Identification and characterization of the egg-laying hormone from the neurosecretary bag cells of Aplysia

This investigation has resulted in the chemical identification and isolation of the egg-laying hormone from Aplysia californica, Aplysia vaccaria, and Aplysia dactylomela. The hormone, which was originally identified as the Bag Cell-Specific protein (BCS protein) on polyacrylamide gels, is a polypeptide of molecular weight ≈ 6000, which is localized in the neurosecretory bag cells of the parietovisceral ganglion and the surrounding connective tissue sheath which contains the bag cell axons. All three species produce a hormone of similar molecular weight, but varying electrophoretic mobility as determined on polyacrylamide gels. As tested, the hormone is completely cross-reactive among the three species.

Although the bag cells of sexually immature animals contain the active hormone, sexual maturation of the animal results in a 10-fold increase in the BCS protein content of these neurons.

A seasonal variation in the BCS protein content was also observed, with 150 times more hormone contained in the bag cells of Aplysia californica in August than in January. This correlates well with the variation in the animals' ability to lay eggs throughout the year (Strumwasser et al., 1969). There are some indications that the receptivity of the animal to the available hormone also fluctuates during the year, being lower in winter than in swmner. The seasonal rhythm of the other species, Aplysia vaccaria and Aplysia dactylomela, has not been investigated.

A polyacrylamide gel electrophoresis analysis of water-soluble proteins in Aplysia californica revealed several other nerve-specific proteins. One of these is also located in the bag cell somas and stains turquoise with Amido Schwarz. The function of this protein has not been investigated.

Biochemical and Biophysical Characterization of Huntingtin

Huntington’s disease (HD) is a fatal autosomal dominant neurodegenerative disease. HD has no cure, and patients pass away 10-20 years after the onset of symptoms. The causal mutation for HD is a trinucleotide repeat expansion in exon 1 of the huntingtin gene that leads to a polyglutamine (polyQ) repeat expansion in the N-terminal region of the huntingtin protein. Interestingly, there is a threshold of 37 polyQ repeats under which little or no disease exists; and above which, patients invariably show symptoms of HD. The huntingtin protein is a 350 kDa protein with unclear function. As the polyQ stretch expands, its propensity to aggregate increases with polyQ length. Models for polyQ toxicity include formation of aggregates that recruit and sequester essential cellular proteins, or altered function producing improper interactions between mutant huntingtin and other proteins. In both models, soluble expanded polyQ may be an intermediate state that can be targeted by potential therapeutics.

In the first study described herein, the conformation of soluble, expanded polyQ was determined to be linear and extended using equilibrium gel filtration and small-angle X-ray scattering. While attempts to purify and crystallize domains of the huntingtin protein were unsuccessful, the aggregation of huntingtin exon 1 was investigated using other biochemical techniques including dynamic light scattering, turbidity analysis, Congo red staining, and thioflavin T fluorescence. Chapter 4 describes crystallization experiments sent to the International Space Station and determination of the X-ray crystal structure of the anti-polyQ Fab MW1. In the final study, multimeric fibronectin type III (FN3) domain proteins were engineered to bind with high avidity to expanded polyQ tracts in mutant huntingtin exon 1. Surface plasmon resonance was used to observe binding of monomeric and multimeric FN3 proteins with huntingtin.

Cultural methods of detection for microorganisms: recent advances and successes

Most microbiological methods require culture to allow organisms to recover or to selectively increase, and target organisms are identified by growth on specific agar media. Many cultural methods take several days to complete and even then the results require confirmation. Alternative techniques include the use of chromogenic and fluorogenic substances to identify bacteria as they are growing, selective capture using antibodies after short periods of growth, molecular techniques, and direct staining with or without flow cytometry for enumeration and identification. Future microbiologists may not use culture but depend on the use of specific probes and sophisticated detection systems.

Altered glutamyl-aminopeptidase activity and expression in renal neoplasms

Background: Advances in the knowledge of renal neoplasms have demonstrated the implication of several proteases in their genesis, growth and dissemination. Glutamyl-aminopeptidase (GAP) (EC. 3.4.11.7) is a zinc metallopeptidase with angiotensinase activity highly expressed in kidney tissues and its expression and activity have been associated wtih tumour development. Methods: In this prospective study, GAP spectrofluorometric activity and immunohistochemical expression were analysed in clear-cell (CCRCC), papillary (PRCC) and chromophobe (ChRCC) renal cell carcinomas, and in renal oncocytoma (RO). Data obtained in tumour tissue were compared with those from the surrounding uninvolved kidney tissue. In CCRCC, classic pathological parameters such as grade, stage and tumour size were stratified following GAP data and analyzed for 5-year survival. Results: GAP activity in both the membrane-bound and soluble fractions was sharply decreased and its immunohistochemical expression showed mild staining in the four histological types of renal tumours. Soluble and membrane-bound GAP activities correlated with tumour grade and size in CCRCCs. Conclusions: This study suggests a role for GAP in the neoplastic development of renal tumours and provides additional data for considering the activity and expression of this enzyme of interest in the diagnosis and prognosis of renal neoplasms.

Pio del Rio Hortega and the discovery of the oligodendrocytes

Rio del Rio Hortega (1882-1945) discovered microglia and oligodendrocytes (OLGs), and after Ramon y Cajal, was the most prominent figure of the Spanish school of neurology. He began his scientific career with Nicolas Achucarro from whom he learned the use of metallic impregnation techniques suitable to study non-neuronal cells. Later on, he joined Cajal's laboratory. and Subsequently, he created his own group, where he continued to develop other innovative modifications of silver staining methods that revolutionized the study of glial cells a century ago. He was also interested in neuropathology and became a leading authority on Central Nervous System (CNS) tumors. In parallel to this clinical activity, del Rio Hortega rendered the first systematic description of a major polymorphism present in a subtype of macroglial cells that he named as oligodendroglia and later OLGs. He established their ectodermal origin and suggested that they built the myelin sheath of CNS axons, just as Schwann cells did in the periphery. Notably, he also suggested the trophic role of OLGs for neuronal functionality, an idea that has been substantiated in the last few years. Del Rio Hortega became internationally recognized and established an important neurohistological school with outstanding pupils from Spain and abroad, which nearly disappeared after his exile due to the Spanish civil war. Yet, the difficulty of metal impregnation methods and their variability in results, delayed for some decades the confirmation of his great insights into oligodendrocyte biology until the development of electron microscopy and immunohistochemistry. This review aims at summarizing the pioneer and essential contributions of del Rio Hortega to the current knowledge of oligodendrocyte structure and function, and to provide a hint of the scientific personality of this extraordinary and insufficiently recognized man.

Histological distribution and ultrastructure of exocrine pancreas in Indian major carp (Labeo rohita Ham.) and its alteration in aflatoxicosis

The distribution pattern of exocrine pancreas in Labeo rohita besides its general location along the course of intestinal mesentery was studied. It is evenly distributed within the liver around portal vessels and also within the spleen near a blood vessel. On ultrastructure, two cell types of different degrees of staining intensities containing abundant rough endoplasmic reticulum, mitochondria, pre-zymogen and zymogen granules were marked. During aflatoxicosis, the mesenteric pancreas and hepatic pancreas were mostly affected revealing necrotic changes to acini. The zymogen granular activities were markedly reduced. Ultra structurally, the rough endoplasmic reticulum was fully dilated and formed whorled pattern. The damage to the exocrine pancreas might be affecting digestive enzymes' secretion which may be one of the causes of aflatoxin-induced anorexia in fish.

Characterisation of neurons in the pedal ganglia of the green-lipped mussel, Perna canaliculus, using antibodies raised against neuropeptides and neurotransmitters involved in gastropod egg-laying behaviour and bivalve reproduction and spawning

To characterise central neurons in the pedal ganglia of both male and female green lipped mussel, Perna canaliculus immunohistochemical techniques were used. Mollusc antibodies were used against neuropeptides and neurotansmitters known to control reproduction and spawning. Anti-ELH and anti-APGWamide showed very strong immunoreactivity in small type of neurons. Anti-5-HT and anti-DA immunoreactivity was mostly in large type of neurons. The labelled neurons are consistent with descriptions of neurosecretory cells implicated in the control of reproduction and spawning on the basis of earlier histological staining techniques used in this species. The use of selective immunological markers for peptides and amines appears to be a, promising tool for further characterisation of neurosecretory cells, and to isolate an'tl characterise neuropeptides and other biologically active materials involved in the control of reproduction in Perna canaliculus.

Neurons characterization in the cerebral ganglia of the green-lipped mussel, Perna canaliculus, using antibodies raised against neuropeptides and neurotransmitters involved in gastropod egg-laying behaviour and bivalve reproduction and spawning

Immunohistochemical techniques were used to characterise central neurons in the cerebral ganglia of both male and female Perna canaliculus. We used mollusc antibodies raised against neuropeptides and neurotransmitters known to control reproduction and spawning. Anti-ELH and anti-APGWamide showed very strong immunoreactivity in small type of neurons. Anti-5-HT and anti-DA immunoreactivity was mostly in large type of neurons. The labelled neurons are consistent with descriptions of neurosecretory cells implicated in the control of reproduction and spawning on the basis of earlier histological staining techniques used in this species. The use of selective immunological markers for peptides and amines appears to be a promising tool for further characterisation of neurosecretory cells, and to isolate and characterise neuropeptides and other biologically active materials involved in the control of reproduction in Perna canaliculus.

KARYOTYPE AND BANDING-PATTERNS OF FEA TREE RAT (CHIROMYSCUS-CHIROPUS)

Fea's tree rat (Chiromyscus chiropus) is a very rare species which there are only a few specimens in the world. The chromosomes of two male specimens, collected from Xishuanbanna, Yunnan, are analysed by several banding technique (G-, C-bands, as well as Ag-staining). The diploid chromosome number is 22, and autosomes comprise 5 pairs of metacentrics, 2 pairs of subacrocentrics, and 3 pairs of acrocentrics. The X chromosome is a acrocentric, and Y is a micro-chromosome, almost a point, which could be a marker chromosome of the species and the genus. The centromeric C-bands are very faint, and C-bands of Nos. 1, 2, 9 and Y chromosome are negative. Only one pair Ag-NORs was found on No. 10 in the silver-stained karyotype. The relationship between morphologic and chromosomal features was discussed, and C-banded karyotype evolutionary trend has also been discussed. Moreover, the conventional karyotype of Niviventer confucianus was described.

Isolating and recognizing crudeoiklegrading [sic] bacteria of Queshm Island shores for biodegradation of crude oil pollutions

This research was carried out for recognizing Natural Flora Bacteria of oil pollution in the coasts of Queshm island. In The First steps, The coasts of this Island were scrutinized as a Field of research and For knowing whether oil stains exist or not. It gets obvious That southern coasts of Queshm have got oil pollution which is created by oil tankers which carry oil of Iran continental shelf. Them oil stains were sampled from to certain stations. In The First step, primary isolation of exisiting bacteria in every oil sample was done and then purification of each bacterium was carried out. Then each purified bacterium that has got strong, recognized, typic growth was enriched oil sample of T5 station. And Bacterium C4 (gram—negative coccobacillus) was chosen as the second priority From oil sample of TA station and Bacterium B1 (gram—positive coccus) was chosen as The third priority From oil sample of TI station. All The above mentioned bacteria were biochemically, physiologically and morphologically experimented For specking The species. According To The tests done and comparing with The tests done and comparing with the reference Berge y' s, bacterium A5 Pelongs to the species pseudomonas sp and becterium C4 belongs to the species Aeromonas sp and bacterium BI belongs to The species micrococcus sp. In The Last stage, bacterium with The First priority (TA5 pseudomonas sp) was used in the planned microcosm. The sake of optimum and adapting to Laboratory conditions Each enriched and purified bacterium was given a code for station and a code For itself . Then This bacterium was studied and it was proved that it has potentiality For using oil as a source of carbon. From oil samples of 10 stations, 30 various Colonies of bacterium were Isolated, of which 20 bacteria had the highest potentiality of growth. And the other bacteria that has no typic growth were omitted From being studied. Since all of These 20 bacterium are able to use oil, a bacterium with maximum rate of growth in the presence of crude oil and Lack of other hydrocarbonic sources and with The code A5 ( gram — negative Bacillus ) was chosen as First priority From The mentioned microcosm contains sea water , suspension oil degrading bacterium , crude oil, azote and various concentrations of carbon and Incubated in 30°` and shook 150 PRA1 According to the results , index oil degrading bacterium (pseudomonas sp) belongs oil sample of T5 stations (east of sheeb draz Gulf) which growth best and have the potentiality of degrading oil in 25 glli malas and 50 glli cheese water and with 5 gill urea .

HORMONAL-REGULATION OF TISSUE-TYPE PLASMINOGEN-ACTIVATOR AND PLASMINOGEN-ACTIVATOR INHIBITOR TYPE-1 IN CULTURED MONKEY SERTOLI CELLS

Sertoli cells play a central role in the control and maintenance of spermatogenesis. Isolated Sertoli cells of mouse and rat testes have been shown to secrete plasminogen activator (PA) and a plasminogen activator inhibitor type-1 (PAI-1) in culture. In this study, we have investigated the hormonal regulation of PA and PAI-1 activities in cultured monkey Sertoli cells. Sertoli cells (5x10(5) cells/well) isolated from infant rhesus monkey testes were preincubated at 35 degrees C for 16 h in 24-well plates precoated with poly(D-lysine) (5 mu g/cm(2)) in 0.5 mi McCoy's 5a medium containing 5% of fetal calf serum and further incubated for 48 h in 0.5 mi serum-free medium with or without various hormones or other compounds, PA as well as PAI-1 activities in the conditioned media were assayed by fibrin overlay and reverse fibrin autography techniques respectively. The Sertoli cells in vitro secreted only tissue-type PA (tPA), no detectable amount of urokinase-type PA (uPA) could be observed, Monkey Sertoli cells were also capable of secreting PAI-1, Immunocytochemical studies indicated that both tPA and PAI-1 positive staining localized in the Sertoli cells, spermatids and residual bodies of the seminiferous epithelium; Northern blot analysis further confirmed the presence of both tPA and PAI-1 mRNA in monkey Sertoli cells. Addition of follicle-stimulating hormone (FSH) or cyclic adenosine monophosphate (cAMP) derivatives or cAMP-generating agents and gonadotrophin-releasing hormone (GnRH) agonist or phorbol ester (PMA) to the cell culture significantly increased tPA activity. PAI-1 activity in the culture was also enhanced by these reagents except 8-bromo-dibutyryl-cAMP, forskolin and 3-isobutyl-1-methylxanthin (MIX) which greatly stimulated tPA activity, whereas decreased PAI-1 activity, implying that neutralization of PAI-1 activity by tile high level of tPA in the conditioned media may occur. These data suggest that increased intracellular signals which activate protein kinase A (PKA), or protein kinase C (PKC) can modulate Sertoli cell tPA and PAI-1 activities, The concomitant induction of PA and PAI-1 by the same reagents in the Sertoli cells may reflect a finely tuned regulatory mechanism in which PAI-1 could limit the excession of the proteolysis.

Localization and distribution of tissue type and urokinase type plasminogen activators and their inhibitors type 1 and 2 in human and rhesus monkey fetal membranes

Fetal membranes consist of 10 distinct layers including components of amnion, chorion and decidua, the latter being of maternal origin. They form mechanically integrated sheets capable of retaining amniotic fluid and play an essential role in protecting fetal growth and development in the pregnant uterus. The extracellular matrix, substrate for plasminogen activators (PAs), is an important supportive framework of the fetal membranes. :Fetal membranes from women with preterm premature rupture of membranes may differ in their protease activity compared with normal membranes. To identify the presence of PAs and their inhibitors (PAI) and their possible role in the process of fetal membrane rupture, this study in investigated the distribution and localization of both protein and mRNA for tissue (t) and urokinase (u) PA and their inhibitors type 1 (PAI-1) and type 2 (PAI-2) in amniochorion of human and rhesus monkey using conventional and. confocal immunofluorescence microscopy. In situ hybridization analysis showed that the distribution and localization of mRNAs for tPA, uPA, PAI-I and PAI-2 were similar in the fetal membranes of human and rhesus monkey; no obvious species difference was observed. Evidence of tPA mRNA was detected in amniotic epithelium, trophoblast cells and nearly all cells of the decidual layer. Strong expression of uPA mRNA was noted in the decidual cells which increased in intensity as the abscission point was approached. Weak staining in chorion laeve trophoblast was also detected. In situ hybridization experiments showed PAI-1 mRNA to be concentrated mainly in the decidual cells, some of which were interposed into the maternal-facing edge of the chorion laeve. Maximal labelling of the decidua occurred towards the zone of abscission. Weak expression of PAI-1 mRNA nas also noted in some cells of the chorion laeve. The distribution of PAI-2 mRNA in amniochorion was also concentrated in the cells of the decidual layer, maximum expression of the mRNA was in the level of abscission. No detectable amount of mRNAs for tPA, uPA, PAI-1 and PAI-2 was found in the fibroblast, reticular and spongy layers. Distribution of the proteins of tPA, uPA and PAI-1 in the fetal membranes of these two species was consistent with the distribution of their mRNA. Anti-PAI-2 immunofluorescence was found to be strongly concentrated in the amniotic epithelium, but PAI-2 mRNA was negative in this layer, suggesting that the epithelium-associated PAI-2 is not of epithelial origin. These findings suggest that a local fibrinolysis in fetal membranes generated by precisely balanced expression of PAs and their inhibitors via paracrine or autocrine mechanisms may play an essential role in fetal membrane development, maturation and in membrane rupture. Following an analysis of the distribution and synthesis of activators and inhibitors it was found that they may play a role in abscission during the third stage of labour. (C) 1998 W. B. Saunders Company Ltd.

Hexabromocyclododecane-induced developmental toxicity and apoptosis in zebrafish embryos

Hexabromocyclododecane (HBCD) is widely used as a brominated flame retardant, and has been detected in the aquatic environment, wild animals, and humans. However, details of the environmental health risk of HBCD are not well known. In this study, zebrafish embryos were used to assess the developmental toxicity of the chemical. Four-hour post-fertilization (hpf) zebrafish embryos were exposed to various concentrations of HBCD (0, 0.05, 0.1, 0.5, and 1.0 mg L-1) until 96 h. Exposure to 0.1, 0.5, and 1.0 mg L-1 HBCD significantly increased the malformation rate and reduced survival in the 0.5 and 1.0 mg L-1 HBCD exposure groups. Acridine orange (AO) staining showed that HBCD exposure resulted in cell apoptosis. Reactive oxygen species (ROS) was significantly induced at exposures of 0.1, 0.5, and 1.0 mg L-1 HBCD. To test the apoptotic pathway, several genes related to cell apoptosis, such as p53, Puma, Apaf-1, caspase-9, and caspase-3, were examined using real-time PCR. The expression patterns of these genes were up-regulated to some extent. Two anti-apoptotic genes, Mdm2 (antagonist of p53) and Bcl-2 (inhibitor of Bax), were down-regulated, and the activity of capspase-9 and caspase-3 was significantly increased. The overall results demonstrate that waterborne HBCD is able to produce oxidative stress and induce apoptosis through the involvement of caspases in zebrafish embryos. The results also indicate that zebrafish embryos can serve as a reliable model for the developmental toxicity of HBCD. (C) 2009 Elsevier B.V. All rights reserved.