Studies on peptides

The heptacosapeptide amide corresponding to the entire amino acid sequence of chicken gastrin-releasing peptide (cGRP) was synthesized similarly to the synthesis of porcine GRP by assembling six peptide fragments followed by depro-tection with 1 M trifluoromethanesulfonic acid-thioanisole in TFA. A new carboxyl-activating reagent, thiazolidine-2-thione, was preferentially adopted for preparation of necessary fragments. The synthetic cGRP, purified by ion-exchange chromatography, followed by partition chromatography, was active as the synthetic porcine GRP, when plasma immunoreactive gastrin level was examined in rats. No obvious difference was observed when synthetic and natural cGRP preparations were compared by HPLC, immunochemical property and biological activity in dogs.     

STUDIES ON PEPTIDES

The octacosapeptide amide corresponding to the entire amino acid sequence of chicken VIP was synthesized in a conventional manner, using a new arginine derivative, N^G -mesitylene-2-sulfonylarginine, Arg(Mts). Treatment of a fragment, Z(OMe)-Thr-Asp-Asn-Tvr-NHNH₂ with methanesulfonic acid or HBr was found to give a product with a low recovery of Asp, after aminopeptidase digestion. Ring closure of the Asp-Asn unit seemed to be responsible for this phenomenon. Deprotection with HF or TFA exhibited definitely less such a tendency. In the final step of the synthesis, all protecting groups, including the Mts group, were removed by HF in the presence of m-cresol and the deprotected peptide was purified by ion-exchange chromatography on CM-cellulose followed by isoelectric focusing in Ampholine pH 9–11. Synthetic peptide exhibited the identical Rf value with that of natural chicken VIP and was active as the natural peptide.     

STUDIES ON PEPTIDES

The tetradecapeptide amide corresponding to the entire amino acid sequence of a wasp venom (polistes mastoparan), isolated from Polistes jadwagae, was synthesized using the trifluoroacetic acid-thioanisole deprotecting procedure.     

New C-protective group for peptide synthesis

To investigate the possibility of using the 2-(1-adamantyl)-2-propyl (Adp) ester group for carboxyl protection during peptide synthesis, the tetrapeptide Boc-Phe-Arg(NO₂)-Phe-Pro-OAdp (IV) was prepared by the carbodiimide method. In this synthesis the Z group was removed by transfer hydrogenation and the Nps group by treatment with 2-thiopyridone. The Adp group was then cleaved with 3 % TFA/DCM, yielding Boc-Phe-Arg(NO₂)-Phe-Pro-OH. For subsequent kinetics studies the decapeptide Boc-Leu— Z-Orn-Arg(NO₂)-Pro-Pro-Gly-Phe-Ser(Bzl)-Pro-Pro-OAdp (VIII) was synthesized by a 9 + 1 scheme. This peptide was also selectively deblocked. Comparing reaction abilities of Z-Pro-OAdp and Z-Pro-OBu^t, it was demonstrated that the Adp ester is cleaved by 3% TFA/DCM 230 times faster than the Bu^t ester. For peptides IV and VIII the ratios between the rates of competitive elimination of the Adp and Boc groups by 3% TFA/DCM are 19 and 64, respectively.     

疟原虫组织期裂殖体杀灭剂的研究:2-取代苯氧基和4-甲基伯氨喹以及喹噁啉类衍生物的合成

合成了2-取代苯氧基伯氨喹及其类似物和4-甲基伯氨喹及其同系物以及7-甲氧基-5-(1-氨基烃基)氨基喹噁啉类化合物。其中以4-甲基伯氨喹的同分异构体25及其同系物24的作用最强。感染约氏疟原虫(Plasmodium yoelii)子孢子的小鼠,用10mg/kg单剂灌胃,全部小鼠的原虫血症均呈阴性,疗效高于对照组伯氨喹。     

SOLID PHASE SYNTHESIS OF THE PROTECTED 27 – 42 HEXADECAPEPTIDE OF THE HEAVY CHAIN FROM MYELOMA IMMUNOGLOBULIN M603 Elimination of Side Reactions Associated with Glycyl-2-oxypropionyl-resin

A fully protected 27–42 hexadecapeptide of the variable region of myeloma immunoglobulin M603 was synthesized on a 2-bromopropionyl-resin by the solid phase method. Side reactions due to cyclization of glycyl-2-oxypropionylresin were studied under different reaction conditions. The loss of peptide chains at the dipeptide and tripeptide stages due to diketopiperazine formation was also examined. These side reactions were circumvented by using a combination of fragment and stepwise coupling methods. The synthesized protected peptide was removed from the resin in 85% yield by photolysis, and purified by crystallization and by chromatography on a Sephadex LH-60 column.     

帕瑞昔布联合右颈交感神经干离断对心肌梗死大鼠心室重构的影响

目的探索帕瑞昔布联合右颈交感神经干离断(TCST)对大鼠心肌梗死后左室重构的影响。方法成年雄性SD大鼠40只,采用结扎左冠状动脉前降支方法制备急性心肌梗死模型,制模成功后,采用右TCST治疗和(或)给予帕瑞昔布8 mg·kg~(-1)·d~(-1)治疗。于建模处置4wk后行右颈总动脉插管测定左心室收缩压(LVSP)、左心室舒张末期压(LVEDP)、左心室收缩压上升最大速率(+dp/dt_(max))和左心室收缩压下降最大速率(-dp/dt_(max));放射免疫法检测血浆血栓素A_2(TXA_2)和前列腺素I_2(PGI_2)的浓度;体视学三维形态定量分析左室重构,测定左室心肌细胞体积密度Vv、心肌组织总体积Vt、心肌细胞总体积Vc。结果心肌梗死制模后各组LVSP、±dp/dt_(max)明显降低,而LVEDP升高(均P<0.01),PGI_2、PGI_2/TXA_2显著降低、TXA_2增加(P<0.01),Vv、Vt、Vc均明显升高(P<0.01);右TCST或帕瑞昔布治疗后LVSP及±dp/dt_(max)升高(P<0.01或P<0.05)而LVEDP降低,PGI_2、PGI_2/TXA_2显著增加而TXA_2降低,Vv、Vt、Vc均降低(P<0.01);右TCST联用帕瑞昔布后心功能改善更显著,Vv、Vt、Vc降低更明显(P<0.05)。结论帕瑞昔布与右TCST单用及合用均能显著提高心肌舒缩功能,调节PGI_2与TXA_2的相对平衡,延缓急性心肌梗死大鼠的左室重构过程,但两者合用效果更佳。     

Chlorpyrifos and its metabolites alter gene expression at non-cytotoxic concentrations in D3 mouse embryonic stem cells under in vitro differentiation: Considerations for embryotoxic risk assessment

The effects of organophosphate insecticide chlorpyrifos (CPF) on development are currently under discussion. CPF and its metabolites, chlorpyrifos-oxon (CPO) and 3,5,6-trichloro-2-pyridinol (TClP), were more cytotoxic for D3 mouse embryonic stem cells than for differentiated fibroblasts 3T3 cells. Exposure to 10 μM CPF and TClP and 100 μM CPO for 12 h significantly altered the in vitro expression of biomarkers of differentiation in D3 cells. Similarly, exposure to 20 μM CPF and 25 μM CPO and TClP for 3 days also altered the expression of the biomarkers in the same model. These exposures caused no significant reduction in D3 viability with mild inhibition of acetylcholinesterase and neuropathy target esterase by CPF and severe inhibition by CPO. We conclude that certain in vivo exposure scenarios are possible, which cause inhibition of acetylcholinesterase but without clinical symptoms that reach high enough systemic CPF concentrations able to alter the expression of genes involved in cellular differentiation with potentially hazard effects on development. Conversely, the risk for embryotoxicity by CPO and TClP was very low because the required exposure would induce severe cholinergic syndrome.     

MAHARISHI AMRIT KALASH REJUVENATES AGEING CENTRAL NERVOUS SYSTEM'S ANTIOXIDANT DEFENCE SYSTEM: AN IN VIVO STUDY

The oxygen-free radical involvement in various deteriorative processes and in ageing is unquestionably established. In the present study age-related changes in antioxidant enzyme activity in the different regions of CNS of 10-month and 32-month-old guinea pigs were studied. Maharishi Amrit Kalash has shown promise in inhibiting the in vitro and in vivo lipid peroxidation. Therefore in the present study the effect of MAK on the activity of antioxidant enzymes was checked. Our results indicate that the activity of superoxide dismutase and glutathione peroxidase, was found to be reduced (P<0.05) in all the regions of CNS studied, The activities of catalase declined significantly only in the cerebral cortex, hypothalamus and the cerebellum. Whereas glutathione reductase activity declined in the cerebral cortex and hypothalamus. It is concluded that the age-related decline in the activities of antioxidant enzymes is region-specific as well as enzyme-specific. The endogenous lipid peroxide was found to be increased significantly (P<0.05) in the 32-month-old animals. Whereas the lipid peroxidation after incubating the tissue homogenate in the air was found to be decreased (P<0.05) in the older animals. The results indicate that the accumulation of lipid peroxides take place with age but the susceptibility of lipid peroxidation decreases in the older animals. The treatment of MAK 500 mg kg⁻¹body wt. for 2 months could augment the activities of antioxidant enzymes (P<0.05). The effect of MAK was more pronounced in older than younger animals. It is concluded that the MAK can be used in compensating the decline in the activities of antioxidant enzymes in CNS and thereby it reduces the risks of lipid peroxidation.pc 1999 Academic Press@p$hr     

The intra- and inter-laboratory reproducibility and predictivity of the KeratinoSens assay to predict skin sensitizers in vitro: Results of a ring-study in five laboratories

Due to regulatory constraints and ethical considerations, research on alternatives to animal testing to predict the skin sensitization potential of novel chemicals has gained a high priority. Accordingly, different in vitro, in silico and in chemico approaches have been described in the scientific literature to achieve this goal. To replace regulatory approved animal tests, these alternatives need to be transferable to other labs, their within and between laboratory reproducibility must be assured, and their predictivity should be high. The KeratinoSens assay is a cell-based reporter gene assay to screen substances with a full dose-response assessment. It is based on a stable transgenic keratinocyte cell line. The induction of a luciferase gene under the control of the antioxidant response element (ARE) derived from the human AKR1C2 gene is determined. Here we report on the results of a ring-study with five laboratories performing the KeratinoSens assay on a set of 28 test substances. The assay was found to be easily transferable to all laboratories. Overall both the qualitative (sensitizer/non-sensitizer categorization) and the quantitative (concentration for significant gene induction) results were reproducible between laboratories. A detailed analysis of the transferability, the within- and between laboratory reproducibility and the predictivity is presented.     

Improving the ex vivo stability of drug ester compounds in rat and dog serum: Inhibition of the specific esterases and implications on their identity

In drug development, it has been noticed that some drug compounds, especially esters, are unstable in serum samples ex vivo. This can lead to a substantial underestimation of the actual drug concentration.     

Pharmaceutical Amorphous Nanoparticles

There has been a tremendous revolution in the field of nanotechnology, resulting in the advent of novel drug delivery systems known as nanomedicines for diagnosis and therapy. One of the applications is nanoparticulate drug delivery systems which are used to improve the solubility and oral bioavailability of poorly soluble compounds. This is particularly important because most of the molecules emerging from the drug discovery pipeline in recent years have problems associated with solubility and bioavailability. There has been considerable focus on nanocrystalline materials; however, amorphous nanoparticles have the advantage of synergistic mechanisms of enhancing dissolution rates (due to their nanosize range and amorphous nature) as well as increasing supersaturation levels (due to their amorphous nature). An example of this technology is Nanomorph^TM, developed by Soliqus/Abbott, wherein the nanosize drug particles are precipitated in an amorphous form in order to enhance the dissolution rate. This along with other simple and easily scalable manufacturing techniques for amorphous nanoparticles is described. In addition, the mechanisms of formation of amorphous nanoparticles and several physicochemical properties associated with amorphous nanoparticles are critically reviewed.     

The glycosylated flavonoids vitexin,isovitexin, and quercetrin isolated from Serjania erecta Radlk (Sapindaceae) leaves protect PC12 cells against amyloid-β25-35 peptide-induced toxicity

The Aβ peptide-mediated toxicity participates in the neuronal death that occurs in Alzheimer's disease. The present study aims to isolate the major compounds of Serjania erecta Radlk leaves and assess whether these compounds protect PC12 cells from Aβ_25-35 peptide-induced toxicity. We isolated three flavonoid glycosides with high purity: quercetrin, vitexin, and isovitexin. The Aβ_25-35 peptide alone decreased the PC12 cell viability in a concentration-dependent manner, as evaluated by the 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT) assay. We selected the Aβ_25-35 peptide concentration of 50 µM for the experiments. Treatment of PC12 cells with the flavonoids before exposure to the Aβ_25-35 peptide increased cell viability, i.e., these compounds protected the cells against Aβ_25-35 peptide-induced toxicity. Vitexin promoted higher protection levels than quercetrin and isovitexin, and reduced the lactate dehydrogenase release and NO production in Aβ_25-35 peptide-treated PC12 cells. Therefore, the glycosylated flavonoids that exist in S. erecta leaves, especially vitexin, protect PC12 cells from Aβ_25-35 peptide-induced toxicity.     

Recent insights of the role and signalling pathways of interleukin-34 in liver diseases

Liver disease is a global health problem and is a primary cause of mortality and morbidity worldwide. Specifically, it accounts for approximately two million deaths per year worldwide. The common causes of mortality are the complications of liver cirrhosis, viral hepatitis and hepatocellular carcinoma (HCC). The mechanism of immune response and infiltration of cellular immunity is essential for promoting hepatic inflammatory, especially when the liver is abundant with lymphocytes and phagocytic cells. The injured and immunity cells secret different types of interleukins (cytokines), which can directly or indirectly amplify or inhibit liver inflammation. Many types of cells can produce interleukin-34 (IL-34) that induces the release of multiple inflammatory factors in patients via interaction with various cytokines. This phenomenon leads to the enlargement of the inflammatory response to liver diseases and induces liver fibrosis. This review highlights the proposed roles of IL-34 in liver diseases and discusses the recent findings of IL-34 that support its emerging role in HCC. Specifically, the facilitating effects of these new insights on the rational development of IL-34 for targeted therapies in the future are explored.     

Understanding the physiological functions of the host xenobiotic-sensing nuclear receptors PXR and CAR on the gut microbiome using genetically modified mice

Pharmacological activation of the xenobiotic-sensing nuclear receptors pregnane X receptor (PXR) and constitutive androstane receptor (CAR) is well-known to increase drug metabolism and reduce inflammation. Little is known regarding their physiological functions on the gut microbiome. In this study, we discovered bivalent hormetic functions of PXR/CAR modulating the richness of the gut microbiome using genetically engineered mice. The absence of PXR or CAR increased microbial richness, and absence of both receptors synergistically increased microbial richness. PXR and CAR deficiency increased the pro-inflammatory bacteria Helicobacteraceae and Helicobacter. Deficiency in both PXR and CAR increased the relative abundance of Lactobacillus, which has bile salt hydrolase activity, corresponding to decreased primary taurine-conjugated bile acids (BAs) in feces, which may lead to higher internal burden of taurine and unconjugated BAs, both of which are linked to inflammation, oxidative stress, and cytotoxicity. The basal effect of PXR/CAR on the gut microbiome was distinct from pharmacological and toxicological activation of these receptors. Common PXR/CAR-targeted bacteria were identified, the majority of which were suppressed by these receptors. hPXR-TG mice had a distinct microbial profile as compared to wild-type mice. This study is the first to unveil the basal functions of PXR and CAR on the gut microbiome.     

Novel molecular events associated with altered steroidogenesis induced by exposure to atrazine in the intact and castrate male rat

Toxicology is increasingly focused on molecular events comprising adverse outcome pathways. Atrazine activates the hypothalamic-pituitary adrenal axis, but relationships to gonadal alterations are unknown. We characterized hormone profiles and adrenal (intact and castrate) and testis (intact) proteomes in rats after 3 days of exposure. The adrenal accounted for most of the serum progesterone and all of the corticosterone increases in intact and castrated males. Serum luteinizing hormone, androstenedione, and testosterone in intact males shared a non-monotonic response suggesting transition from an acute stimulatory to a latent inhibitory response to exposure. Eight adrenal proteins were significantly altered with dose. There were unique proteomic changes between the adrenals of intact and castrated males. Six testis proteins in intact males had non-monotonic responses that significantly correlated with serum testosterone. Different dose–response curves for steroids and proteins in the adrenal and testis reveal novel adverse outcome pathways in intact and castrated male rats.