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Cordyceps is an expensive traditional Chinese medicine, which has anti-tumor activity and significant effects on the immune system. In Southeast Asia, Cordyceps is commonly sold in capsule form as a health food product. Most of these products are derived from cultured Cordyceps mycelia. Because of the price difference, some manufacturers claim their products are from natural Cordyceps. In order to distinguish among various types of Cordyceps in the market, the profiles of water-soluble constituents derived from different sources of Cordyceps were determined by capillary electrophoresis (CE). Both natural and cultured Cordyceps showed three peak clusters migrated at 5-7, 9-11 and 12-13 min, and the height and resolution of these peak clusters were rather distinct. Peak cluster at 9-11 min was identified as adenosine, guanosine and uridine, and shared a similarity between natural and cultured products. In contrast, the peak cluster at 5-7 min was characteristic of natural Cordyceps, regardless of hosts and sources. By using the peak characteristics of CE profiles of different Cordyceps samples, hierarchical clustering analysis was performed. The result shows that those samples of natural Cordyceps were grouped together distinct from the cultured and commercial products. Thus, the CE profiles could serve as fingerprints for the quality control of Cordyceps.

Objective: To retrospectively analyze the experiences and results of the treatment on 62 patients with primary varicose of lower extremities with endovenous laser., Methods: All patients were treated with endovenous laser. The laser treatment could begin when the fiber withdraw with 1 cm/2 s. The laser power was 10 - 12 w with the laser pulse duration and the interval 1 second respectively., Results: The duration of follow-up varied from 2 months to 8 months. After endovenous treatment, the varicose veins and edema disappeared in all cases. The itching and uncomfortable feeling was dissipated. No morphine-like analgesic has been used and no serious complications occurred., Conclusion: Endovenous laser treatment of primary varicose of lower extremities is a safe and effective technique.

Hypoxanthine-xanthine oxidase (HX-XO) system is a classical system that can generate superoxide anions. The inductive effect of the HX-XO system for lambda prophage has been investigated in this study. The results showed that the system can induce lambda prophage from lysogenic state to lytic growth. The inductive effect was directly proportional to the concentration of HX and XO and inversely related to the time of preliminary incubation of HX with XO. The cell density of the lysogenic bacteria also greatly affected the inductive effect. The maximal PFU number of 2.9 x 10(4) PFU/ml was recorded at 0.86 mM HX, 1.6 x 10(-2) U/ml XO, and a cell density of 10(8) cells/ml. The inductive effect of the HX-XO system was inhibited in the suspensions by glutathione, superoxide dismutase, and catalase. The results provide evidence that free radicals are the primary factors in the induction of lambda prophage in lysogenic bacteria.

Cordyceps sinensis, a well-known traditional Chinese medicine, possesses activities in anti-tumour, anti-oxidation and stimulating the immune system; however, the identity of active component(s) is not determined. By using anti-oxidation activity-guided fractionation, a polysaccharide of molecular weight approximately 210 kDa was isolated from cultured Cordyceps mycelia by ion-exchange and sizing chromatography. The isolated polysaccharide, having strong anti-oxidation activity, contains glucose, mannose and galactose in a ratio of 1 : 0.6 : 0.75. The pre-treatment of isolated polysaccharide on the cultured rat pheochromocytoma PC12 cells shows strong protective effect against hydrogen peroxide (H(2)O(2))-induced insult. Treatment of the cells with the isolated polysaccharide at 100 microg/ml prior to H(2)O(2) exposure significantly elevated the survival of PC12 cells in culture by over 60%. In parallel, the H(2)O(2)-induced production of malondialdehyde in cultured cells was markedly reduced by the polysaccharide treatment. Moreover, the pre-treatment of the isolated polysaccharide significantly attenuated the changes of glutathione peroxidase and superoxide dismutase activities in H(2)O(2)-treated cells in a dose-dependent manner. This is the first report in identifying a polysaccharide from Cordyceps, which protects against the free radical-induced neuronal cell toxicity.

The cDNA of guinea pig (Cavia porcellus) growth hormone receptor (gpGHR) was cloned using RT-PCR in our laboratory. By sequence alignment, substitutions of amino acids conserved in other mammalian GHRs were found. For example, histidine-168 and tyrosine-332 equivalent to positions 170 and 333 in other mammalian GHRs, which were considered to be necessary for the dimerization of GHR and the specific GH-stimulated functions respectively, were replaced by tyrosine and serine in gpGHR. Here, we report the functional expression of gpGHR and its mutants, gpGHRY168H and gpGHRS332Y, in COS-7 cells and/or Chinese hamster ovary (CHO) cells. It was shown that the COS-7 cells transfected with pcDNA3-gpGHR possessed high affinity to bovine GH [K(a) = 1.3 x10(9) (mol/L)(-1)] and a protein band with molecular weight around 92 kD was detected by anti-mouse GHR monoclonal antibody (mAb263). When CHO cells were transfected with the expression vectors, pcDNA3-gpGHR, pcDNA3-gpGHRY168H and pcDNA3-gpGHRS332Y, the gpGHR and its mutants were expressed and the ligand binding, phosphorylation of JAK2, protein synthesis, and lipogenesis were studied. It was found that the mutation of serine to tyrosine at position 332 greatly increased the GH-stimulated protein synthesis and the phosphorylation of JAK2, while the mutation of tyrosine to histidine at position 168 increased the protein synthesis and decreased the phosphorylation of JAK2 only weakly. However, both mutations decreased the GH-stimulated lipogenesis. Thus, our study provides the experimental evidence that gpGHR may mediate the metabolic actions of GH and the substitutions of some conserved amino acids in gpGHR result in the changes of post-binding signaling.

A cDNA encoding P2Y(1) receptor was isolated by cross-hybridization with chicken homolog. The deduced amino acid sequence of P2Y(1) receptor with 361 amino residues is 80-85% identical to human, rodent and avian homologs. When the cDNA was expressed in mammalian cells, the activation of P2Y(1) receptor by adenine nucleotides stimulated the accumulation of inositol phosphate, and adenosine 3',5'-bismonophosphate (A3P5P) or other antagonists blocked its action; these pharmacological properties showed resemblance of P2Y(1) receptor family in higher vertebrate. A transcript encoding P2Y(1) receptor at approximately 3.2 kb was revealed in the brain, spinal cord and muscle of adult, and it is strongly expressed in developing brain, spinal cord and myotomal muscles of the embryos by hybridization. P2Y(1) receptor was shown to be restricted to the neuromuscular junctions and co-localized with AChRs in adult muscle. These results support the notion that ATP and its P2Y(1) receptor subtype are effectors in organizing the post-synaptic apparatus.

One of the most important findings in structure-function studies on glucagon by means of chemical synthesis is the discovery that [Lys(17,18),Glu(21)]-glucagon had higher biological activity than native glucagon. This mutant of glucagon was called superactive glucagon (SA-glucagon). In the present work, the possibility to obtain SA-glucagon by means of genetic engineering was studied. The gene of SA-glucagon (SAG) was obtained by PCR from a constructed recombinant glucagon plasmid, pAGluT. A secretory expression vector harboring SAG, pBLSG7, containing P(L) promoter and the gene of phoA signal peptide was constructed. In expression studies after transformation of pBLSG7 into E. coli BL21, it was found that the expression yield of SA-glucagon reached 3.65 mg/L(A(600)=1), about 19.5% of total proteins in the culture medium under shaken flask conditions. In addition, the influence of induction temperature and of E. coli strain on the expression yield of SA-glucagon was also studied.

The expression of acetylcholinesterase (AChE) is markedly increased during myogenic differentiation of C2C12 myoblasts to myotubes; the expression is mediated by intrinsic factor(s) during muscle differentiation. In order to analyze the molecular mechanisms regulating AChE expression during myogenic differentiation, a approximately 2.2-kb human AChE promoter tagged with a luciferase reporter gene, namely pAChE-Luc, was stably transfected into C2C12 cells. The profile of promoter-driven luciferase activity during myogenic differentiation of C2C12 myotubes was found to be similar to that of endogenous expression of AChE catalytic subunit. The increase of AChE expression was reciprocally regulated by a cAMP-dependent signaling pathway. The level of intracellular cAMP, the activity of cAMP-dependent protein kinase, the phosphorylation of cAMP-responsive element binding protein and the activity of cAMP- responsive element (CRE) were down-regulated during the myotube formation. Mutating the CRE site of human AChE promoter altered the original myogenic profile of the promoter activity and its suppressive response to cAMP. In addition, the suppressive effect of the CRE site is dependent on its location on the promoter. Therefore, our results suggest that a cAMP-dependent signaling pathway serves as a suppressive element in regulating the expression of AChE during early myogenesis.

Objective: To explore the changes of plasma endothelin (ET) and nitric oxide (NO) levels in patients with acute pancreatitis., Methods: The level of plasma ET was measured by radioactive-immunoassay, and NO by spectrophotometry., Results: The levels of ET, NO and the ET/NO ratio in patients with severe acute pancreatitis (SAP) within 24 hours in hospital were all significantly higher than those in other groups of patients [(176+/-8) pg/ml, (97+/-11) micromol/L, and 1.83+/-0.12, P<0.01]. Compared to healthy controls (N), the levels of ET and NO in patients without pancreatitis acute abdomen (NAP) and patients with mild acute pancreatitis (MAP) increased significantly (P<0.01). After appropriate treatment, the levels of ET and NO in the MAP group were lower (P<0.01). Compared with those before treatment, the levels of ET and NO in the SAP group on the 3rd and 7th day in hospital dropped significantly (P<0.01). The ET/NO ratio on the 7th day was also lower than that on admission (P<0.01)., Conclusions: The malfunction of endothelial cells and the increased ET/NO ratio may be related to the mechanism of pancreatic microcirculatory disturbance in patients with SAP; early dynamic determination of these parameters may help predict the prognosis of SAP.

Aim: To analyze the related substances in ampicillin and amoxicillin and determine the related substances in products., Methods: By using HPLC, electrospray ionzation and selective ion monitoring mass spectrum method, accelerated samples were analyzed for determination the related substances in ampicillin and amoxicillin., Results: Thirteen related substances in ampicillin and 9 related substances in amoxicillin were determined., Conclusion: The results are useful to quality control and stability study for ampicillin and amoxicillin.

Aim: To explore the importance of B9 and B10 amino acid residues in the insulin molecule., Methods: The [B9Glu, B10Asp] insulin (E,D-insulin) receptor binding activity, glucose upta ke activity, and lipogenesis activity were measured in isolated adipocytes. The translocation of glucose transporter 4 (Glut4) and the phosphorylation of insulin receptor (IR) stimulated by E, D-insulin were determined by Western blotting., Results: Compared with native insulin, the receptor binding activity of E,D-insulin was 31 %; the stimulating activity of E,D-insulin in glucose transport and lipogenesis were 45 % and 40 % respectively; the stimulations of Glut4 translocation and insulin receptor autophosphorylation of E,D-insulin were about 58 % and 46 % respectively., Conclusion: B9-serine residue is crucial for insulin actions in glucose metabolism.

The methylotrophic yeast Pichia pastoris, which proved successful in producing many heterologous proteins, was used to express an insulin precursor. A transformant with a high copy number of the gene integrated into the chromosome was obtained by the dot-blotting method. In high-density fermentation using a simple culture medium composed mainly of salt and methanol, the expression level reached 1.5 g/L. A simple two-step method was established to purify the expression product from the culture medium with an overall recovery of about 80%. After tryptic transpeptidation, human insulin with full receptor binding capacity and biological activity was obtained. In the presence of zinc, the recombinant human insulin could be crystallized in the rhombohedral form., (Copyright 2001 John Wiley & Sons, Inc.)

Cyclic adenosine 3',5'-monophosphate (cAMP)-dependent signalling pathway has been proposed to regulate acetylcholinesterase (AChE) expression in chick muscle; however, its role in mammalian enzyme is not known. We provide several lines of evidence to suggest that the cAMP-mediated AChE expression in myotube is oppositely regulated between avian and mammalian enzymes. Human AChE promoter was tagged with luciferase, namely Hp-Luc, which was transfected into cultured chick myotubes. Application of cAMP and forskolin induced the expression of chick AChE but reduced human AChE promoter-driven luciferase activity. Transfection of cDNAs encoding active mutants of G proteins altered the intracellular cAMP level in myotubes as well as the expression of chick and human AChE. When the constitutively active forms of Activating Transcription Factor-1 (EWS/ATF-1 oncogene) were over expressed in Hp-Luc transfected myotubes, the expression of chick AChE transcript and protein increased from approximately 1.8- to approximately 2.5-fold, but the luciferase activity was decreased by over 60%. Overexpression of cAMP-responsive element binding protein (CREB) in Hp-Luc transfected myotubes markedly enhanced the cAMP-mediated AChE expression in up- and downregulated chick and human enzymes, respectively. In addition, CREB bound the CRE sequence of human AChE promoter. Mutation on the CRE site markedly enhanced the expression of the promoter-driven luciferase; however, its response to cAMP inhibition in cultured myotubes was still retained. These findings suggest that a cAMP-dependent pathway is contrasting activation and repression of AChE expression in chick and human muscles., (Copyright 2001 Academic Press.)

Calcitonin gene-related peptide (CGRP), a neuropeptide synthesized by motor neurons, stimulates the expression of AChR and AChE at the vertebrate neuromuscular junctions. The signaling mechanism of CGRP-induced AChE expression in muscle was determined both in vitro and in vivo. In cultured chick myotubes, the intracellular cAMP-dependent protein kinase (PKA) activity increased to approximately 2-fold after the application of CGRP or PKA activators; the induction was blocked by PKA inhibitors. in vivo transfection analysis on chick gastrocnemius muscles showed that the transfection of cDNA encoding constitutively active mutant Galphas increased the expression of AChE mRNA and protein to approximately 2-fold, while the constitutively active mutant Galphai cDNA transfection showed an opposite effect. The induced catalytic subunit of AChE at approximately 105 kDa was determined by specific antibody. These findings indicate that the CGRP-induced AChE expression in chick muscle is mediated by a PKA-dependent pathway.

cDNA cloning and 1 899 bp sequence of growth hormone receptor (GHR) from guinea pig liver are described. The guinea pig GHR consists of 610 amino acids. The structural feature and homology comparison of guinea pig GHR are also reported.

Divalent cations, Ca(2 ), Mg(2 ) and Mn(2 ) enhance the binding of bream growth hormone (brGH) to snakehead fish liver membrane, and their optimum concentration was found to be 8 12 mmol/L, at which Ca(2 ), Mg(2 ) and Mn(2 ) could increase, respectively, the specific binding to 230%, 180%, and 200%, compared with the binding in the absence of ions. The Eadie-Scatchard plot was used for the dynamic analysis of the Ca(2 ) binding site. A low affinity Ca(2 ) binding site was found in the GH-receptor complex with K(m)=0.384 mmol/L, and the affinity constant (K(a)) was increased from 1.045x10(9) L.mol(-1) to 1.295x10(9) L.mol(-1) by the addition of 10 mmol/L CaCl(2). The effects of disulfide bond reducing agents, DTT and ME, on (125)I-brGH binding to growth hormone receptor (GHR) on snakehead fish liver memebrane were also analyzed. The addition of 0.1 20 mmol/L DTT or 0.01% 1% ME to the radioreceptor assay system caused a significant dose dependent increase in the specific binding for (125)I-brGH. In the presence of 0.8 mmol/L DTT or 0.08% ME, the specific binding of (125)I-brGH was increased from 10.2% to 15.5% and 13.2% respectively, and the affinity constant was also increased from 1.265x10(9) L.mol(-1) to 2.185x10(9) L.mol(-1) and 1.625x10(9) L.mol(-1), respectively but no changes in the binding capacity were observed. Further studies showed that the effects of reductants on the specific binding of brGH were due in part to the ligand itself and in part to GHR. In addition, it was observed that one of the three disulfide bonds of brGH could be reduced by 0.8 mmol/L DTT.

The evolutionary position of guinea pig has not been unequivocally clarified, and it appears to show abnormalities in GH response. The cDNA of GHR cytoplasmic domain from guinea pig was cloned and sequenced. By homology comparison, it was found that the sequence we obtained was different markedly from that of rat or mouse. The results provided not only the molecular biology evidence for clarifying the evolutionary position of guinea pig, but also a basis to determine the complete cDNA sequence of GHR from guinea pig and to understand its cytoplasmic signal transduction.

Aim: To study the binding characteristics of pancreatic polypeptide (PP) receptors on rat hepatic membranes., Methods: 125I-PP suitable to study interaction between ligand and receptors were prepared. 125I-porcine PP and 125I-duck PP were used to study PP receptor binding in the controlled conditions., Results: The binding of 125I-porcine PP to receptors on rat hepatic membranes was time- and temperature-dependent. The specific binding of 125I-porcine PP was inhibited by unlabeled porcine PP in a concentration-dependent manner, whereas duck PP was only partially inhibited in the high concentration (> 500 nmol.L-1). Scatchard analysis produced a curvilinear plot, suggesting multiple affinity binding sites, i.e., high-affinity and low-affinity with dissociation constants (Kd) 5.4 and 158 nmol.L-1, respectively., Conclusion: Rat hepatic membranes possessed specific PP receptors and porcine PP binding activity was much higher than that of duck PP.

This paper describes the preparation and biological activity of an insulin analogue in which the B1-3 sequence (Phe-Val-Asn) of insulin is substituted by Ala-Ala-Lys. [B(1)Ala, B(2)Ala, B(3)Lys]-Insulin retains full in vivo activity and receptor binding activity as insulin, but its lipogenesis activity and immunoactivity are 70 % and 0.88 % of those of insulin respectively. The possible contribution of the N-terminus of B-chain of insulin to the structure and function of insulin is discussed.

The insulin-mimetic effects exhibited by vanadate, hydrogen peroxide, and some peroxovanadates have recently been shown to occur, at least in part, through an activation of the insulin receptor tyrosine kinase activity. In this study, we examine the effects of these compounds on insulin receptor binding using receptor preparations from human placental membranes. Among the 16 vanadium(V)-peroxo complexes studied, the [VO(O2)2(bipy)]- ion, where bipy = 2,2'-bipyridine, was found to increase insulin receptor binding by 24%, whereas the [VO(O2)2(en)]- ion, where en = ethylenediamine, was found to reduce insulin receptor binding by about the same amount under steady-state conditions. Scatchard analysis of the binding data indicates that the observed effect of the [VO(O2)2(bipy)]- ion on insulin receptor binding is exerted mainly at the high-capacity low-affinity sites. Furthermore, this modulatory effect is reversible and requires a continuous presence of the compound. By perturbing the membrane environment of the insulin receptor, we have shown that an intact membrane structure is essential for an observable effect. The observed modulation of insulin receptor binding by peroxovanadates is interpreted in terms of a ternary complex model in which the peroxovanadate acts as an allosteric effector modulating the binding equilibrium between insulin and its receptor.

B9Ser of insulin B chain was substituted by Glu using site-directed mutagenesis to obtain a fast-acting insulin-[B9Glu] human insulin. The receptor binding capacity and in vivo biological activity of [B9Glu] human insulin are 21% and 40% as those of porcine insulin respectively.

The receptor binding properties with human placental membrane (HPM) and the in vitro biological activity of an insulin analogue, [B1-Ala, B2-Ala]-insulin were investigated in detail and compared with those of insulin. It was found that the binding of (125)I-[B1-Ala, B2-Ala]-insulin and (125)I-insulin to HPM was time dependent reaching equilibrium after 6min at 37 degrees in the presence of bacitracin with an equilibrium maximum binding of 6.44 fmol/mg protein for [B1-Ala, B2-Ala]-insulin and 3.47 fmol/mg protein for insulin The half time (T1/2) to reach equilibrium was 19 seconds for [B1-Ala, B2-Ala]-insulin and 25 seconds for insulin. [B1-Ala, B2-Ala]-insulin and insulin competed with specific (125)I-[B1-Ala, B2-Ala]-insulin in a dose-dependent manner. From IC(50), the receptor binding activity of [B1-Ala, B2-Ala]-insulin was 139.6% compared with that of insulin. Scatchard analysis revealed that the receptor association constants of [B1-Ala, B2-Ala]-insulin in HPM at 4 degrees were 5.88x10(8)L/mol and 7.63x10(5)L/mol respectively, while those of insulin were 4.83x10(8)L/mol and 3.39x10(5)L/mol respectively. The in vitro activity of [B1-Ala, B2-Ala]-insulin was 130% of that of insulin in terms of lipogenesis in rat adipocytes.

Binding of porcine 125I-insulin (0.15 nM) to hagfish red blood cells was time-dependent, reaching equilibrium after 1 hr at 10 degrees. The specific 125I-insulin binding to hagfish red blood cells was reversible, and unlabeled insulin accelerated the dissociation of 125I-insulin bound to receptors from a T1/2 of 60 min in cells suspended in medium alone to 23 min in medium containing 8 microM nonradioactive insulin. Porcine insulin and desoctapeptide insulin competed for specific binding of 125I-insulin in a dose-dependent manner, whereas glucagon and somatostatin did not. For porcine insulin, Scatchard analysis produced a curvilinear plot, suggesting multiple affinity binding sites with high-affinity and low-affinity association constants (Ka) 0.2 x 10(9) M-1 and 0.27 x 10(7) M-1, respectively. A total of 2090 binding sites per hagfish red blood cell was calculated. Sixty-two percent of the bound 125I-insulin was found to be internalized into the hagfish red blood cells. Less degradation of 125I-insulin was observed by Sephadex G-50 chromatography compared to human red blood cells.

We measured serum T3, T4, FT3, FT4 and thyrotropin (TSH) by radioimmunoassay in 150 patients with epilepsy. Of the 150 epileptic patients, 120 cases received single antiepileptic drug, including Sod valproate (VPA), carbamazepine (CBZ), phenytoin (PHT), or MixtBrodsky (MB). Each subgroup consisted of 30 cases. The other 30 were untreated epileptics. The results suggested that there was no significant difference in serum thyroid hormone levels among healthy control group, untreated group, and hepatic non-enzyme-inducing anticonvulsant group (P < 0.05). As compared with the three groups mentioned above, the serum T3, T4, FT3, FT4 concentrations of the enzyme inducer groups were decreased, however, significant decrease was found in PHT group and CBZ group (P < 0.05, P < 0.005). All 12 patients with circulating thyroid hormone concentrations below normal reference values were given enzyme inducer. None of them revealed any clinical manifestation of hypothyroidism. There was no significant difference in serum TSH level among the above 6 groups (P < 0.05), and the serum TSH level of all patients was within normal reference limits. Our study showed that the changes of thyroid hormone in epileptics were due to the effect of some AEDs and not the disease itself. The disparity between the effect of enzyme inducer and that of non-enzyme-inducing anticonvulsants indicated that the hepatic microsomal enzyme system metabolizing thyroid hormone to be induced was the main mechanism for decreased serum thyroid hormone concentrations. Though the serum thyroid hormone levels of the enzyme inducer groups were decreased, no significant increase of the mean serum TSH concentration was found. This implies that AEDs may interfere the HPA function in some way.

Insulin, following binding to its receptor, produces a dose- and time-dependent stimulation of entry of 2-deoxy-D-[U-14C] glucose and glycogen synthesis from D-[U-14C] glucose in cultured mouse astrocytes following differentiation. Maximal stimulation of both glucose entry (217% above basal) and of glycogen synthesis (209% above basal) was observed at an insulin concentration of 1.7 x 10(-8) M. Insulin also stimulates the incorporation of leucine into astrocytic proteins with maximal stimulation (156% above basal) at an insulin concentration of 1.7 x 10(-7) M, but no effect on leucine uptake was observed at an insulin concentration of 1.7 x 10(-6) M. These results, together with a previous demonstration that insulin and certain insulin analogues stimulate pyrimidine nucleoside incorporation into nucleic acid, indicate that insulin has diverse actions on biomacromolecular metabolism in cultured mouse astrocytes.

Insulin receptor binding and effects upon uridine incorporation into nucleic acid were studied and compared to 3 insulin analogues, DPI, DPI-amide and DOI, in primary cultures of mouse astrocytes. From half-maximal inhibition of [125I]monoiodoinsulin binding (0.14 nM), the relative binding affinities of DPI, DPI-amide and DOI were 20%, 80% and 4% as compared with the native insulin, respectively. IC50 values were 158.5 nM for insulin, 199.5 nM for DPI-amide, 794.3 nM for DPI and 3980 nM for DOI. The corresponding relative potencies in stimulating [14C]uridine incorporation into TCA-insoluble material were 9% for DPI, 100% for DPI-amide and 1.1% for DOI. These findings are commensurate with data for these analogues in other insulin-sensitive tissues. It is concluded that the astrocyte insulin receptor has similar structural requirements to receptor in tissues outside the CNS, at least in terms of the involvement of the C-terminus of the insulin B-chain.

[Trp]B1 analogs to porcine and bovine insulins have been obtained through selective reaction with Msc-ONSu, and followed by Edman degradation, condensation with Msc-Trp-ONp and deprotection. Through cellulose acetate electrophoresis, amino acid composition analysis, UV absorption spectrum and N-terminal analysis, it has been proved that the products are of purified [Trp]B1 insulin. Mouse convulsion assay shows that the biological activities of porcine and bovine [Trp]B1 insulins are 18 i.u./mg and 17 i.u./mg respectively, corresponding to about 70% of the original activity of native porcine insulin. Their structural details are under investigation in our laboratory.

Binding of [125I]monoiodoinsulin to human astrocytoma cells (U-373 MG) was time dependent, reaching equilibrium after 1 h at 22 degrees C with equilibrium binding corresponding to 2.2 fmol/mg protein: this represents approximately 2,000 occupied binding sites per cell. The t1/2 of 125I-insulin dissociation at 22 degrees C was 10 min; the dissociation rate constant of 1.1 X 10(-2) s-1 was unaffected by a high concentration of unlabeled insulin (16.7 microM). Porcine insulin competed for specific 125I-insulin binding in a dose-dependent manner and Scatchard analysis suggested multiple affinity binding sites (higher affinity Ka = 4.4 X 10(8) M-1 and lower affinity Ka = 7.4 X 10(6) M-1). Glucagon and somatostatin did not compete for specific insulin binding. Incubation of cells with insulin (0.5 microM) for 2 h at 37 degrees C increased [2-14C]uridine incorporation into nucleic acid by 62 +/- 2% (n = 3) above basal. Cyclic AMP, in the absence of insulin, also stimulated nucleoside incorporation into nucleic acid [65 +/- 1% (n = 3)] above basal. Preincubation with cyclic AMP followed by insulin had an additive effect on nucleoside incorporation [160 +/- 4% (n = 3) above basal]. Dipyridamole (50 microM), a nucleoside transport inhibitor, blocked both basal and stimulated uridine incorporation. These studies confirm that human astrocytoma cells possess specific insulin receptors with a demonstrable effect of ligand binding on uridine incorporation into nucleic acid.

Binding of 125I-insulin to primary cultures of differentiated mouse astrocytes was time-dependent, reaching equilibrium after 2 h at 22 degrees C, with equilibrium binding corresponding to 20.79 fmol/mg of protein, representing approximately 5,000 occupied binding sites/cell. The half-life of 125I-insulin dissociation at 22 degrees C was 2 min, with an initial dissociation rate constant of 4.12 X 10(-2) s-1. Dissociation of bound 125I-insulin was not accelerated significantly in the presence of unlabeled insulin (16.7 microM). Porcine and desoctapeptide insulins competed for specific 125I-insulin binding in a dose-dependent manner, whereas growth hormone, glucagon, and somatostatin did not. For porcine insulin, Scatchard analysis suggested multiple-affinity binding sites (high-affinity Ka = 4.92 X 10(8) M-1; low-affinity Ka = 0.95 X 10(7) M-1). After incubation with insulin (0.5 microM) for 2 h at 37 degrees C, increases above basal values of 254 +/- 23 and 189 +/- 34% for [3H]uridine uptake and incorporation, respectively, were observed. After incubation with insulin (0.5 microM) for 24 h at 37 degrees C, there were increases of 145 +/- 6% for [3H]thymidine uptake and 166 +/- 11% for thymidine incorporation. Basal and stimulated uridine and thymidine uptake and incorporation were inhibited by 50 microM dipyridamole. These studies confirm that mouse astrocytes in vitro possess specific insulin receptors and demonstrate an effect of insulin on pyrimidine nucleoside uptake and incorporation.

The C-terminus of the insulin B chain is essential for dimerisation and expression of negative cooperativity. In order to evaluate the possible physiological role of these phenomena, we have studied the properties in vivo and in vitro of despentapeptide insulin (B26-30 deleted), derived from beef insulin, and deshexapeptide insulin (B25-30 deleted), derived from pork insulin. These materials do not dimerise and have 15% and 0% retention of negative cooperativity respective. Lipogenesis potencies in rat adipocytes were: despentapeptide insulin 19.9 +/- 0.3%; deshexapeptide insulin 19.9 +/- 1.5%. Binding potencies in adipocytes were: despentapeptide insulin 22.6 +/- 7.8%; deshexapeptide insulin 13.2 +/- 3.3%. Metabolic clearance rates were reduced compared to insulin (insulin = 19.1 +/- 0.9; despentapeptide insulin = 9.7 +/- 0.8; deshexapeptide insulin = 6.4 +/- 0.6 ml . min-1 . kg-1 at plasma concentration 0.5 nmol/l). Hypoglycaemic potencies were reduced for both analogues (40% and 30%) when calculated on the basis of plasma concentration although both analogues and insulin were equally effective at lowering plasma glucose concentration in equimolar doses. Plasma half-disappearance time was prolonged (despentapeptide insulin = 7.3 +/- 0.5; deshexapeptide insulin = 9.1 +/- 0.2 min). Both analogues were full agonists and conformed to the general relationship between in vitro and in vivo properties seen with a wide range of modified insulins. They resemble other analogues with modifications which reduce receptor affinity without impairing dimerisation or negative cooperativity. The results do not support a physiological role for dimerisation or negative cooperativity.

Despentapeptide (B26-30)-insulinamide (B25) prepared by a semisynthetic procedure was found to have about 65% of the hypoglycaemic activity of natural insulin. In contrast, the binding of the modified insulin analogue to insulin specific receptors was markedly increased. The discrepancy between the loss of biological potency and the apparent increase in binding affinity for membrane receptors reveals that not all of the biological activity of insulin is regulated by the receptor-binding system. The tetrapeptidamide of the B-chain of insulin (Arg-Gly-Phe-Phe-NH2) was clearly shown to have both insulin-like and insulin-potentiating actions in vivo although it had no effect on insulin receptor function in vitro. Evidence suggests that the small peptide fragment of insulin may be internalized and acts at the post-binding site(s) of the glucose metabolic pathway in target tissues. The present data support the general concept that insulin may exert its complex molecular actions through internalized hormonal fragment as well as the transmembrane mediators generated from receptor binding.

The semisynthesis of a series of B23-D-Ala deshexapeptide and despentapeptide analogues with B22-arginine replaced are described. The following semisynthetic scheme is used: desnonapeptide insulin pentamethyl ester is prepared from insulin hexamethyl ester through the action of trypsin and carboxypeptidase B, the amino groups are protected with Boc-groups, the protected product is condensed with synthetic peptides, and the protecting groups are removed with trifluoroacetic acid treatment and by saponification. The biological activities of these analogues indicate that the B22-Arg is non-essential and can be replaced by Lys or even Asp without any influence on the biological activity. The activity is reduced to one half when it is replaced by valine or to a very low level when replaced by Gly or D-Arg. The reason for the low activity of guinea pig insulin is discussed.