Purification and properties of a lytic enzyme from the cell wall of Chlorella ellipsoidea C-87

Cell wall lytic activity was detected in the culture medium and cell wall of 1AM Chlorella ellipsoidea C-87. The enzymes of both fractions had their highest activity at pH 5. The lytic activity bound to the cell wall consisted of a polysaccharide releasing enzyme, an exo-type enzyme releasing disaccharide, and glucosidase; but only the polysaccharide releasing enzyme was solubilized by lithium chloride. A polysaccharide releasing enzyme with a molecular weight around 40 kDa was isolated from the culture medium. Hemicellulose is degraded by the polysaccharide releasing enzyme, and the rigid wall by the exo-type enzyme.     

Cation-dependent gonadotropin releasing hormone activation of guanylate cyclase

Summary Gonadotropin releasing hormone enhanced guanylate cyclase [E.C.4.6.1.2] two- to threefold in pituitary, testis, liver and kidney. Dose response relationships revealed that at a concentration of 1 nanomolar, gonadotropin releasing hormone caused a maximal augmentation of guanylate cyclase activity and that increasing its concentration to the millimolar range caused no further enhancement of this enzyme. There was an absolute cation requirement for gonadotropin releasing hormone's enhancement of guanylate cyclase activity as there was no increase without any cation present. Gonadotropin releasing hormone could increase guanylate cyclase activity with either calcium or manganese in the incubation medium but more augmentation was observed with manganese. The data in this investigation suggest that guanylate cyclase may play a role in the mechanism of action of gonadotropin releasing hormone.     

Human follicular gonadotropin releasing peptide analogs. Evaluation of biological (in vitro) and immunological activity

Human follicular gonadotropin releasing peptide (hF-GRP) has been shown to stimulate pituitary LH and FSH secretion in vitro. Six hF-GRP analogs have been synthesized and evaluated for gonadotropin releasing activity in a rat anterior pituitary primary cell culture system. A tyrosine analog of hF-GRP, [Tyr4]-hF-GRP, retained comparable biological activity in releasing gonadotropins. However, acetylation of hF-GRP in Ac-hF-GRP greatly reduced the in vitro activity. The shorter segments of hF-GRP, hF-GRP-(5-14), and hF-GRP-(10-14), were tested for LH and FSH releasing activity, and it was found that the decapeptide retained moderate activity while the activity of the pentapeptide was markedly lower than hF-GRP. The baboon alpha 1 antitrypsin-(27-40) peptide, b-alpha 1 AT-(27-40), is relatively less potent in releasing LH than hF-GRP. Interestingly, the baboon peptide is more potent (2.5-fold) in releasing FSH under identical conditions. The effect of hF-GRP in releasing LH and FSH was not affected by the presence of LHRH antagonists in cell culture systems. When these peptides were tested for immunological activity in a hF-GRP radioimmunoassay, it was found that hF-GRP and [Tyr4]-hF-GRP have comparable activities. The C-terminal decapeptide of hF-GRP is more active (1.5-fold) in the RIA, and the C-terminal pentapeptide had only one third of the immunoreactivity. The b-alpha 1-AT-(27-40) failed to cross-react in the RIA even at a concentration of 20 micrograms per tube.     

Bioactive forms of gonadotropin releasing hormone in the brain of an Indian major carp,Catla catla (Ham.)

Two forms of biologically active gonadotropin releasing hormones were isolated from the hypothalami ofCatla catla. Gonadotropin releasing hormone activity was studiedin vitro using enzymatically dispersed carp pituitary cell incubation system. Gonadotropin released into the medium was measured by carp gonadotropin-radio immuno assay. Acetic acid extracted hypothalamic material was subjected to acetone fractionation. Among the three protein pellets obtained at different time periods (ACI, ACII and ACIII), AC II exhibited the gonadotropin releasing hormone activity. Gel filtration of AC II through Sephadex G-25 column showed three protein peaks (SG I, SG II SGIII) and only S G II demonstrated strong gonadotropin releasing hormone activity. Elution of SG II through FPLC Mono Q column (an anion exchanger) in NaCl gradient programme showed one unadsorbed (MQ I) and three adsorbed (MQ II, MQ III and MQ IV) protein peaks. MQ III, which was eluted with 51% NaCl, exhibited gonadotropin releasing hormone activity. Surprisingly, unadsorbed fractions, MQ I, also showed gonadotropin releasing hormone activity. MQ 1 was therefore subjected to FPLC Mono S (a cation exchanger) column chromatography where a highly active gonadotropin releasing hormone enriched peak, i.e., MS III, could be eluted with 45% NaCl. These findings show thatCatla catla hypothalamus has two forms of gonadotropin releasing hormones one anionic (carp gonadotropin releasing hormone I) and another cationic (carp gonadotropin releasing hormone II). These two forms of gonadotropin releasing hormones were also active in heterologous carp species, rohu(Labeo rohita), mrigal(Cirrhinus mrigala) and an exotic common carp(Cyprinus carpio). Combined activity of two forms of gonadotropin releasing hormones was significantly greater as compared to any of the single form.     

Desensitization of testicular ornithine decarboxylase to gonadotropin releasing hormone and gonadotropic hormones

Prior exposure of the testis to gonadotropin releasing hormone, luteinizing hormone or follicle stimulating hormone caused the testis refractory to these hormones in terms of ornithine decarboxylase activity at 24 h. Luteinizing hormone caused desensitization in the Leydig cells while the levels of ornithine decarboxylase in the seminiferous tubules were unaltered. In gonadotropin releasing hormone desensitized testis all the other treated compounds namely, luteinizing hormone, follicle stimulating hormone, prostaglandin F2 alpha, norepinephrine and cyclic AMP caused stimulation of ornithine decarboxylase activity. The testis desensitized with LH responded to cyclic AMP and norepinephrine whereas prostaglandin E2 or gonadotropin releasing hormone caused less stimulation of ornithine decarboxylase activity. These results indicate that testicular desensitization to gonadotropin releasing hormone and luteinizing hormone is not due to a post cyclic AMP block.     

Alternative strategy for converting an inverting glycoside hydrolase into a glycosynthase

The tyrosine residue Y198 is known to support a nucleophilic water molecule with the general base residue, D263, in the reducing-end xylose-releasing exo-oligoxylanase (Rex). A mutation in the tyrosine residue changing it into phenylalanine caused a drastic decrease in the hydrolytic activity and a small increase in the F(-) releasing activity from alpha-xylobiosyl fluoride in the presence of xylose. In contrast, mutations at D263 resulted in the decreased F(-) releasing activity. As a result of the high F(-) releasing activity and low hydrolytic activity, Y198F of Rex accumulates a large amount of product during the glycosynthase reaction. We propose a novel method for producing a glycosynthase from an inverting glycoside hydrolase by mutating a residue that holds the nucleophilic water molecule with the general base residue while keeping the general base residue intact.     

Endocrine and non-endocrine activities of growth hormone secretagogues in humans

Growth hormone (GH) secretagogues (GHS) are synthetic peptidyl and non-peptidyl molecules which possess strong, dose-dependent and reproducible GH releasing effects as well as significant prolactin (PRL) and adrenocorticotropic hormone (ACTH) releasing effects. The neuroendocrine activities of GHS are mediated by specific receptors mainly present at the pituitary and hypothalamic level but also elsewhere in the central nervous system. GHS release GH via actions at the pituitary and (mainly) the hypothalamic level, probably acting on GH releasing hormone (GHRH) secreting neurons and/or as functional somatostatin antagonists. GHS release more GH than GHRH and the coadministration of these peptides has a synergistic effect but these effects need the integrity of the hypothalamo-pituitary unit. The GH releasing effect of GHS is generally gender-independent and undergoes marked age-related variations reflecting age-related changes in the neural control of anterior pituitary function. The PRL releasing activity of GHS probably comes from direct pituitary action, which indeed is slight and independent of both age and gender. The acute stimulatory effect of GHS on ACTH/cortisol secretion is similar to that of corticotropin releasing hormone (CRH) and arginine vasopressin (AVP). In physiological conditions, the ACTH releasing activity of GHS is mediated by central mechanisms, at least partially, independent of both CRH and AVP but probably involving GABAergic mechanisms. The ACTH releasing activity of GHS is gender-independent and undergoes peculiar age-related variations showing a trend towards increase in ageing. GHS possess specific receptors also at the peripheral levels in endocrine and non-endocrine human tissues. Cardiac receptors are specific for peptidyl GHS and probably mediate GH-independent cardiotropic activities both in animals and in humans.     

Inhibition of 5′-nucleotidase activity after growth of Dictyostelium discoideum

The specific activity of 5′-nucleotidase activity in cell-free extracts of Dictyostelium discoideum at both exponential and stationary growth phases was determined. The 5′-nucleotidase activity of both membrane and soluble fractions was determined. The results show that at exponential growth more activity is found in the soluble fraction. Furthermore, the results show that stationary phase cells contain about 10-fold less activity than cells at exponential growth. To determine if stationary phase cells contained an inhibitor of 5′-nucleotidase, purified membranes were incubated with a high speed supernatant (S-100) prepared from cells at this stage. The results showed not only a time and concentration dependent loss of membrane bound activity, but also that most of the lost activity could be recovered in a soluble form. This result suggested that the 5′-nucleotidase was being released by a factor in the S-100. Additional studies showed inactivation of the releasing factor by a protease and further, that this inactivation could be prevented by serine protease inhibitors. The specificity of releasing factor with respect to two other membrane bound activities was determined. The results indicated no loss of either 3′5′-cyclic phosphodiesterase or adenylate cyclase. In addition, the results of a comparison of the activity of the releasing factor at two stages of growth showed similar values at both exponential and stationary growth phase. This latter finding suggests that the loss of 5′-nucleotidase activity at stationary phase is not due to modulation of the releasing factor activity. An alternative mechanism is proposed.     

Preliminary studies of plasma growth hormone releasing activity during medical therapy of acromegaly.

The in vitro growth hormone releasing activity of plasma obtained from six acromegalic subjects was measured before and during therapy. In five subjects, plasmas were obtained before and during successful medical therapy with medroxyprogesterone acetate (MPA). The sixth subject was sampled before and after transphenoidal Sr90-induced hypopituitarism. All subjects had a decrement in fasting growth hormone levels with respective therapies (29-88%). The in vitro growth hormone released from Rhesus monkey anterior pituitaries was assessed after incubating one lateral half in control plasma (pre-therapy) and the contralateral pituitary half in plasma obtained during or after therapy. Studies with plasmas obtained from the five patients successfully treated with MPA showed a decreased in growth hormone releasing activity during therapy in all (18-57%). Plasma obtained after Sr90 pituitary ablation in the sixth subject had 35% more growth hormone releasing activity than obtained before therapy. These results suggest that active acromegalics who respond to MPA with significantly lowered growth hormone levels may actually achieve this response because of a decrease in growth hormone releasing factor measured peripherally. The opposite response in one acromegalic subject, following Sr90 pituitary ablation and hypopituitarism, suggests that growth hormone releasing factor secretion may increase when growth hormone levels are lowered by ablative therapy.     

In vivo and in vitro release of ACTH by synthetic CRF

The 41-residue corticotropin releasing factor (CRF) was synthesized by the solid phase method. The synthetic CRF and arginine vasopressin (AVP) were examined for ACTH releasing activity and effects on the release of 5 other pituitary hormones in vivo and in vitro. Injection of the CRF into pharmacologically blocked rats increased plasma corticosterone levels in a dose-related manner. The minimum effective dose was 1.6 x 10(-12) mol/100 g body weight. CRF also significantly stimulated release of ACTH-like immunoreactivity in a dose-related manner from rat pituitary quarters beginning at a concentration of 10(-9) M. AVP, a peptide known to have CRF activity, exhibited slightly lower corticotropin releasing activity than the CRF at equimolar dose levels. Secretion of other pituitary hormones was not appreciably altered by either the CRF or AVP.     

Generation of hypophysiotropic activity in hypothalamic extracts in vitro.

The luteinizing hormone (LH) and follicle stimulating hormone (FSH) releasing activity, as well as the prolactin (PRL) release-inhibiting activity were measured in both neutral aqueous, and acid ethanolic extracts of rat hypothalami. LH and FSH-releasing activities were detectable only in the latter type of extract, whereas PRL release-inhibiting activity appeared in both. Neutral ultrafiltrates of the neutral extracts contained no gonadotropin releasing activity, however, acidification of the filtration medium induced its appearance. PRL release was inhibited by both neutral and acid filtrates. These results suggest that LH and FSH releasing factor(s) may be stored in the hypothalamus in an inactive form from which the active peptide is generated in vitro under acid conditions; however, this does not appear to be true for the component(s) responsible for the inhibition of PRL release.     

17‐β estradiol attenuates the pro‐oxidant activity of corticotropin‐releasing hormone in macroendothelial cells

Corticotropin‐releasing hormone, which is the predominant regulator of neuroendocrine responses to stress, attenuates inflammation through stimulation of glucocorticoid release. Enhanced corticotropin‐releasing hormone expression has been detected in inflammatory cells of the vascular endothelium, where it acts as a local regulator of endothelial redox homeostasis. Estrogens have beneficial effects on endothelial integrity and function, though the mechanism underlying their antioxidative effect remains as yet largely unknown. We therefore investigated the effect of 17β‐estradiol on pro‐oxidant action of corticotropin‐releasing hormone in vitro in macroendothelial cells, and, more specifically, the role of 17β‐estradiol on corticotropin‐releasing hormone‐induced activities/release of the antioxidant enzymes namely, endothelial nitric oxide synthase, superoxide dismutase, catalase, and glutathione. We observed that 17β‐estradiol abolished the stimulatory effect of corticotropin‐releasing hormone on intracellular reactive oxygen species levels and counteracted its inhibitory effect on endothelial nitric oxide synthase activity and nitric oxide release. In addition, 17β‐estradiol significantly induced superoxide dismutase and catalase activity, an effect that was not significantly influenced by corticotropin‐releasing hormone. Finally, 17β‐estradiol significantly increased glutathione levels and the glutathione/glutathione + glutathione disulfide ratio, an action that was partially blocked by corticotropin‐releasing hormone. Our results reveal that 17β‐estradiol counterbalances corticotropin‐releasing hormone‐mediated pro‐inflammatory action and thereby maintains the physiological threshold of the endothelial cell redox environment. These observations may be of importance, considering the protective role of estrogen in the development of atherosclerosis.     

通络救脑注射液对大鼠脑微血管内皮细胞活性 及其条件培养液影响的初步研究

目的：观察通络救脑注射液对正常及拟缺血大鼠脑微血管内皮细胞的活性影响,并初步探讨细胞条件培养液内蛋白分泌的时效特征、奠定可溶性蛋白深入分析的技术基础。方法：通络救脑注射液作用于正常夏拟缺血脑微血管内皮细胞之后,用MTS／PMS比色分析法测定细胞的活性,Bradford法测定细胞培养液总蛋白含量,比色分析法测定细胞培养液乳酸脱氢酶（LDH）漏出值,同步观察了5个时间点的细胞活性及条件培养液总蛋白量及LDH释放量。结果：通络救脑注射液能够提高拟缺血细胞的活性,且抑制LDH释放量;正常组分泌总蛋白量3h达到高峰,此时细胞活性最佳,LDH释放量亦少。拟缺血组分泌总蛋白量是6h达到高峰,此时LDH释放量最少,但细胞活性与3h比较有所下降。结论：通络救脑注射液对拟缺血细胞损伤具有保护作用：以3h至6h的细胞条件培养液做为收集目标是研究条件培养液的最佳时间段。     

Design,synthesis and evaluation of phenylfuroxan nitric oxide-donor phenols as potential anti-diabetic agents

Both nitric oxide (NO) dysfunction and oxidative stress have been regarded as the important factors in the development and progression of diabetes and its complications. Multifunctional compounds with hypoglycemic, NO supplementation and anti-oxidation will be the promising agents for treatment of diabetes. In this study, six phenylfuroxan nitric oxide (NO) donor phenols were synthesized, which were designed via a combination approach with phenylfuroxan NO-donor and natural phenols. These novel synthetic compounds were screened in vitro for α-glucosidase inhibition, NO releasing, anti-oxidation, anti-glycation and anti-platelet aggregation activity as well as vasodilatation effects. The results exhibited that compound T5 displayed more excellent activity than other compounds. Moreover, T5 demonstrated significant hypoglycemic activity in diabetic mice and oral glucose tolerance test (OGTT) mice. T5 also showed NO releasing and anti-oxidation in diabetic mice. Based on these results, compound T5 deserves further study as potential new multifunctional anti-diabetic agent with antioxidant, NO releasing, anti-platelet aggregation and vasodilatation properties.     

Corticotropin releasing factor activity of CRF 41 in normal man is potentiated by angiotensin II and vasopressin but not by desmopressin

Multiple hypothalamic factors seem to influence ACTH release. In vitro and/or in vivo animal models have shown that angiotensin II, vasopressin and some of its analogs are ACTH secretagogues capable of potentiating the corticotropin releasing activity of CRF41. Since these effects are controversial in man, we investigated in 3 groups of volunteers the corticotropin releasing activity of a 2h-infusion of angiotensin II (7 ng/kg/min), vasopressin (1 ng/kg/min) and desmopressin (1 ng/kg/min) given alone or in combination with a bolus injection of 100 micrograms CRF41 by measuring plasma concentrations of ACTH, cortisol, dehydroepiandrosterone and delta 4-androstenedione. Given alone angiotensin II and desmopressin had no significant effect in contrast to vasopressin which increased significantly the ACTH and steroid levels. Angiotensin II and vasopressin were both able to potentiate the corticotropin releasing activity of CRF41, whereas desmopressin was unable to produce such a potentiation. These results suggest that in man vasopressin and angiotensin II may well regulate the responsiveness of the pituitary-adrenal axis in various physiological or pathophysiological situations.     

Differential effects of several dopamine uptake inhibitors and releasing agents on locomotor activity in normal and in reserpinized mice

Several drugs that were potent dopamine uptake inhibitors in vitro (including GBR 13,069 and GBR 13,098, amfonelic acid, mazindol, Win 35,065 and Win 35,482) caused large increases in locomotor activity in normal mice but not in reserpinized mice. Additionally, these drugs given to reserpinized mice prior to the dopamine releasing agent d-amphetamine, were able to prevent the normal increase in activity caused by d-amphetamine. The data demonstrate markedly different in vivo effects of dopamine uptake inhibitors and dopamine releasing agents.     

Effects of corticotropin releasing factor on locomotor activity in hypophysectomized rats

Corticotropin releasing factor (CRF) injected intracerebroventricularly to hypophysectomized and sham hypophysectomized rats produced a dose dependent increase in locomotor activity, but in untreated hypophysectomized rats 10× more CRF was needed to produce a significant increase in activity. Concomitant daily supplements of rat growth hormone, thyroxine, and corticosterone to the hypophysectomized rats eliminated locomotor activity differences between the two groups. There was no statistically significant difference in locomotor response to either saline, 0.1 μg CRF, 1.0 μg CRF or 10.0 μg CRF in the group of animals receiving hormonal supplements. These results demonstrate that CRF can produce behavioral activation in rats independently of its effects on releasing hormones from the pituitary gland.     

Activity of vertebrate gonadotropin-releasing hormones and analogs with variant amino acid residues in positions 5, 7 and 8 in the goldfish pituitary.

All non-mammalian vertebrates as well as marsupial mammals have two or more forms of gonadotropin-releasing hormone (GnRH) in the brain. Goldfish brain and pituitary contains two molecular forms of GnRH, salmon GnRH ([Trp7, Leu8]m-GnRH; s-GnRH) and chicken GnRH-II ([His5, Trp7, Tyr8]m-GnRH; cII-GnRH). Both sGnRH and cII-GnRH stimulate gonadotropin (GtH) as well as growth hormone (GH) release from the goldfish pituitary. The purpose of the present study was to study the activity of the five known forms of GnRHs as well as analogs of mammalian GnRH (m-GnRH) with variant amino acid residues in positions 5, 7 and 8 in terms of binding to GnRH receptors, and release of GTH and GH from the perifused fragments of goldfish pituitary in vitro. All five vertebrate GnRH peptides stimulated both GtH and GH release in a dose-dependent manner, although their potencies were very different. cII-GnRH was somewhat more active than s-GnRH in releasing GtH, whereas s-GnRH tended to have a greater potency than cII-GnRH in terms of GH release. Both chicken GnRH-I (cI-GnRH) and lamprey GnRH (l-GnRH) were significantly less potent than mGnRH, s-GnRH and cII-GnRH in releasing GtH and GH. cII-GnRH binds with higher affinity for the high affinity binding sites compared to all other native peptides. The activity of [Trp7]-GnRH was similar to both s-GnRH and cII-GnRH in releasing GtH and GH. Substitution of His5 resulted in a significant decrease in GtH releasing potencies compared to mGnRH, sGnRH and cII-GnRH. [His5]-GnRH also had lower GH releasing potency than mGnRH and sGnRH. Tyr8, His8 and Leu8 substitutions caused significant decreases in GtH releasing potencies compared to mGnRH, s-GnRH and cII-GnRH, but did not cause a significant change in GH releasing potency. The combination of [His5, Trp7]-GnRH had GtH and GH releasing activities similar to m-GnRH, s-GnRH and cII-GnRH.(ABSTRACT TRUNCATED AT 400 WORDS)     

Insect cell conditioned medium contains an endoglycosidase able to liberate chondroitin sulfate chains from aggrecan.

Culture medium obtained from baculovirus-infected High Five insect cells contains an endoglycosidase activity capable of releasing chondroitin sulfate chains from aggrecan, decorin and biglycan. Release appears to occur by cleavage within the linkage region of the chondroitin sulfate chain, but not all chains are amenable to release. Culture medium from Sf9 insect cells does not contain this activity. The endoglycosidase may become a useful reagent for biochemical research for releasing intact chondroitin sulfate chains from proteoglycans. It may also represent an impediment to such research when baculovirus systems are used to generate recombinant proteoglycans.     

Isolation and structure of several peptides from porcine hypothalami

Ten peptides were isolated from porcine hypothalami and structurally elucidated. These included four dipeptides Arg-Phe, Phe-Tyr, Val-Trp, and Tyr-Phe; a tripeptide Lys-Phe-Tyr; two tetrapeptides Gly-Lys-Val-Asn and Phe-Glu-His-Glu, a nonapeptide Val-Val-Tyr-Pro-Trp-Thr-Gln-Arg-Phe; a decapeptide Leu-Val-Val-Tyr-Pro-Trp-Thr-Gln-Arg-Phe and a hexadecapeptide Phe-Leu-Gly-Phe-Pro-Thr-Thr-Lys-Thr-Tyr-Phe-Pro-His-Phe-Asn-Leu. The tetrapeptide Gly-Lys-Val-Asn, the nonapeptide, the decapeptide and the hexadecapeptide most probably represent artifact fragments of alpha- and beta-chains of porcine hemoglobin. The natural or synthetic Gly-Lys-Val-Asn and Phe-Glu-His-Glu had some growth hormone releasing activity while Val-Trp, Tyr-Phe and Lys-Phe-Tyr had slight prolactin releasing activity. The biological activities of other peptides have not been determined yet.