparation reveals the presence of at least eight polypeptides. The major polypeptide, which is a glycotprotein of apparent mol wt of 100,000, contains most of the radioactivity introduced by (125)I-labeling of the intact eggs. Lysis of the cortical granules is observed under hypotonic conditions, or under isotonic conditions if Ca(2+) ion is present. When lysis is under isotonic conditions is induced by addition of Ca(2+) ion, the electron-dense contents of the granules remain insoluble. In contrast, hypotonic lysis results in release of the contents of the granule in a soluble form. However, in both cases the (125)I-labeled glycoprotein remains insoluble, presumably because it is a component of either the plasma membrane or the vitelline layer. All these findings indicate that, using this purified preparation, it should be possible to carry out in vitro studies to better define some of the initial, surface-related events observed in vivo upon fertilization.     

Phylogenetic utility of the nuclear gene arginine decarboxylase: an example from Brassicaceae

Arginine decarboxylase (ADC) is an important enzyme in the production of putrescine and polyamines in plants. It is encoded by a single or low-copy nuclear gene that lacks introns in sequences studied to date. The rate of Adc amino acid sequence evolution is similar to that of ndhF for the angiosperm family studied. Highly conserved regions provide several target sites for PCR priming and sequencing and aid in nucleotide and amino acid sequence alignment across a range of taxonomic levels, while a variable region provides an increased number of potentially informative characters relative to ndhF for the taxa surveyed. The utility of the Adc gene in plant molecular systematic studies is demonstrated by analysis of its partial nucleotide sequences obtained from 13 representatives of Brassicaceae and 3 outgroup taxa, 2 from the mustard oil clade (order Capparales) and 1 from the related order Malvales. Two copies of the Adc gene, Adc1 and Adc2, are found in all members of the Brassicaceae studied to data except the basal genus Aethionema. The resulting Adc gene tree provides robust phylogenetic data regarding relationships within the complex mustard family, as well as independent support for proposed tribal realignments based on other molecular data sets such as those from chloroplast DNA.      

Intestinal barrier function of Atlantic salmon (<Emphasis Type="Italic">Salmo salar</Emphasis> L.) post smolts is reduced by common sea cage environments and suggested as a possible physiological welfare indicator

Background  

Fish farmed under high intensity aquaculture conditions are subjected to unnatural environments that may cause stress. Therefore awareness of how to maintain good health and welfare of farmed fish is important. For Atlantic salmon held in sea cages, water flow, dissolved oxygen (DO) levels and temperature will fluctuate over time and the fish can at times be exposed to detrimentally low DO levels and high temperatures. This experimental study investigates primary and secondary stress responses of Atlantic salmon post smolts to long-term exposure to reduced and fluctuating DO levels and high water temperatures, mimicking situations in the sea cages. Plasma cortisol levels and cortisol release to the water were assessed as indicators of the primary stress response and intestinal barrier integrity and physiological functions as indicators of secondary responses to changes in environmental conditions.     

Three forms of GnRH in the brain and pituitary of the turbot, Scophthalmus maximus: immunological characterization and seasonal variation

Three forms of GnRH, chicken (c) GnRH-II, salmon (s) and seabream (sb) GnRH, were immunologically characterized in the brain and pituitary of turbot by ELISA. cGnRH-II and sGnRH were detected in the brain, while sbGnRH and sGnRH (but not cGnRH-II) were detected in the pituitary. In females, the levels of cGnRH-II in the turbot brain extracts increased from May to July, concomitant with an increase in oocyte diameter. In the pituitary, sbGnRH was found to be the dominant form, with levels 100-600-fold those of sGnRH. Both sGnRH and sbGnRH in the pituitary showed variation during the spawning season; sbGnRH increased from May to July and correlated with the increase in oocyte diameter, while sGnRH decreased. The overall patterns were the same for male turbot, although levels were generally lower. These findings suggest that sbGnRH could be controlling reproduction in the turbot. However, the seasonal variation in sGnRH indicates a potential physiological role in turbot reproduction. This study gives the first immunological indications that sbGnRH is present in the pituitary of a pleuronectiform fish, and will provide the basis for further studies on the endocrine regulation of reproduction in flatfish.     

Chromosome numbers and meiotic analysis in the pre-breeding of <Emphasis Type="BoldItalic">Brachiaria decumbens</Emphasis> (Poaceae)

A total of 44 accessions of Brachiaria decumbens were analysed for chromosome count and meiotic behaviour in order to identify potential progenitors for crosses. Among them, 15 accessions presented 2n = 18; 27 accessions, 2n = 36; and 2 accessions, 2n = 45 chromosomes. Among the diploid accessions, the rate of meiotic abnormalities was low, ranging from 0.82% to 7.93%. In the 27 tetraploid accessions, the rate of meiotic abnormalities ranged from 18.41% to 65.83%. The most common meiotic abnormalities were related to irregular chromosome segregation, but chromosome stickiness and abnormal cytokinesis were observed in low frequency. All abnormalities can compromise pollen viability by generating unbalanced gametes. Based on the chromosome number and meiotic stability, the present study indicates the apomictic tetraploid accessions that can act as male genitor to produce interspecific hybrids with B. ruziziensis or intraspecific hybrids with recently artificially tetraploidized accessions.     

Spermatogenesis and related plasma androgen levels in Atlantic halibut (Hippoglossus hippoglossus L.)

Spermatogenesis in male Atlantic halibut (Hippoglossus hippoglossus L.) was investigated by sampling blood plasma and testicular tissue from 15-39-month-old fish. The experiment covered a period in which all fish reached puberty and completed sexual maturation at least once. The germinal compartment in Atlantic halibut testis appears to be organized in branching lobules of the unrestricted spermatogonial type, because spermatocysts with spermatogonia were found throughout the testis. Spermatogenesis was characterized histologically, and staged according to the most advanced type of germ cell present: spermatogonia (Stage I), spermatogonia and spermatocytes (Stage II), spermatogonia, spermatocytes and spermatids (Stage III), spermatogonia, spermatocytes, spermatids and spermatozoa (Stage IV), and regressing testis (Stage V). Three phases could be distinguished: first, an initial phase with low levels of circulating testosterone (T; quantified by RIA) and 11-ketotestosterone (11-KT; quantified by ELISA), spermatogonial proliferation, and subsequently the initiation of meiosis marked by the formation of spermatocytes (Stage I and II). Secondly, a phase with increasing T and 11-KT levels and with haploid germ cells including spermatozoa present in the testis (Stage III and IV). Thirdly, a phase with low T and 11-KT levels and a regressing testis with Sertoli cells displaying signs of phagocytotic activity (Stage V). Circulating levels of 11-KT were at least four-fold higher than those of T during all stages of spermatogenesis. Increasing plasma levels of T and 11-KT were associated with increasing testicular mass throughout the reproductive cycle. The absolute level of, or the relation between, testis growth and circulating androgens were not significantly different in first time spawners compared to fish that underwent their second spawning season. These results provide reference levels for Atlantic halibut spermatogenesis.