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One of the amino H atoms of L-phenylalanyl-L-valine, C(14)H(20)N(2)O(3), participates in a rare secondary interaction in being accepted by the aromatic ring of the phenylalanine side chain. The phenyl group is also a donor in a weak hydrogen bond to the peptide carbonyl group.
The title peptide, C7H14N2O3, crystallizes with seven independent molecules in the asymmetric unit. All have essentially the same overall conformation, but some flexibility is exhibited by the glycine residue. It appears that the high Z' value, observed only three times before for an organic compound, permits formation of shorter hydrogen bonds in one of the two head-to-tail chains involving the N-terminal amino groups and the C-terminal carboxylate groups than found in a hypothetical model structure of glycyl-L-valine with Z' = 1, and that it furthermore alleviates strain associated with an eclipsed orientation of the amino group.
In each of N-(6-amino-3, 4-dihydro-3-methyl-5-nitroso-4-oxopyrimidin-2-yl)valine, C(10)H(15)N(5)O(4) (3) (orthorhombic, P2(1)2(1)2(1)), N-(6-amino-3, 4-dihydro-3-methyl-5-nitroso-4-oxopyrimidin-2-yl)serine monohydrate, C(8)H(11)N(5)O(5).H(2)O (4) (orthorhombic, P2(1)2(1)2(1)), and N-(6-amino-3, 4-dihydro-3-methyl-5-nitroso-4-oxopyrimidin-2-yl)threonine, C(9)H(13)N(5)O(5)(5) (monoclinic, P2(1)), the C-nitroso fragments exhibit almost equal C-N and N-O bond lengths: the C-N range is 1. 315 (3)-1.329 (3) A and the N-O range is 1.293 (3)-1.326 (3) A. In each compound there are also very short intermolecular O-H.O hydrogen bonds, in which carboxyl groups act as hydrogen-bond donors to the nitrosyl O atoms: the O.O distances range from 2.440 (2) to 2. 504 (4) A and the O-H.O angles lie between 161 and 163 degrees. An interpretation of the relationship between the unusual intramolecular bond lengths and the very short intermolecular hydrogen bonds has been developed based on database analysis and computational modelling. In each of (3)-(5) there is an extensive network of intermolecular hydrogen bonds, generating three-dimensional frameworks in (3) and (5), and two-dimensional sheets in (4).
A convergent synthesis of the peptide [1-(beta-mercapto-beta, beta-cyclopentamethylenepropionic acid)- 2-(O-ethyl-D-tyrosine)-4-valine-9-desglycine]arginine vasopressin (1), based on the classical solution phase method, was developed. The molecule is assembled by a 3 + 4 coupling via the azide method; then the disulfide bridge is installed by iodine treatment of the bis-acetamidomethyl protected thiols, and the terminal arginine amide added by a 7 + 1 coupling. The method has been used to prepare gram quantities of 1 in more than 98% purity and in 13% yield (based on tetrapeptide intermediate 13) after a single stage purification. The method appears to be particularly suitable for the large scale preparation of 1 and other vasopressin congeners. A novel, albeit low level, transfer of acetamidomethyl group from the sulfur of cysteine to the asparagine amide side-chain was detected following hydrogen chloride treatment of Boc-containing intermediates.
Cell-free cyclization of delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine to isopenicillin N by lytic enzyme extracts of Cephalosporium acremonium M-0198 was stimulated by ferrous ions. The optimum concentration of FeSO4 was 80 microM. No additional stimulation was observed with ascorbate, adenosine 5'-triphosphate, or alpha-ketoglutarate, but Triton X-100 and sonication of the extracts increased activity. ZnSO4 was very inhibitory to enzyme activity; CuSO4 was somewhat less inhibitory, and the least effective of the three was MnCl2. The dimer of the tripeptide was converted to a penicillin that has the biological spectrum of isopenicillin N, and this reaction was also stimulated by FeSO4. We found that sonication can be used directly to prepare extracts with cyclization activity from mycelia, without preparing protoplast lysates. The kinetics of cyclase appearance and disappearance during fermentation were similar to those of ring-expansion activity, i.e., enzyme appeared and peaked 13 h after growth ceased and then disappeared.
Saturation mutagenesis of the 2,4-dinitrotoluene dioxygenase (DDO) of Burkholderia cepacia R34 at position valine 350 of the DntAc alpha-subunit generated mutant V350F with significantly increased activity towards o-nitrophenol (47 times), m-nitrophenol (34 times), and o-methoxyphenol (174 times) as well as an expanded substrate range that now includes m-methoxyphenol, o-cresol, and m-cresol (wild-type DDO had no detectable activity for these substrates). Another mutant, V350M, also displays increased activity towards o-nitrophenol (20 times) and o-methoxyphenol (162 times) as well as novel activity towards o-cresol. Products were synthesized using whole Escherichia coli TG1 cells expressing the recombinant R34 dntA loci from pBS(Kan)R34, and the initial rates of product formation were determined at 1 mM substrate by reverse-phase high-pressure liquid chromatography. V350F produced both nitrohydroquinone at a rate of 0.75 +/- 0.15 nmol/min/mg of protein and 3-nitrocatechol at a rate of 0.069 +/- 0.001 nmol/min/mg of protein from o-nitrophenol, 4-nitrocatechol from m-nitrophenol at 0.29 +/- 0.02 nmol/min/mg of protein, methoxyhydroquinone from o-methoxyphenol at 2.5 +/- 0.6 nmol/min/mg of protein, methoxyhydroquinone from m-methoxyphenol at 0.55 +/- 0.02 nmol/min/mg of protein, both methylhydroquinone at 1.52 +/- 0.02 nmol/min/mg of protein and 2-hydroxybenzyl alcohol at 0.74 +/- 0.05 nmol/min/mg of protein from o-cresol, and methylhydroquinone at 0.43 +/- 0.1 nmol/min/mg of protein from m-cresol. V350M produced both nitrohydroquinone at a rate of 0.33 nmol/min/mg of protein and 3-nitrocatechol at 0.089 nmol/min/mg of protein from o-nitrophenol, methoxyhydroquinone from o-methoxyphenol at 2.4 nmol/min/mg of protein, methylhydroquinone at 1.97 nmol/min/mg of protein and 2-hydroxybenzyl alcohol at 0.11 nmol/min/mg of protein from o-cresol. The DDO variants V350F and V350M also exhibited 10-fold-enhanced activity towards naphthalene (8 +/- 2.6 nmol/min/mg of protein), forming (1R,2S)-cis-1,2-dihydro-1,2-dihydroxynaphthalene. Hence, mutagenesis of wild-type DDO through active-site engineering generated variants with relatively high rates toward a previously uncharacterized class of substituted phenols for the nitroarene dioxygenases; seven previously uncharacterized substrates were evaluated for wild-type DDO, and four novel monooxygenase-like products were found for the DDO variants V350F and V350M (methoxyhydroquinone, methylhydroquinone, 2-hydroxybenzyl alcohol, and 3-nitrocatechol).
avtA, which encodes the alanine-valine transaminase, transaminase C, was cloned in vivo with high- and low-copy-number mini-Mu cloning vectors. The phenotype conferred by the cloned avtA+ gene usually depended upon the plasmid copy number; most high-copy-number avtA+ plasmids permitted isoleucine-requiring ilvE strains to grow in the absence of isoleucine (multicopy suppression), while low-copy-number avtA+ plasmids did not. avtA was mapped to a 1.25-kilobase segment by comparison of the restriction maps of 24 independent mini-Mu plasmids and then by gamma-delta (Tn1000) mutagenesis of a pBR322-avtA+ plasmid. The direction of transcription of avtA on the cloned fragment was determined with fusions to a promoterless lac gene.
delta-(L-alpha-Aminoadipyl)-L-cysteinyl-D-valine synthetase (ACVS) was purified from Streptomyces clavuligerus by a combination of salt precipitation, ultrafiltration, and anion-exchange chromatography. The final purified material gave two protein bands with molecular weights of 283,000 and 32,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Electrophoresis in nondenaturing gels gave a single protein band with an estimated molecular weight of 560,000. These results suggest that ACVS is a multimer composed of nonidentical subunits.
Lysine epsilon-aminotransferase (LAT) in the beta-lactam-producing actinomycetes is considered to be the first step in the antibiotic biosynthetic pathway. Cloning of restriction fragments from Streptomyces clavuligerus, a beta-lactam producer, into Streptomyces lividans, a nonproducer that lacks LAT activity, led to the production of LAT in the host. DNA sequencing of restriction fragments containing the putative lat gene revealed a single open reading frame encoding a polypeptide with an approximately Mr 49,000. Expression of this coding sequence in Escherichia coli led to the production of LAT activity. Hence, LAT activity in S. clavuligerus is derived from a single polypeptide. A second open reading frame began immediately downstream from lat. Comparison of this partial sequence with the sequences of delta-(L-alpha-aminoadipyl)-L-cysteinyl-D valine (ACV) synthetases from Penicillium chrysogenum and Cephalosporium acremonium and with nonribosomal peptide synthetases (gramicidin S and tyrocidine synthetases) found similarities among the open reading frames. Since mapping of the putative N and C termini of S. clavuligerus pcbAB suggests that the coding region occupies approximately 12 kbp and codes for a polypeptide related in size to the fungal ACV synthetases, the molecular characterization of the beta-lactam biosynthetic cluster between pcbC and cefE (approximately 25 kbp) is nearly complete.
A 24-kb region of Cephalosporium acremonium C10 DNA was cloned by hybridization with the pcbAB and pcbC genes of Penicillium chrysogenum. A 3.2-kb BamHI fragment of this region complemented the mutation in the structural pcbC gene of the C. acremonium N2 mutant, resulting in cephalosporin production. A functional alpha-aminoadipyl-cysteinyl-valine (ACV) synthetase was encoded by a 15.6-kb EcoRI-BamHI DNA fragment, as shown by complementation of an ACV synthetase-deficient mutant of P. chrysogenum. Two transcripts of 1.15 and 11.4 kb were found by Northern (RNA blot) hybridization with probes internal to the pcbC and pcbAB genes, respectively. An open reading frame of 11,136 bp was located upstream of the pcbC gene that matched the 11.4-kb transcript initiation and termination regions. It encoded a protein of 3,712 amino acids with a deduced Mr of 414,791. The nucleotide sequence of the gene showed 62.9% similarity to the pcbAB gene encoding the ACV synthetase of P. chrysogenum; 54.9% of the amino acids were identical in both ACV synthetases. Three highly repetitive regions occur in the deduced amino acid sequence of C. acremonium ACV synthetase. Each is similar to the three repetitive domains in the deduced sequence of P. chrysogenum ACV synthetase and also to the amino acid sequence of gramicidin synthetase I and tyrocidine synthetase I of Bacillus brevis. These regions probably correspond to amino acid activating domains in the ACV synthetase protein. In addition, a thioesterase domain was present in the ACV synthetases of both fungi. A similarity has been found between the domains existing in multienzyme nonribosomal peptide synthetases and polyketide and fatty acid synthetases. The pcbAB gene is linked to the pcbC gene, forming a cluster of early cephalosporin-biosynthetic genes.
The capacity of the synthetic elastase inhibitor, succinyl alanyl alanyl prolyl valine chloromethyl ketone (CMK), to moderate elastase-induced emphysema in hamsters was examined using morphometric and physiologic measurements. When 0.5 mg of CMK in saline was injected intratracheally 1 h before the intratracheal injection of 0.1 mg of porcine pancreatic elastase, the hamsters did not develop emphysema. When CMK was injected intratracheally 1 h after elastase, the severity of emphysema was reduced by approximately 50% compared with control animals receiving saline 1 h after elastase. The CMK was ineffective when administered intratracheally 4 h after elastase.
An abnormal hemoglobin, termed Hb Savannah, was found in red cell hemolysate of a young Caucasian girl with severe hemolytic anemia. The presence of this unstable variant became evident when inclusion bodies appeared rapidly upon exposure of red cells to redox dyes and a large percentage of hemoglobin in hemolysate precipitated on warming to 65 degrees C. Treatment of the hemoglobin with p-hydroxymercuribenzoate (PMB) caused a rapid dissociation into monomers; starch-gel electrophoresis of PMB-treated hemoglobin showed the presence of abnormal beta-chains. Data from structural studies of isolated beta-chains indicated substitution of a valyl residue for the normally occurring glycyl residue at position 24, which corresponds to helical residue B6. A similar substitution but with an arginine replacing the glycyl residue has been observed in Hb Riverdale-Bronx. The glycine to valine substitution will change the relationship of the B and the E helices which results in extensive conformational changes in the beta-chain. This change presumably causes an increased dissociation of the hemoglobin molecule into dimers and probably monomers, and a decreased stability of the alphabeta-dimers. The hemoglobin abnormality may be the result of a fresh mutation because the abnormality is not present in the parents nor in any of the seven siblings.
R1518 is a valine ester prodrug of levovirin as an investigational new drug for the treatment of hepatitis C virus. Two phase 1, single- and multiple-dose studies were conducted to investigate the pharmacokinetics of R1518 in healthy volunteers. After oral dosing, R1518 was rapidly and exclusively converted to levovirin. Levovirin plasma concentrations peaked at 2 hours, with T(1/2) ranging from 6 to 8 hours. The T(1/2) of R1518 was less than 1 hour, with relative exposures (R1518/levovirin) less than 6%. A high-fat meal did not affect the pharmacokinetics. The female groups in both studies had higher plasma levels than males did due to age and renal function difference. An accumulation ratio of 1.3 to 1.5 was observed with the twice-daily regimen. About 75% to 90% of the levovirin equivalent dose was recovered in urine. Increase in exposure was slightly disproportionate to increase in dose. Significantly improved oral absorption of levovirin was achieved following administration of R1518.
Peptide histidine isoleucine (PHI), peptide histidine valine (PHV), and vasoactive intestinal polypeptide (VIP) are cosynthesized from the same precursor and share high levels of structural similarities with overlapping biological functions. In this study, the first PHI/PHV receptor was isolated and characterized in goldfish. To study this receptor using homologous peptides, we have also characterized the goldfish prepro-PHI/VIP, and, surprisingly, a shorter transcript lacking the VIP coding region was isolated. A PHI/VIP precursor without the VIP coding sequence has never before been reported. Initial functional expression of the PHI/PHV receptor in Chinese hamster ovary cells revealed that it could be activated by human PHV [50% effective concentration (EC(50)): 43 nM] and to a lesser extent human PHI (EC(50): 133 nM) and helodermin (EC(50): 166 nM) but not fish and mammalian pituitary adenylate cyclase-activating polypeptides and VIPs. Subsequent studies indicated that, similar to the pituitary adenylate cyclase-activating polypeptide receptors (PAC1-R, VPAC1-R, and VPAC2-R), the receptor isolated in this study is able to interact with goldfish PHI and its C-terminally extended form, PHV with EC(50) values 93 and 43 nM, respectively. Northern blot and RT-PCR/Southern blot analyses revealed that the PHI/VIP gene is expressed in the intestine, brain, and gall bladder and the PHI/PHV receptor gene is primarily expressed in the pituitary and to a lesser extend in the intestine and gall bladder, suggesting that PHI/PHV may play a role, notably in the regulation of pituitary function. In conclusion, our results demonstrate for the first time the existence of a PHI/PHV receptor, indicating that the functions of PHI and PHV could be mediated by their own receptor in addition to VIP receptors.
We have identified two different single nucleotide missense substitutions at valine-865 in exon 7 of the human androgen receptor (AR) gene in two families with androgen resistance. Val-->methionine is associated with the complete syndrome; Val-->leucine is associated with the partial form. In genital skin fibroblasts, both alterations yield a normal maximum binding capacity, but an increased apparent equilibrium dissociation constant for all androgens tested. In genital skin fibroblasts, Val865-Met A-R complexes have increased rate constants of dissociation with 5 alpha-dihydrotestosterone, and the nonmetabolized ligands methyltrienolone or mibolerone (MB); their Val865-Leu counterparts have increased rates with methyltrienolone and MB, but not with 5 alpha-dihydrotestosterone. In transiently transfected COS-1 or PC-3 cells, Met865 AR is more severely impaired than Leu865 AR in transactivating two different androgen-responsive reporter constructs, thereby correlating with clinical phenotype. In COS-1 cells exposed to MB for 74 h, this relative impairment correlates with the relative instability of the MB-binding activity of each mutant AR, suggesting that their respective intrinsic transcriptional regulatory competence is normal. Notably, these mutant ARs lose significantly more MB-binding activity than immunoreactivity, suggesting that prolonged MB exposure induces them to adopt a nonbinding state. The position homologous to Val865 in the AR is occupied by Leu or Met in the three steroid receptors closely related to the AR. This indicates the structural subtlety that underlies the steroid-binding activity of different steroid receptors.
A gramicidin S analog ([Orn1,1']GS.4HCl) containing L-ornithine in place of L-valine at the 1,1' positions was synthesized by the conventional solution method in order to examine whether this analog had antibacterial activity toward Gram-negative bacteria. In the synthesis of [Orn1,1']GS.4HCl, two intermediate analogs ([Orn1,1', Orn(For)2,2']GS.2HCl and [Orn(Z)1,1']GS.2HCl) were obtained. [Orn1,1']GS.4HCl and [Orn1,1', Orn(For)2,2']GS.2HCl showed no activity toward either Gram-negative or Gram-positive bacteria, whereas [Orn(Z)1,1']GS.2HCl showed appreciable activity toward only Gram-positive bacteria.
Thrombospondin-1 (TSP-1) is a matrix protein implicated in mechanisms of wound healing. TSP-1 contains the sequence cysteine-serine-valine-threonine-cysteine-glycine (CSVTCG) that has been shown to function primarily as a cell adhesion domain. Our laboratory has isolated a novel receptor specific for the CSVTCG adhesive domain of TSP-1. Immunohistochemical staining techniques and computerized image analysis were used to identify and quantitate TSP-1 and its CSVTCG receptor in surgically created colon anastomotic wounds. Histopathologic and quantitative examination demonstrated increased expression of TSP-1 and its CSVTCG receptor in areas of wound healing. These findings suggest a role for TSP-1 and its CSVTCG receptor in wound healing. The control of expression and activity of these molecules may eventually be the basis for the development of wound healing agents that could significantly reduce the morbidity from surgical intervention.
Thrombospondin-1 (TSP-1) is a 450 kDa matrix bound glycoprotein involved in tumor invasion, metastasis, and angiogenesis. One of the receptors involved in TSP-1 mediated tumor cell adhesion and metastasis is the cysteine-serine-valine-threonine-cysteine-glycine (CSVTCG) receptor. One mechanism of TSP-1 in promoting tumor cell metastasis involves the up-regulation of matrix metalloproteinase-9 (MMP-9) expression, specifically through the CSVTCG TSP-1 receptor. TSP-1 and its CSVTCG receptor has been implicated in tumor progression in a variety of cancers including breast adenocarcinomas, head and neck squamous cell carcinomas, and pancreatic carcinomas. In this study, we examined 99 cases of colorectal cancer by immunohistochemical analysis to investigate 1) the localization of TSP-1 and CSVTCG TSP-1 receptor, 2) the relationship with MMP-9, and 3) the correlation of expression with clinical staging. Strong expression of TSP-1 was observed in the submucosa or the serosa adjacent to the tumor. Positive staining for CSVTCG TSP-1 receptor was observed in tumor cells and microvessels. MMP-9 was also expressed in tumor cells. In addition, staining intensity of CSVTCG TSP-1 receptor was higher in poorly differentiated adenocarcinoma than well or moderately differentiated adenocarcinoma. Tumors in which inflammatory cells stained strongly for CSVTCG TSP-1 receptor correlated with decreased incidence of distant metastasis and angiogenesis. These data were consistent with our previous studies for breast, pancreatic, and head and neck carcinoma. They suggest an important role for TSP-1 and CSVTCG TSP-1 receptor in tumor progression in colorectal cancer.