Separation of canavanine and canaline by high performance liquid chromatography

Summary The plant amino acid canavanine and its hydrolytic product canaline were successfully separated and identified by Reverse Phase High Performance Liquid Chromatograph (RP-HPLC). This procedure was used to demonstrate the arginasemediated cleavage of canavanine to yield canaline and urea, and the subsequent formation of a Schiff's base complex between canaline and pyridoxal phosphate. Both aforementioned reactions were demonstrated by RP-HPLC.     

Aberrant, canavanyl protein formation and the ability to tolerate or utilize L-canavanine

L-Canavanine, 2-amino-4-(guanidinooxy)butyric acid, and L-arginine incorporation into de novo synthesized proteins was compared in six organisms. Utilizing L-[guanidinooxy14C]canavanine and L-[guanidino14C]arginine at substrate saturation, the canavanine to arginine incorporation ratio was determined in de novo synthesized proteins. Caryedes brasiliensis and Sternechus tuberculatus, canavanine utilizing insects; Canavalia ensiformis, a canavanine storing plant; and to a lesser extent Heliothis virescens, a canavanine resistant insect, failed to accumulate significant canavanyl proteins. By contrast, Manduca sexta, a canavanine-sensitive insect, and Glycine max, a canavanine free plant, readily incorporated canavanine into newly synthesized proteins. This study supports the contention that the incorporation of canavanine into proteins in place of arginine contributes significantly to canavanine's antimetabolic properties.     

Aberrant,canavanyl protein formation and the ability to tolerate or utilize L-canavanine

Summary L-Canavanine, 2-amino-4-(guanidinooxy)butyric acid, and L-arginine incorporation into de novo synthesized proteins was compared in six organisms. Utilizing L-[guanidinooxy¹⁴C]canavanine and L-[guanidino¹⁴C]arginine at substrate saturation, the canavanine to arginine incorporation ratio was determined in de, novo synthesized proteins.Caryedes brasiliensis andSternechus tuberculatus, canavanine utilizing insects;Canavalia ensiformis, a canavanine storing plant; and to a lesser extentHeliothis virescens, a canavanine resistant insect, failed to accumulate significant canavanyl proteins. By contrast,Manduca sexta, a canavanine-sensitive insect, andGlycine max, a canavanine free plant, readily incorporated canavanine into newly synthesized proteins. This study supports the contention that the incorporation of canavanine into proteins in place of arginine contributes significantly to canavanine's antimetabolic properties.     

Attenuation of diabetic retinopathy by the molecular chaperone-inducer amino acid analogue canavanine in streptozotocin-diabetic rats

The effect of canavanine treatment on the electroretinograms of healthy and streptozotocin-diabetic rats was studied. The characteristic amplitudes of the a-wave, W₂ and W₃ oscillatory potentials were markedly diminished in the 2-week streptozotocin-diabetic rats compared with those of the control rats. In contrast, the amplitudes of all the responses of the canavanine-pretreated streptozotocin-diabetic rats were practically indistinguishable from those of the control animals. Our results prompt further investigations for the use of amino acid analogues and other inducers of molecular chaperones in easing the chronic consequences of diabetes such as retinopathy. Received 3 June 1998; received after revision 14 August 1998; accepted 14 August 1998     

反相高效液相色谱测定庆大霉素效价方法的研究

建立了反相高效液相色谱（Reverse Phaseliquid Chromatography,RP—HPLC）测定庆大霉素效价的方法,结合管碟法所做的生物效价进行比较,其相关度R。：0．9823,RSD：1．29,证明RP—HPLC可直接测定庆大霉素的效价,从而解决了生测效价程序复杂,人为误差大的问题。RP—HPLC测定方法：采用岛津高效液相色谱SPD．IOAVP、UV．VIS、LC．IOATVP,PepMapC18Trs4．6×150mmSilicaC18（5μm300A）PhenomenexODS色谱柱,流动相MILLI-Q水：冰醋酸：甲醇（比例为25：5：70）配置0．02mol／L庚烷磺酸钠溶液,流速1．0mL／min,检测波长330nm,检测灵敏度0．020AUFS,柱温为室温,进样量20μL,在10min内可对样品液中C1、C1a、C2a、C24种物质同时进行定性定量测定。     

Transesterification of juvenile hormone occurs in vivo in locust when injected in alcoholic solvents

Catabolism of juvenile hormone was studied in vivo in the African locust by injection of the labelled natural enantiomer (10R) (12-³H) JH-III. Due to the poor solubility of JH in aqueous solution, it was injected in a water-miscible solvent. Ethanol was chosen for its apparently low toxicity towards the locust. In these experimental conditions, reverse phase liquid chromatography procedure (RP-HPLC) coupled with on line radiodetection, revealed an apolar metabolite of JH-III. This compound was found both in adult females and in fifth stadium larvae. We demonstrate that this metabolite resulted from substitution of the carboxyl methyl group of JH-III by some hydrophobic moiety. This compound co-migrates in our RP-HPLC system with the JH analog epoxy-ethyl farnesoate (JH-III ethyl ester) obtained by KCN-catalysed transesterification of JH-III in ethanol. Both JH-III ethyl ester of chemical origin and biological compounds extracted from locusts give the same spectra when analyzed by gas chromatography-mass spectroscopy (GC-MS). Transesterification of JH-III was not observed with locust tissues incubated in vitro but occurred in vivo even if JH was injected in other alcoholic solvents such as propanol. Our data suggest that transesterification of JH-III occurred in vivo and underline the role of injecting solvent in in vivo studies.     

Cryo-bioorganic chemistry: freezing effect on stereoselection of l- and dl-leucine cooligomerization in aqueous solution

The chirality of l-/dl-leucine (50–50%) cooligomerization was investigated in liquid and frozen aqueous solutions. Cooligomerization was carried out by carbonyldiimidazole activation without initiator at an ambient (+22°C) and frozen (−18°C) temperature, respectively. The separated samples obtained after different time intervals of treatment were completely hydrolyzed (HCl) and the diastereomeric l- and d-leucine derivates of Marfey's reagent (1-fluoro-2,4-dinitrophenyl-5-l-alanine amide) were then traced and evaluated by RP-HPLC analysis. After 9 days of oligomerization, the l-Leu content was slightly enhanced in the liquid (57%) and somewhat more enhanced in the frozen (64%) samples. After 17 days, however, the l-Leu content had decreased in the liquid (53%) and frozen (56%) conditions. These l-enantiomer amplifications indicate that an l-antipode is preferentially incorporated into the α-helical turn of the oligomer in the earlier stage of cooligomerization, while, later, the d-antipode is also incorporated. The role of ice in the improved stereoselection is discussed. This is the first recorded example of the effect of freezing on stereoselection. Received 27 October 2000; revised 11 December 2000; accepted 4 January 2000     

CSTX-9, a toxic peptide from the spider Cupiennius salei: amino acid sequence, disulphide bridge pattern and comparison with other spider toxins containing the cystine knot structure

CSTX-9 (68 residues, 7530.9 Da) is one of the most abundant toxic polypeptides in the venom of the wandering spider Cupiennius salei. The amino acid sequence was determined by Edman degradation using reduced and alkylated CSTX-9 and peptides generated by cleavages with endoproteinase Asp-N and trypsin, respectively. Sequence comparison with CSTX-1, the most abundant and the most toxic polypeptide in the crude spider venom, revealed a high degree of similarity (53% identity). By means of limited proteolysis with immobilised trypsin and RP-HPLC, the cystine-containing peptides of CSTX-9 were isolated and the disulphide bridges were assigned by amino acid analysis, Edman degradation and nanospray tandem mass spectrometry. The four disulphide bonds present in CSTX-9 are arranged in the following pattern: 1-4, 2-5, 3-8 and 6-7 (Cys₆-Cys₂₁, Cys₁₃-Cys₃₀, Cys₂₀-Cys₄₈, Cys₃₂-Cys₄₆). Sequence comparison of CSTX-1 with CSTX-9 clearly indicates the same disulphide bridge pattern, which is also found in other spider polypeptide toxins, e.g. agatoxins (ω-AGA-IVA, ω-AGA-IVB, μ-AGA-I and μ-AGA-VI) from Agelenopsis aperta, SNX-325 from Segestria florentina and curtatoxins (CT-I, CT-II and CT-III) from Hololena curta. CSTX-1/CSTX-9 belong to the family of ion channel toxins containing the inhibitor cystine knot structural motif. CSTX-9, lacking the lysine-rich C-terminal tail of CSTX-1, exhibits a ninefold lower toxicity to Drosophila melanogaster than CSTX-1. This is in accordance with previous observations of CSTX-2a and CSTX-2b, two truncated forms of CSTX-1 which, like CSTX-9, also lack the C-terminal lysine-rich tail. Received 23 July 2001; accepted 31 July 2001     

Heterogeneous responses of canine basilar and middle cerebral arteries to serotonin at normal and high CO2 tension.

The responses of basilar arteries (BAs) to serotonin were attenuated by high PCO2 (86 +/- 1 mm Hg) and the pH matched acidotic solution (PCO2 37 +/- 1 mm Hg), whereas the responses of middle cerebral arteries (MCAs) were not. High PCO2 decreased the basal tone of both arteries, and the changes in basal tone due to high PCO2 were not influenced by 3 x 10(-7) M imipramine, 10(-5) M pargyline or 10(-4) M aspirin. The responses of BAs to serotonin were attenuated by high PCO2 in the presence of imipramine, pargyline and aspirin. The responses of MCAs to serotonin were not influenced by high PCO2 in the presence of pargyline and aspirin, but attenuated by high PCO2 in the presence of imipramine.     

枸杞多糖对人肺腺癌 A549细胞增殖和凋亡的影响

目的探讨枸杞多糖(LBP)对体外培养的人肺腺癌细胞 A549的增殖抑制作用及其可能的作用机制.方法用不同浓度的LBP处理 A549细胞,MTT法检测24、48、72h时间点 LBP对 A549细胞的生长抑制率,实验设为对照组和实验组(1/2IC50作用48小时),MTT法绘制生长曲线、细胞计数计算倍增时间、流式细胞仪检测凋亡率及其细胞周期、RT PCR检测 SurvivinmRNA的变化、Westernblot检测 CyclinB1蛋白的变化,transwell体外侵袭实验观察药物对细胞体外侵袭的影响.结果 MTT显示不同浓度的LBP均能明显抑制 A549细胞的增殖且成剂量 效应关系,实验组细胞的倍增时间、凋亡率与对照组相比,均有统计学意义(P<0.05);LBP使细胞阻滞在 G2期,SurvivinmRNA表达和 CyclinB1蛋白的表达均降低,与对照组相比差异有显著性(P<0.05).结论 LBP可抑制 A549细胞的增殖,其机理可能与 LBP使 SurvivinmRNA表达下降引起细胞凋亡及 CyclinB1蛋白的表达降低造成细胞周期阻滞及抑制细胞的侵袭能力有关     

Correlation between the susceptibility of E. coli to phagocytosis and their ability to invade HeLa cells in vitro.

The susceptibility of several strains of E. coli to phagocytic killing by polymorphonuclear lencocytes and the ability of the same strains to invade HeLa cells were studied. It was found that only the strains resistant to killing by leucocytes were able to penetrate and multiply within HeLa cells.     

Phylogenetic relationship of organisms obtained by ribosomal protein comparison

The evolutionary relationships of ribosomal proteins from eubacteria, archaea, eukaryotes, chloroplasts and mitochondria were examined by their degree of conservation, their structural and functional properties and by the occurrence of conserved structural elements. The structural domains formed by the different protein families were studied. The occurrence of monophyletic groups was investigated for each protein family within the archaea. Phylogenetic trees were constructed between these organisms and together with the homologous sequences of the other phylogenetic domains. All organisms belonging to the archaea clearly formed a monophyletic group. The conserved sequence motifs were checked for the potential to form similar secondary structural elements. Received 24 October 1996; accepted 30 October 1996     

Correlation between the susceptibility of E. coli to phagocytosis and their ability to invade HeLa cells in vitro

Summary The susceptibility of several strains of E. coli to phagocytic killing by polymorphonuclear leucocytes and the ability of the same strains to invade HeLa cells were studied. It was found that only the strains resistant to killing by leucocytes were able to penetrate and multiply within HeLa cells.Acknowledgments. We thank Dr Bianca Pani who supplied us with HeLa cells. Supported by grant No. 75.00695.04 from the National Research Council of Italy (CNR).     

Neuroma formation and abnormal afferent nerve discharges after partial beak amputation (beak trimming) in poultry

Following partial amputation of the beak recordings were taken of the electrical activity from single afferent fibers of the intramandibular nerve. A total of 192 single afferent fiber units were isolated of which 47 were classified as nociceptors, with an abnormal pattern of discharge, and 89 were abnormal spontaneously active units. Following amputation neuromas were developing by 15 days after surgery and they were well formed by 20 to 30 days. The presence of neuromas together with abnormal spontaneous activity originating from them raise serious welfare questions concerning beak trimming.     

Studies on chemically induced tumors in rats: I. Heterogeneity of tumor cells and establishment of syngeneic, tumor-specific cytotoxic T cell clones

Sarcoma P1 was induced in DA rats by DMBA. Anti-P1 antibodies were produced in DA rats, purified via fixed tumor cells and used to induce anti-idiotypic antibodies in syngeneic rats. The anti-idiotypic antibodies were used to generate cytotoxic, P1 specific DA T cells in vitro. These cytotoxic T cells and P1 tumor cells were cloned by limiting dilution. Using the DA anti-P1 specific cytotoxic T cell clones, we were able to characterize 2 types of P1 tumor cell clones, namely those which were susceptible and those which were resistant to the P1 specific cytotoxic T cells. Cytotoxic T cell injected i.v. into syngeneic DA rats could not prevent the development of lethal P1 tumors.     

The effect of postnatal undernourishment on epileptiform kindling of dorsal hippocampus

Summary Rats were undernourished postnatally from birth through 20 days of age. They were subsequently tested for susceptibility to motor seizures kindled in hippocampus in adulthood. Compared to littermate control animals the postnatally undernourished rats were more susceptible to the kindling treatment. We conclude that early postnatal undernourishment has a permanent effect on susceptibility of the hippocampus to electrically-induced seizures.This work was supported by Public Health Service grant NS 13799, National Science Foundation grant GB 35532, and by Hoffman-LaRoche, Inc.     

Occurrence,distribution and nature of neuropeptide Y in the rat pancreas

Summary Significant quantities of a newly discovered peptide, neuropeptide Y, were found in the rat pancreas, where they were localized to nerves in the exocrine parenchyma and around arterial and ductal structures. Although unaffected by surgical parasympathectomy, the periarterial and periductal nerves were abolished by chemical sympathectomy, suggesting that NPY is partially costored with sympathetic transmitters in nerve fibers.     

Detection, by direct lymphocyte immunocytotoxicity, of antigens in cells transformed by Rous virus]

The hamster cells transformed by the Rous Sarcoma Virus (V.S.R.) evidenced surface antigenic alterations that were detected by a method of cellular mediated immunocytotoxicity. Immune hamster lymphocytes were added to tritiated proline prelabeled target cells. These lymphocytes were able to lyse specifically the V.S.R. transformed cells.     

Monolayer culture of adult mouse hepatic cells secreting albumin and alpha fetoprotein]

Adult mouse liver cells obtained by enzymatic dispersion were maintained in primary cultures for up to 4 weeks. They retained some of the typical morphological and ultrastructural characteristics of hepatocytes. After 2 days of culture, structures similar to bile canaliculi were found and after 6 days clusters of small proliferating cells were noticed in the gaps of the monolayer formed by large well-spread hepatocytes. Almost all 3-day cultures began to secrete alphafetoprotein for about 2 weeks, whereas albumin was secreted throughout the period of culture.     

Effect of phospholipids on purified lipoamidase

Summary Phospholipids were added to purified lipoamidase from porcine brain microsomal membranes, and changes in lipoamidase activity were examined. Approximately twofold activation of lipoamidase activity occurred upon the addition of phosphatidylethanolamine. On the other hand, phosphosphatidylserine, cardiolipin, and phosphatidic acid reduced the enzyme activity by approximately 80%. This pattern of the activation of lipoamidase by phosphatidylethanolamine and its inhibition by phosphatidylserine is similar to the pattern for adenylate cyclase, and contrasts with the pattern for ATPase.