Reversible, slow, tight-binding inhibition of human leukocyte elastase

CBz-Ala-Ala-Pro-ambo-Val-CF3 (1) was synthesized. The compound inhibits human Leucocyte elastase with Ki = 1.0 x 10(-9) M. This inhibitor is reversible, slow, tight-binding inhibitor with k on = 2 x 10(4) M-1 s-1 k off = 1.9 x 10(-5) s-1. For the solubilization of elastin by HLE by 1 I.C. 50 = 110 nM. This inhibitor is the most effective aldehyde or ketone inhibitor of a serine proteinase yet described.     

Subcloning,expression, purification,and characterization of recombinant human leptin-binding domain

A subdomain of the human leptin receptor encoding part of the extracellular domain (amino acids 428 to 635) was subcloned, expressed in a prokaryotic host, and purified to homogeneity, as evidenced by SDS-PAGE, with over 95% monomeric protein. The purified leptin-binding domain (LBD) exhibited the predicted beta structure, was capable of binding human, ovine, and chicken leptins, and formed a stable 1:1 complex with all mammalian leptins. The binding kinetics, assayed by surface plasmon resonance methodology, showed respective k(on) and k(off) values (mean +/- S.E.) of 1.20 +/- 0.23 x 10(-5) mol(-1) s(-1) and 1.85 +/- 0.30 x 10(-3) s(-1) and a K(d) value of 1.54 x 10(-8) m. Similar results were achieved with conventional binding experiments. LBD blocked leptin-induced, but not interleukin-3-induced, proliferation of BAF/3 cells stably transfected with the long form of human leptin receptor. The modeled LBD structure and the known three-dimensional structure of human leptin were used to construct a model of 1:1 LBD.human leptin complex. Two main residues, Phe-500, located in loop L3, and Tyr-441, located in L1, are suggested to contribute to leptin binding.     

A novel spectrofluorimetric method for determination of imatinib in pure,pharmaceutical preparation,human plasma,and human urine

The following paper represents a simple, highly sensitive, responsive validated and developed spectrofluorimetric method for estimation of imatinib (IMB) in its pure, commercial preparation, human urine and human blood plasma. The calibration curve was in the range 4–900 ng ml^?1 for pure form and urine and 8–900 ng ml^?1 for plasma in a medium contains carboxymethyl cellulose (CMC) and acetate buffer (pH 5) with excitation wavelength (λ_ex) 230 nm and emission wavelength (λ_em) 307 nm. The limit of detection (LOD) was 0.37 ng ml^?1 for the pure form, 0.64 ng ml^?1 for human urine, and 0.70 ng ml^?1 for human plasma, while the limit of quantitation (LOQ) was 1.2 for pure form, 1.91 for urine and 2.1 for plasma. The suggested method was successfully applied for evaluation of IMB in tablets within 99% mean percentage recovery. The excipients that are usually used as additives in pharmaceutical dosage form did not interfere with the suggested method. The method was efficiently used for estimation of IMB in human urine and human plasma. The effect of some cations that might be present in urine and plasma was also studied. The method was also focused on human volunteers and in vitro drug release.     

Cloning, genomic organization, and characterization of a human cholinephosphotransferase

A cholinephosphotransferase activity catalyzes the final step in the de novo synthesis of phosphatidylcholine via the transfer of a phosphocholine moiety from CDP choline to diacylglycerol. Ethanolaminephosphotransferase activity catalyzes a similar reaction substituting CDP ethanolamine as the phosphobase donor. We report the identification and cloning of a human cDNA (human cholinephosphotransferase (hCPT1)) that codes for a cholinephosphotransferase-specific enzyme. This was demonstrated using in vitro enzyme assays and in vivo measurement of the reconstitution of the phosphatidylcholine and phosphatidylethanolamine biosynthetic pathways in yeast cells devoid of their own endogenous cholinephosphotransferase and ethanolaminephosphotransferase activities. This contrasted with our previously cloned human choline/ethanolaminephosphotransferase cDNA that was demonstrated to code for a dual specificity choline/ethanolaminephosphotransferase. The hCPT1 and human choline/ethanolaminephosphotransferase (hCEPT1) predicted amino acid sequences possessed 60% overall identity and had only one variation in the amino acid residues within the CDP-alcohol phosphotransferase catalytic motif. In vitro assessment of hCPT1 and hCEPT1 derived cholinephosphotransferase activities also revealed differences in diradylglycerol specificities including their capacity to synthesize platelet-activating factor and platelet-activating factor precursor. Expression of the hCPT1 mRNA varied greater than 100-fold between tissues and was most abundant in testis followed by colon, small intestine, heart, prostate, and spleen. This was in marked contrast to the hCEPT1 mRNA, which has been found in similar abundance in all tissues tested to date. Both the hCPT1 and hCEPT1 enzymes were able to reconstitute the synthesis of PC in yeast to levels provided by the endogenous yeast cholinephosphotransferase; however, only hCEPT1-derived activity was able to complement the yeast CPT1 gene in its interaction with SEC14 and affect cell growth.     

Gangliosides of human, bovine, and rabbit plasma

Gangliosides were isolated from human, bovine, and rabbit plasma and were quantified by gas-liquid chromatography. Purification was achieved by sequential use of partitioning in solvents, DEAE-Sephadex chromatography, base treatment, and silicic acid chromatography. Human and bovine plasma yielded slightly more than 1 micro mole of lipid-bound sialic acid/100 ml; for rabbit plasma the value was 0.28 micro mole/100 ml. The total bovine plasma ganglioside fraction contained equal amounts of N-acetylneuraminic and N-glycolylneuraminic acids, rabbit plasma gangliosides had about 1% of the latter, and the human plasma sample contained only the former. Thin-layer chromatography revealed important differences among the plasmas from the three species, but all possessed hematosides and hexosamine-containing gangliosides. The approximate ratios of these two categories, based on sialic acid content, were (hematosides: hexosamine-type): human, 2:1; rabbit, 3:2; and bovine, 2:3. The fatty acid compositions of both categories were characteristic of extraneural gangliosides and included six major acids: palmitic, stearic, behenic, tricosanoic, lignoceric, and nervonic. The major long-chain base in each sample was sphingosine, while only a trace of the C(20) isomer was detected.     

Expression, purification, and characterization of human acetyl-CoA carboxylase 2

The full-length human acetyl-CoA carboxylase 1 (ACC1) was expressed and purified to homogeneity by two separate groups (Y.G. Gu, M. Weitzberg, R.F. Clark, X. Xu, Q. Li, T. Zhang, T.M. Hansen, G. Liu, Z. Xin, X. Wang, T. McNally, H. Camp, B.A. Beutel, H.I. Sham, Synthesis and structure-activity relationships of N-{3-[2-(4-alkoxyphenoxy)thiazol-5-yl]-1-methylprop-2-ynyl}carboxy derivatives as selective acetyl-CoA carboxylase 2 inhibitors, J. Med. Chem. 49 (2006) 3770-3773; D. Cheng, C.H. Chu, L. Chen, J.N. Feder, G.A. Mintier, Y. Wu, J.W. Cook, M.R. Harpel, G.A. Locke, Y. An, J.K. Tamura, Expression, purification, and characterization of human and rat acetyl coenzyme A carboxylase (ACC) isozymes, Protein Expr. Purif., in press). However, neither group was successful in expressing the full-length ACC2 due to issues of solubility and expression levels. The two versions of recombinant human ACC2 in these reports are either truncated (lacking 1-148 aa) or have the N-terminal 275 aa replaced with the corresponding ACC1 region (1-133 aa). Despite the fact that ACC activity was observed in both cases, these constructs are not ideal because the N-terminal region of ACC2 could be important for the correct folding of the catalytic domains. Here, we report the high level expression and purification of full-length human ACC2 that lacks only the N-terminal membrane attachment sequence (1-20 and 1-26 aa, respectively) in Trichoplusia ni cells. In addition, we developed a sensitive HPLC assay to analyze the kinetic parameters of the recombinant enzyme. The recombinant enzyme is a soluble protein and has a K(m) value of 2 microM for acetyl-CoA, almost 30-fold lower than that reported for the truncated human ACC2. Our recombinant enzyme also has a lower K(m) value for ATP (K(m)=52 microM). Although this difference could be ascribed to different assay conditions, our data suggest that the longer human ACC2 produced in our system may have higher affinities for the substrates and could be more similar to the native enzyme.     

Comparison of metabolic activation of carcinogens in human, rat, and hamster hepatocytes

The activities to activate and detoxify procarcinogens were compared in intact hepatocytes from humans, Sprague-Dawley rats and Syrian golden hamsters. Mutagenic metabolites that were released from the isolated hepatocytes were detected by mutation induction in co-cultivated Salmonella typhimurium TA98. Hepatocytes from the 3 animal species all activated aflatoxin B1 (AFB1), acetylaminofluorene (AAF) and aminofluorene (AF) and released active metabolites to induce mutation in the indicator S. typhimurium T98. Hamster hepatocytes were more effective than were human and rat hepatocytes to mediate mutation of Salmonella TA98 by AFB1, AAF and AF. Hepatocytes of human and rat failed to mediate mutation by 1-aminopyrene (1-AP). Indeed, at low concentration of 1-AP and 1-nitropyrene (1-NP), the presence of the hepatocytes decreased the number of TA98 revertants. Only at higher concentrations of 1-aminopyrene and 1-nitropyrene did hamster hepatocytes increase mutation frequencies of indicator cells over the control groups. It seems that hepatocytes, particularly human hepatocytes, are better able to absorb and detoxify 1-AP and 1-NP than to activate them.     

Purification of human interleukin 1 from human monocyte culture supernatants and identity of thymocyte comitogenic factor, fibroblast-proliferation factor, acute-phase protein-inducing factor, and endogenous pyrogen

Human interleukin 1 (IL-1) in lipopolysaccharide and silica-stimulated human peripheral blood monocyte culture supernatants was purified to apparent homogeneity by sequential chromatography using DEAE-Sephacel, Sephacryl S-200, CM-high-performance liquid chromatography (HPLC), and hydroxyapatite-HPLC. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) yielded only one band detectable by silver staining with an apparent molecular weight (MW) of 19,000 under nonreducing conditions. IL-1 activity was eluted from a single site from PAGE performed in the absence of SDS. About 4.4 micrograms of IL-1 was purified from 5.0 liters of culture supernatant of lipopolysaccharide- and silica-stimulated human peripheral blood monocytes, with 46.6% recovery of biological activity. The specific activity of the purified IL-1 was 4.3 X 10(7) U/mg protein. Amino acid composition analysis of the purified human IL-1 was similar to that previously described for murine IL-1. The purified IL-1 exhibited the biological activities previously attributed to IL-1, including thymocyte comitogenic activity, fibroblast proliferation activity, acute-phase protein (haptoglobin)-inducing activity, and endogenous pyrogen activity.     

SLLP1, a unique,intra-acrosomal,non-bacteriolytic,c lysozyme-like protein of human spermatozoa

We report the presence of a unique, non-bacteriolytic, c (chicken or conventional type) lysozyme-like protein, SLLP1, in the acrosome of human sperm. C lysozymes are bacteriolytic and can also bind to N-acetylglucosamines linked by beta-1,4 glycosidic bonds. Most of the invariant residues (17 out of 20), including all the cysteines, were conserved in SLLP1, but the two catalytic residues E35 and D52 of c lysozymes were replaced with T and N, respectively. The full-length cDNA encodes a protein of 215 aa with a predicted protease cleavage site between A87 and K88. The processed form of SLLP1, which showed an exon-intron organization similar to human c lysozyme, was the major isoform in the acrosome of ejaculated sperm. As expected, based on its sequence, the mature protein secreted from yeast showed no bacteriolytic activity. A significant decrease (54%, P < or = 0.001) in the number of sperm bound to zona-free hamster eggs was observed in the presence of antisera to recombinant SLLP1. SLLP1 mRNA (size, approximately 1 kb) appeared to be expressed only in the testis and in the Burkitt lymphoma Raji cell line. The gene SPACA3 encodes SLLP1 and contains five exons at locus 17q11.2. Because of its typical c lysozyme-like sequence, genomic organization, conservation of putative substrate-binding sites even in the absence of catalytic residues, and localization in the acrosomal matrix, we hypothesize that, after acrosome reaction, SLLP1 could be a potential receptor for the egg oligosaccharide residue N-acetylglucosamine, which is present in the extracellular matrix over the egg plasma membrane, within the perivitelline space, pores of zona pellucida, and cumulus layers.     

Expression,purification, and characterization of human tyrosyl-tRNA synthetase

Human tyrosyl-tRNA synthetase is a homodimeric enzyme and each subunit is near 58 KD. It catalyzes the aminoacylation of tRNA(Tyr) by L-tyrosine. The His(6)-tagged human TyrS gene was obtained by RT-PCR from total RNA of human lung giant-cell cancer strain 95 D. It was confirmed by sequencing and cloned into the expression vector pET-24 a (+) to yield pET-24 a (+)-HTyrRS, which was transfected into Escherichia coli BL21-CodonPlus-RIL. The induced-expression level of His(6)-tagged human TyrRS was about 24% of total cell proteins under IPTG inducing. The recombinant protein was conveniently purified in a single step by metal (Ni(2+)) chelate affinity chromatography. About 22.3mg purified enzyme could be obtained from 1L cell culture. The k(cat) value of His(6)-tagged human TyrRS in the second step of tRNA(Tyr) aminoacylation was 1.49 s(-1). The K(m) values of tyrosine and tRNA(Tyr) were 0.3 and 0.9 microM. Six His residues at the C terminus of human TyrRS have little effect on the activities of the enzyme compared with other eukaryotic TyrRSs.     

Resolution, detection, and characterization of redox conformers of human HSF1

We describe here an experimental protocol for the resolution, detection, and quantitation of the reduced and oxidized conformers of human heat shock factor 1 (hHSF1) and report on the effects in vitro and in vivo of redox-active agents on the redox status, structure, and function of hHSF1. We showed that diamide, a reagent that promotes disulfide bond formation, caused a loss of immunorecognition of the monomeric hHSF1 protein in a standard Western blot detection procedure. Modification of the Western blot procedure to include dithiothreitol in the equilibration and transfer buffers after gel electrophoresis allowed for the detection of a compact, intramolecularly disulfide cross-linked oxidized hHSF1 (ox-hHSF1) in the diamide-treated sample. The effect of diamide was blocked by pretreatment with N-ethylmaleimide and was reversed by dithiothreitol added to the sample prior to gel electrophoresis. Incubation with nitrosoglutathione at 42 degrees C also promoted the conversion of HSF1 to ox-HSF1; at 25 degrees C, however, nitrosoglutathione was by itself without effect but blocked the formation of ox-hHSF1 in the presence of diamide. The disulfide cross-linked ox-hHSF1 was monomeric and resistant to the in vitro heat-induced trimerization and activation. The possibility that ox-HSF1 may occur in oxidatively stressed cells was evaluated. Treatment of HeLa cells with 2 mm l-buthionine sulfoximine promoted the formation of ox-HSF1 and blocked the heat-induced activation of HSF DNA binding activity. Our result suggests that hHSF1 may have integrated redox chemistry of cysteine sulfhydryl into its functional responses.     

Specific binding, internalization, and degradation of human neutrophil activating factor by human polymorphonuclear leukocytes

The interaction of 125I-labeled recombinant human neutrophil activating factor (NAF) with polymorphonuclear leukocytes (PMN) was studied by means of a radioreceptor assay. The binding was characterized by a rapid transition (t1/2 less than or equal to 1 min) from a pH 3-sensitive state at 4 degrees C to pH 3 resistance at 37 degrees C. This was not caused by internalization of NAF since pH 3-resistant bound iodinated NAF could still be exchanged by an excess of nonlabeled NAF, i.e. was dissociable. Internalized iodinated NAF was processed into trichloroacetic acid-soluble forms. Scatchard transformation of binding isotherms at 4 and 37 degrees C led to nonlinear curves, a finding which is consistent with the expression of two receptor populations, one with high (KD = 11-35 pM) and the other with lower affinity (KD = 640-830 pM) at 4 degrees C. Numbers of the low affinity binding sites were approximately 34,000, and those with high affinity were 5,200/PMN when estimated at 4 degrees C. Binding of iodinated NAF to PMN was specific since it could be competed by an excess of nonlabeled NAF but not by two other activators of PMN function, formylmethionyl-leucyl-phenylalanine or human recombinant granulocyte-macrophage colony-stimulating factor. In addition to human PMN, NAF also bound specifically to two human monocytic cell lines; however, only the low affinity binding site could be detected on these cells.     

Structural comparison of human, bovine, rat, and Walker 256 carcinosarcoma asparaginyl-tRNA

The complete nucleotide sequences of human placenta, human liver, and bovine liver tRNAAsn have been determined. A comparison of these tRNA structures with the previously reported nucleotide sequences of rat liver and Walker 256 carcinosarcoma tRNAAns reveals that the primary nucleotide sequences of the major species of mammalian cytoplasmic tRNAasn are conserved in higher eucaryotes. The complete nucleotide sequence of these tRNAs is: pG-U-C-U-C-U-G-U-m1G-m2G-C-G-C-A-A-D-C-G-G-D-X-A-G-C-G-C-m2(2)G-psi-psi-C-G-G-C-U-Q(G)-U-U-t6A-A-C-C-G-A-A-A-G-m7G-D-U-G-G-U-G-G-Z-psi-C-G-m1A-G-C-C-C-A-C-C-C-A-G-G-G-A-C-G-C-C-AOH where X is 3-(3-amino-3-carboxyl-n-propyl)uridine, Q is 7-(4,5-cis-dihydroxyl-1-cyclopenten-3-yl-aminomethyl)-7-deazaguanosine, Z is an unknown modified nucleotide, and Q(G) represents the replacement of Q nucleoside by G nucleoside in Walker 256 carcinosarcoma tRNAAsn. These primary structures were determined by combined use of the 3H- and 32P-post-labeling techniques. Sequences were compared by tritium nucleoside trialcohol analysis, completed RNAase T1 digestion followed by 3H-labeled fingerprinting on polyethylenimine-impregnated cellulose by two-dimensional thin-layer chromatography (TLC), and polyacrylamide gel electrophoresis of either 5'-32P- and/or 3'-[32P]pCp-labeled tRNA after partial ribonuclease digestions.     

Inactivation of a human norovirus surrogate, human norovirus virus-like particles, and vesicular stomatitis virus by gamma irradiation

Gamma irradiation is a nonthermal processing technology that has been used for the preservation of a variety of food products. This technology has been shown to effectively inactivate bacterial pathogens. Currently, the FDA has approved doses of up to 4.0 kGy to control food-borne pathogens in fresh iceberg lettuce and spinach. However, whether this dose range effectively inactivates food-borne viruses is less understood. We have performed a systematic study on the inactivation of a human norovirus surrogate (murine norovirus 1 [MNV-1]), human norovirus virus-like particles (VLPs), and vesicular stomatitis virus (VSV) by gamma irradiation. We demonstrated that MNV-1 and human norovirus VLPs were resistant to gamma irradiation. For MNV-1, only a 1.7- to 2.4-log virus reduction in fresh produce at the dose of 5.6 kGy was observed. However, VSV was more susceptible to gamma irradiation, and a 3.3-log virus reduction at a dose of 5.6 kGy in Dulbecco's modified Eagle medium (DMEM) was achieved. We further demonstrated that gamma irradiation disrupted virion structure and degraded viral proteins and genomic RNA, which resulted in virus inactivation. Using human norovirus VLPs as a model, we provide the first evidence that the capsid of human norovirus has stability similar to that of MNV-1 after exposure to gamma irradiation. Overall, our results suggest that viruses are much more resistant to irradiation than bacterial pathogens. Although gamma irradiation used to eliminate the virus contaminants in fresh produce by the FDA-approved irradiation dose limits seems impractical, this technology may be practical to inactivate viruses for other purposes, such as sterilization of medical equipment.     

Expression, purification, and kinetic characterization of full-length human fibroblast activation protein

Human fibroblast activation protein (FAP), an integral membrane serine protease, was produced in insect cells as a hexa-His-tagged protein using a recombinant baculovirus expression system. Two isoforms of FAP, glycosylated and nonglycosylated, were identified by Western blotting using an anti-His-tag antibody and separated by lectin chromatography. The glycosylated FAP was purified to near homogeneity using immobilized metal affinity chromatography and was shown to have both postprolyl dipeptidyl peptidase and postgelatinase activities. In contrast, the nonglycosylated isoform demonstrated no detectable gelatinase activity by either zymography or a fluorescence-based gelatinase activity assay. The kinetic parameters of the dipeptidyl peptidase activity for glycosylated FAP were determined using dipeptide Ala-Pro-7-amino-trifluoromethyl-coumarin as the substrate. The k(cat) is 2.0 s(-1) and k(cat)/K(m) is 1.0 x 10(4) M(-1) s(-1) at pH 8.5. The pH dependence of k(cat) reveals two ionization groups with pK(a1) of 7.0 and pK(a2) of 11.0. The pH profile of k(cat)/K(m) yields similar results with pK(a1) 6.2 and pK(a2) 11.0. The neutral pK(a1) is associated with His at the active site. The basic pK(a2) might be contributed from an ionization group that is not involved directly in catalysis, instead associated with the stability of the active site structure.     

CJX1, an amlodipine derivative, interacts with ATPase of human P-glycoprotein

Our aim has been to elucidate the possible mechanism of CJX1, an amlodipine derivative, in the modulation of P-gp function by determining its effect on P-gp ATPase activity. Basal P-gp ATPase activity was increased by CJX1 with half-maximal activity concentration (Km) of 8.6 ± 1.4 μM. Kinetic analysis indicated a non-competitive inhibition of Verapamil (Ver)-stimulated P-gp ATPase activity by CJX1 and competitive inhibition of CJX1-stimulated P-gp ATPase activity by tetrandrine (Tet). The effect of CsA on CJX1-stimulated and Ver-stimulated P-gp ATPase activity was non-competitive and competitive inhibition, respectively. These findings implying that CJX1 and Tet can bind P-gp either on overlapping sites or distinct but interacting sites, while CJX1 and Ver as well as CsA can bind P-gp on separated sites in K562/DOX cells. Furthermore, the combined effect of CJX1 and Ver has been evaluated isobolographically in numerous fixed-ratio combinations of 1:1, 1:2, 1:4, 1:8, 1:10 in K562/DOX cells. The results show that mixtures of both drugs at these fixed-ratios exerted synergistic interactions, indicating that when the two reverses that bind P-gp on separated sites are combined, each can contribute to the overall interaction with P-gp, leading to the greater effect than that by either agent alone.     

Expression, purification, and characterization of recombinant human serum albumin fusion protein with two human glucagon-like peptide-1 mutants in Pichia pastoris

Glucagon-like peptide-1 (GLP-1) is a 30-residue peptide hormone secreted by intestinal L-cells in response to nutrient ingestion. In the present study, overlapping PCR technology was employed to construct two GLP-1 mutants (GLP-1(A2G))2 and human albumin (HSA) genes in vitro without linker. The spliced gene, (GLP-1(A2G))2-HSA, was over expressed under the control of promoter AOX1 and Mat alpha signal peptide in Pichia pastoris. SDS-PAGE and Western blotting were applied to assay the recombinant fusion protein in the culture broth. The results demonstrated that the recombinant (GLP-1(A2G))2-HSA concentration in the broth could reach a level of 245.0 mg/L and the expressed fusion protein was capable of cross-reacting with anti-human GLP-1 and anti-human albumin antibody. The recombinant (GLP-1(A2G))2-HSA protein was purified by ultrafiltration, columns of Q-sepharose fast flow and Superdex 75 size-exclusion. The recombinant (GLP-1(A2G))2-HSA protein obtained could lower in vivo glucose concentration in blood and stimulate in vitro islet cell proliferation. In mouse model, the fusion protein was detectable in plasma even 308 h after a single subcutaneous dose of 1.25 mg/kg. The result showed that the terminal biological half-time of the protein was about 54.2 h which is 650-fold longer than that of GLP-1. The pharmacokinetic analysis of the protein suggests its promising application in clinical medicine.