Rapid purification of human complement receptor type 1 (CD35, CR1)

BACKGROUND: Migraine is a prevalent disorder whose relationship to other conditions remains poorly understood. METHODS: Associations between migraine and physiological, behavioral, and demographic characteristics were assessed in a retrospective cohort study of 79,588 enrollees in a large prepaid health maintenance organization who underwent a multiphasic preventive health checkup in 1971-1973. RESULTS: Migraine was found to be inversely associated with age and education and strongly associated with the female sex. The likelihood of migraine was significantly higher among blacks, smokers, those who drink more than six cups of coffee per day, those with Raynaud's syndrome, and those with a family history of migraine. The magnitude of associations between migraine and other factors was, in general, reduced among those with a self-reported physician diagnosis of migraine compared to those whose migraine status was defined on the basis of reported symptoms. CONCLUSIONS: Migraine prevalence was found to be higher in blacks and other unspecified minorities than in the white population. The magnitude of the associations between migraine and behavioral risk factors was strongly influenced by the method of migraine ascertainment. The inverse association with level of education suggests that social causation or drift may have been operating in this disease in the early 1970s, 15 to 20 years earlier than recent population-based studies would suggest. Further research is needed to fully appreciate the spectrum of disease and behaviors associated with migraine.     

Rapid purification and properties of betaine aldehyde dehydrogenase from Pseudomonas aeruginosa

Betaine aldehyde dehydrogenase (BADH) (EC 1.2.1.8) catalyzes the last, irreversible step in the synthesis of the osmoprotectant glycine betaine from choline. In Pseudomonas aeruginosa this reaction is also an obligatory step in the assimilation of carbon and nitrogen when bacteria are growing in choline or choline precursors. We present here a method for the rapid purification to homogeneity of this enzyme by the use of ion-exchange and affinity chromatographies on 2',5'-ADP-Sepharose, which results in a high yield of pure enzyme with a specific activity at 30 degreesC and pH 7.4 of 74.5 U/mg of protein. Analytical ultracentrifugation, gel filtration, chemical cross-linking, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis suggest that BADH from P. aeruginosa is a homodimer with 61-kDa subunits. The amino acid composition and the N-terminal sequence of 21 amino acid residues showed significant similarity with those of the enzymes from Xanthomonas translucens and Escherichia coli. Neither BADH activity nor BADH protein was found in cell extracts from bacteria grown in the absence of choline. In contrast to other BADHs studied to date, the Pseudomonas enzyme cannot use positively charged aldehydes other than betaine aldehyde as substrates. The oxidation reaction has an activation energy of 39.8 kJ mol-1. The pH dependence of the velocity indicated an optimum at pH 8.0 to 8.5 and the existence of two ionizable groups with macroscopic pK values of 7.0 +/- 0.1 and 9. 7 +/- 0.1 involved in catalysis and/or binding of substrates. The enzyme is inactivated at 40 degreesC, but activity is regained when the heated enzyme is cooled to 30 degreesC or lower. At the optimum pH of 8.0, the enzyme is inactivated by dilution, but it is stable at pH 6.5 even at very low concentrations. Also, P. aeruginosa BADH activity is rapidly lost on removal of K+. In all cases studied, inactivation involves a biphasic process, which was dependent on the enzyme concentration only in the case of inactivation by dilution. NADP+ considerably protected the enzyme against these inactivating conditions.     

Maturation of peroxisomes in differentiating human hepatoblastoma cells (HepG2): possible involvement of the peroxisome proliferator-activated receptor alpha (PPAR alpha)

Although several variant alleles at the human NAT1 gene locus have been reported, their relationship to phenotypic variations in NAT1 function remains unclear. We have used in-vivo and invitro phenotyping tests, along with PCR-based cloning and heterologous expression, to investigate the extent of variation in NAT1 function and to characterize novel allelic variants at the NAT1 gene locus. The NAT1-selective substrate p-aminosalicylic acid (PAS) was used as a probe for NAT1 function. In-vivo PAS acetylation rates were estimated by determining the ratio of PAS to N-acetylated PAS (AcPAS) in urine and plasma following the oral ingestion of Nemasol Sodium. Excluding outliers, a 65-fold variation in the urinary AcPAS:PAS ratio was observed (n = 144), while a 5.6-fold variation in the plasma AcPAS:PAS ratio was seen in a subset (n = 19) of this sample. Urinary and plasma ratios correlated moderately (r = 0.74, p < 0.0005). One individual (case 244) had a marked impairment of PAS N-acetylation, with 10-fold lower urinary and plasma AcPAS:PAS ratios compared with other subjects. Biochemical investigations in whole blood lysates from case 244 suggested a NAT1 kinetic defect, with a 20-fold increased apparent K(m) for PAS and a 90-fold decreased Vmax for AcPAS formation. We subcloned, sequenced and expressed the protein-coding regions of the NAT1 alleles from case 244 and from seven other selected probands. Sequence analysis revealed the presence of two new variant alleles, designated as NAT1 x 14 and NAT1 x 15, in case 244, as well as one variant, NAT1 x 11, which has been observed in previous investigations. NAT1 x 14 contained a missense mutation (G560-->A) that is predicted to change a single amino acid (Arg187-->Gln), as well as two 3' non-coding region mutations (T1088-->A and C1095-->A) that have previously been observed in the NAT1 x 10 allelic variant. NAT1 x 15 had a single nonsense mutation (C559-->T; Arg187-->stop) and, thus, encodes a truncated protein. The activity of recombinant NAT1 14 mirrored the defective enzyme function in whole blood lysates from case 244, while NAT1 15 was completely inactive. Expressed NAT1 11, on the other hand, had identical activity to the wild type NAT1 4 allele, suggesting that the coding region mutations in this variant are functionally silent. The frequencies of NAT1 x 11, NAT1 x 14 and NAT1 x 15 were 0.021, 0.028 and 0.014 (n = 288 alleles), respectively, suggesting that they are relatively rare in our predominantly Caucasian sample.     

Rapid purification of cationic granule proteases: application to human granzymes

This report describes a simple scheme for the simultaneous purification of cationic human granzymes A, B, and 3 from human interleukin 2 (IL-2)-activated lymphocytes. The process, which requires approximately 8 h, includes: (1) cell cavitation, (2) two centrifugation steps, (3) four granule solubilization steps, and (4) cation-exchange chromatography. Granule solubilization consists of three extractions with a hypotonic buffer (25 mM NaCl) that contained Triton X-100 followed by a final extraction in hypertonic detergent-free buffer (390 mM NaCl). We recovered approximately 35% of the trypsin-like (tryptase) activities mediated by granzymes A and 3, respectively, and approximately 25% of the asp-ase activity of granzyme B. The granzymes were identified after elution from the Mono S column by Western blot with a polyclonal antibody that reacts with a conserved amino acid sequence (9-16) of lymphoid/myeloid serine proteases. By amino-terminal sequencing, eluted granzyme A and B were indeed homogeneous. Granzyme 3, although highly enriched, appears to be contaminated with an uncharacterized granzyme. Although we have developed this scheme to rapidly isolate the granzymes, the procedure should assist the purification of secretory granule-associated cationic proteins that reside in neutrophils and mast cells as well as other cells that possess secretory function.     

Partial purification and some properties of human liver alkaline phosphatase

1. Alkaline phosphatase (EC 3.1.3.1) from human liver was solubilized from the homogenate using 0.2% Triton X-100 containing 0.2 M lithium 3,5-diiodosalicylate, and the pellet obtained was resolubilized with 20% n-butanol. The procedure resulting in 3842-fold purification included acetone fractionation, ammonium sulfate precipitation, DEAE-cellulose chromatography, Sephadex G-200 gel filtration, hydroxyapatite gel chromatography and further concanavalin A/Sepharose 4B affinity chromatography. 2. The highly purified enzyme showed one major protein band on acrylamide gel electrophoresis at pH 8.6, and exhibited one-seventh of the alkaline p-nitrophenylphosphatase activity in the hepatic enzyme preparation contains of the alkaline pyrophosphatase activity. 3. The highly purified enzyme was a sialic-acid containing glycoprotein. 4. Sialidase-treated hepatic enzyme clearly presented the phenomenon of delayed mobility, and the delayed enzyme fraction stained more strongly than that of non-treated hepatic alkaline phosphatase. 5. In order to investigate the role of the carbohydrate region(s) of the hepatic alkaline phosphatase molecule on substrate binding, the effect of sialidase treatment on the rate of substrate inhibition of alkaline phosphatase was studied. In the case of hepatic enzyme without sialidase, substrate inhibition of alkaline phosphatase activity was clearly shown, while in the case of the hepatic enzyme with sialidase, there was hardly any substrate inhibition in the range of 1-8 mM p-nitrophenylphosphate.     

Antitumor activity of recombinant human tumor necrosis factor-alpha (rH-TNF alpha) and liposome-entrapped rH-TNF alpha

The antitumor activities of recombinant human tumor necrosis factor-alpha (rH-TNF alpha) and liposome-entrapped rH-TNF alpha were evaluated in various glioma cell lines and a rat brain T9 gliosarcoma model. rH-TNF alpha had a direct cytotoxic activity against various glioma cell lines in vitro, and indirect cytotoxic activity against gliosarcoma (T9) in vivo. Liposome-entrapped rH-TNF alpha had increased direct cytotoxic activity in vitro, and against experimentally induced brain tumors in vivo. The effects in vivo were probably due to vascular damage of the tumor vessels as shown by histological examination and activation of cytotoxic macrophages as shown in vitro. These results indicate that the general or local administration of liposome-entrapped rH-TNF alpha may become a useful adjunct treatment for malignant brain tumor.     

Cloning, purification, and properties of a phosphotyrosine protein phosphatase from Streptomyces coelicolor A3(2)

We have investigated by restriction fragment analysis genomic abnormalities involving the c-myc gene in DNA isolated from adenomas and hepatocellular carcinomas (HCCs). Adenomas and HCCs were induced by the "resistant hepatocyte" protocol in diethylnitrosamine-initiated male F344 rats. Southern-blot analysis of EcoRI-restricted DNA from normal liver, early and late adenomas, 12 weeks (EAs) and 30 weeks (LAs) after initiation, and HCCs, showed 2 bands of 18 and 3.2 kb hybridizing with c-myc, in all tissues. c-myc amplification occurred in almost all HCCs, and in the majority of EAs and LAs. These results were confirmed by dilution analysis. c-myc amplification was also seen in adenomas and HCCs by Southern analysis with HindIII-restricted DNA, and in HCCs by differential PCR. c-myc mRNA increase occurred in all adenomas and HCCs, but it was higher in the lesions showing gene amplification. Moreover, a 13-kb DNA extraband, hybridizing with c-myc, was found in the HindIII-restricted DNA from HCCs, but not in normal liver and adenomas, and a 7.1-kb extra band was present in EcoRI-digested DNA from one LA. EcoRI-restricted DNA from some adenomas exhibited a decrease in intensity of the 18-kb fragment, and an increase in intensity of the 3.2-kb fragment. No alteration in banding pattern occurred in the beta-actin gene in adenomas. These results provide evidence of amplification and some other rearrangements involving the c-myc gene, in pre-malignant and malignant liver lesions, induced by the RH protocol, and suggest a role of c-myc rearrangement in the progression of adenomas to malignancy.     

Activation kinetics and single channel properties of recombinant alpha1beta2gamma2L GABA(A) receptor channels

Recombinant alpha1beta2gamma2L GABA(A) receptor channels, transiently expressed in HEK 293 cells, were investigated using the patch-clamp technique in combination with a device for ultra-fast solution exchange. The dose-response relationship revealed an EC50 of 11.6 +/- 0.9 microM and saturated with 3 mM GABA. The slope between 0.001 and 0.01 mM GABA was 2.2 +/- 0.4, indicating at least three binding sites for GABA. The rise time decreased from about 120 ms at 0.001 mM GABA to about 0.8 ms at 10 mM GABA. Single channel openings were grouped in bursts with an average duration of 10.3 +/- 3.0 ms. More than 95% of the current was represented by a single channel slope conductance of about 29 pS.     

Affinity purification and some molecular properties of human liver alkaline phosphatase

Alkaline phosphatase from human liver was purified to homogeneity. The purification procedure included solubilization with butanol, fractionation with acetone, and chromatography on concanavalin A-Sepharose, DEAE-cellulose, Sephadex G-200 and DEAE-Sephadex. Purity was established by standard and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. The isoelectric point of the protein was determined to be 4.0. Sephadex-gel filtration gave a mol.wt. of 146000, although a higher value was obtained in the presence of 100mM-NaC1. The subunit mol.wt. 76700, was determined by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Neuraminidase treatment resulted in two enzyme-activity bands on isoelectric-focused gels with isoelectric points of 6.6 and 6.8. The desialylated enzyme gave only one protein band on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis with a subunit molecular weight indistinguishable from that of the non-neuraminidase-treated protein. The desialylated enzyme was more readily denatured by sodium dodecyl sulphate in the presence of mercaptoethanol than was the native enzyme.     

Expression, purification, and inhibitory properties of human proteinase inhibitor

In a previous report, the cDNA for human proteinase inhibitor 8 (PI8) was first identified, isolated, and subcloned into a mammalian expression vector and expressed in baby hamster kidney cells. Initial studies indicated that PI8 was able to inhibit the amidolytic activity of trypsin and form an SDS-stable approximately 67-kDa complex with human thrombin [Sprecher, C. A., et al. (1995) J. Biol Chem. 270, 29854-29861]. In the present study, we have expressed recombinant PI8 in the methylotropic yeast Pichia pastoris, purified the inhibitor to homogeneity, and investigated its ability to inhibit a variety of proteinases. PI8 inhibited the amidolytic activities of porcine trypsin, human thrombin, human coagulation factor Xa, and the Bacillus subtilis dibasic endoproteinase subtilisin A through different mechanisms but failed to inhibit the Staphylococcus aureus endoproteinase Glu-C. PI8 inhibited trypsin in a purely competitive manner, with an equilibrium inhibition constant (Ki) of less than 3.8 nM. The interaction between PI8 and thrombin occurred with a second-order association rate constant (kassoc) of 1.0 x 10(5) M-1 s-1 and a Ki of 350 pM. A slow-binding kinetics approach was used to determine the kinetic constants for the interactions of PI8 with factor Xa and subtilisin A. PI8 inhibited factor Xa via a two-step mechanism with a kassoc of 7.5 x 10(4) M-1 s-1 and an overall Ki of 272 pM. PI8 was a potent inhibitor of subtilisin A via a single-step mechanism with a kassoc of 1.16 x 10(6) M-1 s-1 and an overall Ki of 8.4 pM. The interaction between PI8 and subtilisin A may be of physiological significance, since subtilisin A is an evolutionary precursor to the intracellular mammalian dibasic processing endoproteinases.