Short Analogues Research Articles

Internal ribosome-binding site directs expression of parathyroid hormone analogue (8–84) in Escherichia coli

Expression of the human parathyroid hormone (PTH) gene in E. coli yielded intact PTH and PTH-(8-84). To determine if PTH-(8-84) is the result of a competing translation initiated from methionine codon-8 or degradation of the intact PTH, twelve new gene constructs with or without an internal ribosome-binding site (iRBS) in the PTH-(1-5) region were prepared via substitution with degenerate codons. Expression of constructs without iRBS produced only intact PTH. Constructs with weak iRBS, including one that resembles the cDNA sequence, yielded PTH-(8-84) as a minor product. In contrast, constructs with strong iRBS produced predominantly or exclusively this shorter analogue.

Dearylation of α,ω-diphenylpermethylated oligosilanes with triflic acid

The relative rates of displacement of phenyl groups for a series of α,ω-diphenylpermethylated oligosilanes with the formula Ph(SiMe 2) n Ph ( n = 2−5) were studied. Triflic acid was utilized in the displacement reactions which occur as a two-step process with protonation at the ipso-carbon atom as the rate limiting step. The results showed the displacement of the firs phenyl group is more facile than the second group. The largest difference in reactivities is found for the disilane. Competitive displacement reactions between various oligomers were analyzed to establish the influence of the chain length of oligosilanes on the relative reactivities of the terminal phenyl groups. Both the first and second phenyl group displacement reactions are faster for the longer chain oligomers than the shorter analogs. The 13C NMR spectra of the oligosilanes indicate the highest election density on the ipso-carbon atoms in the disilane, in contrast to its lowest reactivity. This is interpreted by the increased stabilization of the positive charge int he transition state with an increase in the chain length. Thus, the reactivities of oligosilanes are governed by the structures of the transition states rather than the ground states.

Low temperature impact properties of long fiber thermoplastic composite molding materials

AbstractFour long fiber thermoplastic resin matrices, nylon 6, polypropylene, polyethylene terephthalate, and styrene maleic anhydride, containing differing amounts of long fiber glass reinforcement, were tested for notched Izod impact strength over the temperature range of 22 to −32°C. The notched impact properties of the long fiber thermoplastic composite molding materials are substantially greater than literature values for short fiber analogues. The fiber dominant performance of the long fiber materials is evidenced by increasing impact values with corresponding increases in weight percent fiber content. No apparent ductile/brittle transition in the fracture mode was observed for the long fiber materials that were tested.

Combination of long and short term GnRH analogue protocols: a new therapeutic approach to persistent high progesterone levels in IVF cycles

Thirty-four patients who had had persistently high progesterone (P) levels during menotrophin therapy in previous in-vitro fertilization (IVF) cycles, despite pretreatment with a long acting gonadotrophin-releasing hormone analogue (GnRHa), were randomly divided into two groups according to the mode of ovulation induction used. In 16 women (group 1), induction of ovulation was performed with decapeptyl (DTRP6)/pure follicle-stimulating hormone (PFSH)/human menopausal gonadotrophin (HMG)/human chorionic gonadotrophin (HCG). In 18 patients (group II), the protocol was identical to the former group, except for the addition of a short-acting GnRHa (buserelin) that was started when P levels were persistently high. The combination of long and short acting GnRH analogues (group II) resulted in an improved follicular phase with significantly lower P levels on the day of HCG administration (P less than 0.001). Furthermore, a significantly higher number of oocytes was retrieved, fertilized and cleaved (P less than 0.005; P less than 0.001; P less than 0.005, respectively) and, as a consequence, significantly higher pregnancy rates were achieved (22.2 versus 12.5%; P less than 0.005). These results indicate that a combination of long- and short-acting GnRH agonists may be of value in cases of persistent high P levels during menotrophin therapy in IVF cycles. Further larger studies must be performed before the true efficacy of this mode of treatment can be determined.

Antibacterial Activity and Inhibition of Protein Synthesis inEscherichia coliby Antisense DNA Analogs

Protein synthesis, which takes place within ribosomes, is essential for the survival of any living organism. Ribosomes are composed of both proteins and RNA. Specific interaction between the 3' end CCUCC sequence of prokaryotic 16S rRNA and a partially complementary sequence preceding the initiating codon of mRNA is believed to be a prerequisite for initiation of protein synthesis. Here we report the use of short (three to six nucleotides) synthetic DNA analogs complementary to this sequence to block protein synthesis in vitro and in vivo in Escherichia coli. In the DNA analogs the normal phosphodiester bond in the antisense DNA was replaced by methylcarbamate internucleoside linkages to enhance transport across plasma membranes. Of the analogs tested, those with the sequence AGG and GGA inhibit protein synthesis and colony formation by E. coli strains lacking an outer cell wall. Polyethylene glycol 1000 (PEG 1000) was attached to the 5' end of some of the test methylcarbamate DNAs to enhance solubility. Analogs of AGG and GGAG with PEG 1000 attached inhibited colony formation in normal E. coli. These analogs may be useful food additives to control bacterial spoilage and biomedically as antibiotics.

Modelling of substrate binding to 3-phosphoglycerate kinase with analogues of 3-phosphoglycerate

Two short analogues of 3-phosphoglycerate, -OOC-CHOH-CH2-O-PO32-, phosphonolactate, (-OOC-CHOH-CH2-PO32-) and arsonolactate (-OOC-CHOH-CH2-AsO32-) have been tested with 3-phosphoglycerate kinase. None of these served as substrate for the kinase reaction, unlike the previously studied [Orr, G. A. & Knowles, J. R. (1974) Biochem. J. 141, 721-723] analogues -OOC-CHOH-CH2-CH2-PO32- and -OOC-CHOH-CH2-CH2-AsO32-, which are isosteric with 3-phosphoglycerate. Thus, a decrease in the substrate size and the accompanying stereochemical changes cannot be tolerated by the catalytic mechanism. Instead, both analogues acted as relatively poor competitive inhibitors with respect to both 3-phosphoglycerate and MgATP. AT pH 8.5 and 20 degrees C, the inhibitory constants (Ki) of phosphonolactate and arsnolactate against both substrates are 17 +/- 5 mM and 30 +/- 7 mM, respectively. Surprisingly, however, both analogues proved to be more effective than either 3-phosphoglycerate or its isosteric analogues in protecting the enzyme against modification of its fast-reacting thiols. This comparison suggests that the shorter analogues bind differently, and that the catalytic mechanism demands a precise fitting of the -CH2-O-PO32- segment of the substrate.

Cysteinyl methyl ester of AVP(4–8), a potent agonist on the maintenance of passive avoidance in rats

A series of short AVP analogs with D- or L-arginine forming the C-terminal was synthesized, and their peripheral effects, i.e., vasoconstrictor and antidiuretic activities, and behavioral effects in enhancing retention in passive avoidance in rats were evaluated. We found that: 1) AVP(4–8) and its cysteinyl methyl ester were more potent in the behavioral response than its D-Arg homologs; 2) the methyl ester derivative was the most effective analog in this behavioral test among the peptides we synthesized, with a potency 40 times as high as AVP; 3) neither the D- nor the L-Arg short derivatives of AVP showed any peripheral effects up to a dose thousands of times greater than AVP. The results support the contention that arginine in the short analogs plays an important role in their behavioral response associated with a relatively steady peptide conformation.

A Synthetic Linear Decapeptide Binds to the Atrial Natriuretic Peptide Receptors and Demonstrates Cyclase Activation and Vasorelaxant Activity

A linear decapeptide, [cyclohexylalanine 106]ANP-(105-114)NH2 (1), where ANP is atrial natriuretic peptide, was prepared by solid phase synthesis and purified by reverse-phase liquid chromatography. This novel peptide was found to bind to ANP receptors in rabbit lung membranes, to stimulate cGMP production in various tissues, and to fully relax precontracted rabbit aorta in a dose-dependent fashion. The potency of 1 in the various in vitro assays varies between one-twentieth and one-eightieth of the potency of the reference peptide, the 24-mer rat ANP-(103-126). The linear decapeptide 1, which encompasses amino acid residues from the rat ANP sequence (105-114), features a cyclohexylalanine residue instead of the phenylalanine 106 residue in the hormone sequence, a free sulfhydryl function at the N-terminal cysteine 105, and a carboxamide C terminus. Its disulfide dimer 6 was active in the rabbit aorta assay while the S-methyl cysteine 7 analogue was not active in the same assay at similar concentrations. The decapeptide 1 is of particular significance because it is the shortest analogue reported to date endowed with agonistic activity at the guanylate cyclase-coupled ANP receptor. In particular, it is interesting to compare its structure to the structures of other short linear analogues of ANP which are totally devoid of the ability to stimulate particulate guanylate cyclase activity.

Chemical synthesis of echistatin, a potent inhibitor of platelet aggregation from Echis carinatus: synthesis and biological activity of selected analogs.

Echistatin, a polypeptide from the venom of the saw-scaled viper, Echis carinatus, containing 49 amino acids and 4 cystine bridges was synthesized by solid-phase methodology in 4% yield. In the final step, air oxidation of the octahydroderivative was found to be optimal at pH 8. The synthetic product was shown to be physically and biologically indistinguishable from native material. It inhibits fibrinogen-dependent platelet aggregation stimulated by ADP with IC50 = 3.3 x 10(-8) M and also prevents aggregation initiated by thrombin, epinephrine, collagen, or platelet-activating factor. Reduction of purified synthetic echistatin to octahydroechistatin with dithiothreitol followed by air oxidation regenerated homogeneous echistatin in quantitative yield. This highly specific refolding strongly suggests that the linear sequence of octahydroechistatin contains all of the information that is required for the proper folding of the peptide. The sequence Arg24-Gly-Asp of echistatin occurs also in adhesive glycoproteins that bind to the platelet fibrinogen receptor--a heterodimeric complex composed of glycoproteins IIb and IIIa. In an effort to evaluate the role of this putative binding site we have synthesized analogs of echistatin with substitution of Arg-24. Replacement with ornithine-24 (Orn-24) resulted in an analog having a platelet aggregation inhibitory activity with IC50 = 1.05 x 10(-7) M. Substitution with Ala-24 gave IC50 = 6.1 x 10(-7) M. The inhibitory activity of the corresponding short sequence analogs Arg-Gly-Asp-Phe (IC50 = 6 x 10(-6) M), Orn-Gly-Asp-Phe (IC50 = 1.3 x 10(-4) M), and Ala-Gly-Asp-Phe (IC50 = 5.0 x 10(-4) M) was also determined. These results suggest that arginine plays a more important role in the binding of the tetrapeptide than in that of echistatin.

Probing the role of substrate conformation in phospholipase A2 action on aggregated phospholipids using constrained phosphatidylcholine analogues.

Phospholipase A2s hydrolyze aggregated phospholipid substrates much more rapidly than dispersed monomeric ones. Whether this is a consequence of interface-associated conformational changes of the enzyme or of the substrate, or of both, remains a key question in lipid enzymology. This problem is addressed herein using a rationally designed probe of substrate conformation. (1,3/2)-1-O-(phosphorylcholine)-2,3-O-dihexanoylcyclopentane-1,2,3 -triol is a novel short chain phosphatidylcholine analogue in which the glycerol-like backbone is part of a five-membered ring and therefore covalently constrained within a small defined range of conformations. To the extent that the constrained analogue resists aggregation-associated conformational changes, it provides a means for assessing the contribution of such changes to phospholipase A2 action on aggregated phospholipids. The monomeric (-)-cyclopentanoid analogue is a substrate for phospholipase A2s from Naja naja naja venom. However, when this constrained phospholipid is aggregated, its hydrolysis rate is not enhanced, in contrast to its unconstrained counterpart, 1,2-dihexanoyl-sn-glycero-3- phosphorylcholine. This lack of activation was not caused by a failure of the enzyme to bind the micellar, constrained analogue. While the constrained analogue does not show interfacial activation, it does show the activation of phosphatidylethanolamine hydrolysis typical of phosphorylcholine-containing lipids. Hence, these results strongly support the contention that specific packing-induced conformations of aggregated substrate play a substantial role in the large interfacial activations observed with phospholipase A2.

Immunochemistry of scorpion toxins

Peptide 19-28, a model of an antigenic region of Androctonus australis Hector toxin II, has a rigid alpha-helix structure in the native protein. It was used as immunogen either in the free form, or bound to bovine serum albumin (BSA) or linked to its synthesis support (macroporous polyacrylamide resin). Only the anti-(peptide-BSA) and the anti-(peptide-resin) antibodies recognized the native toxin. The use of short synthetic analogues of peptide 19-28 suggests a specificity difference in the two antipeptides. Anti-(peptide-BSA) recognizes probably two determinants localized at the C and N terminals of the peptide chain. Anti-(peptide-resin) preferentially recognizes the N-terminal extremity. Finally we showed that the alpha-helix region remains accessible to antipeptide 19-28 when the toxin is bound to its receptor.

Peptide substrates for chymosin (rennin). Interaction sites in kappa-casein-related sequences located outside the (103-108)-hexapeptide region that fits into the enzyme's active-site cleft.

The role of individual amino acid residues in the 98-102 and 111-112 regions of bovine kappa-casein in its interaction with the milk-clotting enzyme chymosin (rennin) was investigated. to this end the tryptic 98-112 fragment of kappa-casein was modified in its N- and/or C-terminal part by chemical (guanidation, ethoxyformylation, repeated Edman degradation) and enzymic (carboxypeptidase) treatments. Further, use was made of short synthetic kappa-casein analogues in which His-102 had been replaced by Pro or Lys. All peptides and their derivatives were tested comparatively at various pH values for their ability to act as chymosin substrates via specific cleavage of the peptide bond at position 105-106. The results indicate that in the alternating 98-102 sequence (His-Pro-His-Pro-His) the His as well as the Pro residues contribute to the substrate activity with no predominant role of any one of these groups. Another interaction site is formed by the Lys residue at position 111 of the substrate. A model of the enzyme-substrate complex is proposed. Herein the 103-108 fragment of the substrate, to be accommodated within the enzyme's active-site cleft, is brought into position by electrostatic binding (via His-98, His-100, His-102 and Lys-111) near the entrance of the cleft. These interactions are strongly supported by Pro residues at positions 99, 101, 109 and 110 of the substrate, which act as stabilizers of the proper conformation of the substrate in the enzyme-substrate complex.

Mechanism of Uptake of l‐Arginine by Sugar‐Cane Cells

Suspension cells of sugar cane were used as a model system for cells of higher plants to study the mechanism of L-arginine uptake. The uptake system is specific for the L-arginine molecule in the fully ionized state, i.e. delta-guanidino group and alpha-amino group positively charged and carboxyl group negatively charged. This was concluded because the Km value for uptake increased strongly for: (a) L-arginine analogues which lack the charged carboxyl group (L-arginine methyl ester, agmatin); (b) L-arginine analogues, which lack the charged alpha-amino group (L-arginine acid, gamma-guanidinobutyric acid); (c) L-arginine analogues, which lack the charged delta-guanidino group or gamma-guanidinoxy group (L-citrulline, L-canavanine at neutral and alkaline pH-values). The importance of the positive charge of the delta-guanidino group or gamma-guanidinoxy group was further documented by Km values for L-arginine and L-canavanine at different pH values. Only at pH values where the gamma-guanidinoxy group is protonated, was there an effective uptake of L-canavanine and effective competition of L-canavanine with L-arginine. The length of the L-arginine molecule was less important: slightly larger (L-homoarginine) or shorter analogues (L-lysine) were taken up rather well. A spatial rearrangement at the alpha-carbon (D-ariginine) was, however, not tolerated. The uptake of L-arginine proceeds by electrogenic uniport, there is no evidence for symport or antiport of another molecule (though L-canavanine uptake at neutral pH value causes a transient alkalinization of the suspension medium). Charge equilibration is brought about by efflux of protons and potassium ions.

Acute influences of some ACTH-related peptides on fighting and adrenocortical activity in male laboratory mice

A number of corticotrophin preparations when given acutely enhanced fighting behavior, an event which may be consequent upon elevation of circulating corticosterone. A series of short ACTH analogues (ACTH 11–24, ACTH 4–10) had slight influences on circulating plasma corticosterone values and on fighting behavior. However, 20 μg ACTH 1–10 given 12 hr before testing did increase fighting c.f. controls. As this compound only caused a slight stimulation of plasma corticosterone titers, the possibility exists of an extra-adrenal influence. One cannot, it appears, eliminate glucocorticoid influences using these preparations in the mouse. Acutely, ACTH 1–24 had virtually no influences on Animex-assessed motor activity but did enhance fighting in castrated mice maintained with either androgen or estrogen. It is tentatively concluded that the ACTH influences on aggression in the mouse are partially mediated via glucocorticoid influences on the CNS combined with an extra adrenal influence of part of the ACTH molecule. A possible reason for the differing acute and chronic influences of ACTH on fighting in mice is presented.

Affinity chromatography of renin using inhibitory renin substrate analogs.

SummarySeveral short peptide analogs of renin substrate were tested for their ability to inhibit renin in solution and to bind renin in affinity chromatography. The peptides Leu-Leu-Val-Tyr-OCH3, Leu-Leu-Val-Phe-OCH3 and Leu-Val-Phe-OCH3 had in vitro inhibitory activity against both hog and human renin. Kinetic studies of the prototype inhibitor Leu-Leu-Val-Phe-OCH3 demonstrated classical competitive inhibition, with a Ki of 180 μM. When impure hog renin was eluted from succinylated aminoethyl agarose columns containing co-valently bound Leu-Leu-Val-Phe-OCH3 or Leu-Val-Phe-OCH3, the elution of renin activity was delayed in comparison with the elution of inactive contaminating proteins. The binding of human renin to agarose bound inhibitors could also be demonstrated if a longer connecting arm was interposed between the peptide ligand and the supporting polysaccharide.

Statistical sampling in community noise measurement

The statistical theory of community noise sampling is investigated and used to derive the optimum sampling procedures. General expressions are given for the sampling errors in L10 and other percentiles. The correlation between percentile sampling errors is discussed in relation to the sampling error in TNI and to the correct procedure of data analysis. The traffic noise models of Weiss and Kurze are used to derive a sampling theory for Leq. It is shown that, for continuous sampling, the sampling error, ΔLeq, can be estimated by ΔLeq~7⋅4/mdB(A) where m is the number of vehicles passing during the sample. A sampling rate of 2 per second is shown to be a good approximation to continuous sampling. The model is used to demonstrate that, when sampling with a sound level meter, using a fixed number of readings, the readings should be at least 5 seconds apart. The use of very short analogue tape samples (microsampling) is shown to lead to improved sampling accuracy on theoretical grounds.

Synthesis of highly active corticotropic (1-D-serine,17,18-dilysine)-beta-corticotropin-(1-18)-octadecapeptide amide

AbstractA synthesis is described of [1‐D‐serine, 17, 18‐dilysine]‐β‐corticotropin‐(1–18)‐octadecapeptide amide (I), a short chain ACTH analogue which has been found to possess in animals and also in man high and prolonged corticotropic activity. Synthesis was carried out by the fragment condensation approach involving, in the last build‐up step, coupling of the protected sequences 1–10 and 11–18. From the protected octadecapeptide derivative 1–18 the free peptide was obtained in high yield by acidolysis.

Synthetic Studies of Structure-Function Relationships in Staphylococcal Nuclease: SYNTHETIC ANALOGUES OF FRAGMENT P2

Abstract The solid phase technique of peptide synthesis has been used to study structural features related to the activity of Fragment P2 (Residues 6 through 48) of nuclease-T. Binding of synthetic analogues to the complimentary native Fragment P3 (Residues 49 through 149) may be detected by affinity chromatography upon an agarose column containing covalently bound P3. Binding in free solution was detected by changes in the fluorescence spectrum of the single tryptophan residue in P3. Previous studies have shown that the synthetic 6-47 peptide, after purification by specific binding to an agarose P3 column, can generate approximately 30% of the maximum enzymic activity when added in an equivalent molar quantity to Fragment P3 Deletion of the NH2-terminal tripeptide from synthetic P2 (yielding synthetic 9-47) leaves both binding and activating abilities intact. Two shorter analogues, synthetic 18-47 and synthetic 33-47, have no activity, although the 18-47 analogue appears to retain some binding affinity for native P3. The methionine residues at positions 26 and 32 may be replaced by norleucine to give an analogue capable of both binding to and activating P3. Replacement of glutamic acid by glutamine at position 43 leads to an inactive derivative which retains its ability to bind specifically to Fragment P3.

A novel method for analysing singly-occurring pulses with nanosecond resolution

The system is designed to record pulse-shape information of singly occurring pulses with rise-times down to 1 ns. The basic theory of the ‘interrogate pulse’ sampling scheme is described and a system to implement this scheme is outlined. The proposed system employs short term analogue storage of the sampled input, followed by a fast multiplexed analogue to digital converter and digital storage. Some results from an experimental instrument using this system are given.