Quantitative, competitive PCR assay for HIV-1 using a microplate-based detection system

OBJECTIVE: Amylin, a secretory peptide of beta-cells, is the constituent peptide of islet amyloid, which is characteristic of NIDDM, and changes in amylin secretion in response to therapies may influence the rate of production of islet amyloid. The primary objective of this study was to determine whether therapy with sulfonylurea or basal insulin in NIDDM would alter amylin secretion in a way that might affect the formation of islet amyloid. RESEARCH DESIGN AND METHODS: In a randomized crossover design, eight subjects with NIDDM underwent three 8-week periods of therapy with diet alone, sulfonylurea, or exogenous basal insulin, with evaluation of amylin, amylin-like peptide (ALP), and glucose and C-peptide concentrations, both during fasting and after a standard breakfast. Changes in beta-cell function (% beta) were assessed, in the basal state by homeostasis model assessment (HOMA) and in the stimulated state by hyperglycemic clamps. Seven nondiabetic control subjects each underwent a meal profile and hyperglycemic clamp. RESULTS: Both sulfonylurea and insulin therapy reduced basal glucose concentrations compared with diet alone, but neither reduced the increased postprandial glucose increments. Both sulfonylurea and insulin therapy increased basal % beta, assessed by HOMA, but only sulfonylurea increased the second-phase C-peptide responses to the hyperglycemic clamp. Sulfonylurea increased time-averaged mean postprandial amylin and ALP concentrations compared with diet alone (geometric mean [1-SD range] for amylin, 4.9 [2.0-11.8] vs. 3.0 [1.4-6.2] pmol/l, P = 0.003; for ALP, 16.4 [8.5-31.7] vs. 10.1 [4.9-20.8] pmol/l, P = 0.001). Insulin therapy reduced basal ALP concentrations compared with diet alone (2.9 [1.5-5.6] vs. 6.0 [2.6-13.6] pmol/l, P = 0.03), but had no effect on postprandial concentrations of amylin (3.0 [1.3-6.5] pmol/l) or ALP (10.0 [5.5-18.1] pmol/l). CONCLUSIONS: By increasing postprandial concentrations of the constituent peptides of islet amyloid, sulfonylurea therapy might increase the rate of deposition of islet amyloid and thereby accelerate the decline of % beta in NIDDM, compared with diet therapy alone.     

Tuning the activity of an enzyme for unusual environments: sequential random mutagenesis of subtilisin E for catalysis in dimethylformamide

Random mutagenesis has been used to engineer the protease subtilisin E to function in a highly nonnatural environment--high concentrations of a polar organic solvent. Sequential rounds of mutagenesis and screening have yielded a variant (PC3) that hydrolyzes a peptide substrate 256 times more efficiently than wild-type subtilisin in 60% dimethylformamide. PC3 subtilisin E and other variants containing different combinations of amino acid substitutions are effective catalysts for transesterification and peptide synthesis in dimethylformamide and other organic media. Starting with a variant containing four effective amino acid substitutions (D60N, D97G, Q103R, and N218S; where, for example, D60N represents Asp-60-->Asn), six additional mutations (G131D, E156G, N181S, S182G, S188P, and T255A) were generated during three sequential rounds of mutagenesis and screening. The 10 substitutions are clustered on one face of the enzyme, near the active site and substrate binding pocket, and all are located in loops that connect core secondary structure elements and exhibit considerable sequence variability in subtilisins from different sources. These variable surface loops are effective handles for "tuning" the activity of subtilisin. Seven of the 10 amino acid substitutions in PC3 are found in other natural subtilisins. Great variability is exhibited among naturally occurring sequences that code for similar three-dimensional structures--it is possible to make use of this sequence flexibility to engineer enzymes to exhibit features not previously developed (or required) for function in vivo.     

Quantitative Limulus lysate assay for endotoxin activity: aggregation of radioiodinated coagulogen monomers

This communication describes a modification of the Limulus lysate assay which allows precise quantitation of picograms of bacterial lipopolysaccharide activity. The method measures the incorporation of 125I-labeled coagulogen monomers into the lysate clot as a function of lipopolysaccharide concentration. The method is more precise and requires less lysate than the previously described quantitative assays for endotoxin activity.     

Quantitative assessment of enzyme specificity in vivo: P2 recognition by Kex2 protease defined in a genetic system

The specificity of the yeast proprotein-processing Kex2 protease was examined in vivo by using a sensitive, quantitative assay. A truncated prepro-alpha-factor gene encoding an alpha-factor precursor with a single alpha-factor repeat was constructed with restriction sites for cassette mutagenesis flanking the single Kex2 cleavage site (-SLDKR downward arrowEAEA-). All of the 19 substitutions for the Lys (P2) residue in the cleavage site were made. The wild-type and mutant precursors were expressed in a yeast strain lacking the chromosomal genes encoding Kex2 and prepro-alpha-factor. Cleavage of the 20 sites by Kex2, expressed at the wild-type level, was assessed by using a quantitative-mating assay with an effective range greater than six orders of magnitude. All substitutions for Lys at P2 decreased mating, from 2-fold for Arg to >10(6)-fold for Trp. Eviction of the Kex2-encoding plasmid indicated that cleavage of mutant sites by other cellular proteases was not a complicating factor. Mating efficiencies of strains expressing the mutant precursors correlated well with the specificity (kcat/KM) of purified Kex2 for comparable model peptide substrates, validating the in vivo approach as a quantitative method. The results support the conclusion that KM, which is heavily influenced by the nature of the P2 residue, is a major determinant of cleavage efficiency in vivo. P2 preference followed the rank order: Lys > Arg > Thr > Pro > Glu > Ile > Ser > Ala > Asn > Val > Cys > AsP > Gln > Gly > His > Met > Leu > Tyr > Phe > Trp.     

The 0.78 A structure of a serine protease: Bacillus lentus subtilisin

Ultrahigh-resolution X-ray diffraction data from cryo-cooled, B. lentus subtilisin crystals has been collected to a resolution of 0.78 A. The refined model coordinates have a rms deviation of 0.22 A relative to the same structure determined at room temperature and 2.0 A resolution. Several regions of main-chain and side-chain disorder have been identified for 21 out of 269 residues in one polypeptide chain. Hydrogen atoms appear as significant peaks in the Fo - Fc difference electron density map, and carbon, nitrogen, and oxygen atoms can be differentiated. The estimated standard deviation (ESD) for all main-chain non-hydrogen bond lengths is 0.009 A and 0.5 degrees for bond angles based on an unrestrained full-matrix least-squares refinement. Hydrogen bonds are resolved in the serine protease catalytic triad (Ser-His-Asp). Electron density is observed for an unusual, short hydrogen bond between aspartic acid and histidine in the catalytic triad. The hydrogen atom, identified by NMR in numerous serine proteases, appears to be shared by the heteroatoms in the bond. This represents the first reported correlation between detailed chemical features identified by NMR and those in a cryo-cooled crystallographic structure determination at ultrahigh resolution. The short hydrogen bond, designated "catalytic hydrogen bond", occurs as part of an elaborate hydrogen bond network, involving Asp of the catalytic triad. While unusual, these features appear to have conserved analogues in other serine protease families although specific details differ from family to family.     

Quenched BODIPY dye-labeled casein substrates for the assay of protease activity by direct fluorescence measurement

We have prepared casein conjugates of two BODIPY dyes for use as fluorogenic protease substrates in homogeneous assays. Both conjugates are labeled to such an extent that the dyes are efficiently quenched in the protein, yielding virtually nonfluorescent substrate molecules. These fluorogenic substrates release highly fluorescent BODIPY dye-labeled peptides upon protease digestion, with fluorescence increases proportional to enzyme activity. These quenched substrates are suitable for the continuous assay of enzymatic activity using standard fluorometers, filter fluorometers, or fluorescence microplate readers using either fluorescein excitation and emission wavelengths to measure BODIPY FL casein hydrolysis or Texas Red wavelengths to detect proteolysis of BODIPY TR-X casein. Most current techniques for detecting protease activity, such as the fluorescein thiocarbamoyl casein (FTC-casein) protease assay, require extensive manipulation, including separation steps, and are therefore labor intensive and error-prone. In comparison, we found the BODIPY dye-labeled casein protease assays to be simple and precise and to have greater sensitivity and a broader dynamic range of detection than the FTC-casein assay. We were able to sensitively detect the activities of a wide variety of enzymes with these new substrates, including serine, acid, sulfhydryl, and metalloproteases. We also found the assay suitable for quantitating protease inhibitor concentrations and for real-time analysis of proteolysis.     

Standardization of an opsonophagocytic assay for the measurement of functional antibody activity against Streptococcus pneumoniae using differentiated HL-60 cells

Dopamine (DA) has been reported to depolarize neurons in the prefrontal cortex (PFC). To further characterize this effect of DA, we made whole cell recordings from PFC pyramidal cells in rat brain slices. As reported previously, DA depolarized most PFC cells tested. This effect of DA was concentration-dependent and persisted in the presence of synaptic blockade, indicating a direct effect of DA on the recorded cell. During DA-induced depolarization, PFC neurons consistently showed an increase in excitability, suggesting that the depolarization is not directly related to DA-induced inhibition of PFC neurons previously observed in vivo. Surprisingly, the effect of DA was not mimicked or blocked by several commonly used DA agonists and DA antagonists. The alpha and beta antagonists phentolamine and alprenolol and the atypical antipsychotic drug clozapine also showed no significant effect on DA-induced depolarization. These results suggest that DA-induced depolarization may be mediated by a nonspecific mechanism. However, it remains possible that there exists a new type of DA receptors in the PFC not sensitive to classical DA agonists and antagonists, particularly given the fact that DA applied in the same manner depolarized only PFC neurons but not those in the striatum or the substantia nigra.     

Development of a sensitive and specific assay system for cholecystokinin tetrapeptide

Cholecystokinin is a gastrointestinal and neuropeptide which has been implicated in a wide range of physiological and behavioral processes. We have developed a sensitive and specific assay system to measure the various forms of cholecystokinin (CCK) in human plasma. This 3-step system involves i) extraction of CCK fragments from plasma using reverse phase chromatography; ii) separation of peptides by high performance liquid chromatography; and iii) detection and quantification of peptides with a double-antibody radioimmunoassay, using an antibody raised against cholecystokinin tetrapeptide (CCK-4) coupled to thyroglobulin and 125I Bolton-Hunter CCK-4 as tracer. The antibody detects CCK-4, sulfated CCK-8 (CCK-8S) and nonsulfated CCK-8 (CCK-8ns) with equal affinity. The lower limit of detection is 2.7 fmol, with an ED50 of 10.6 +/- 2.2 fmol. Mean CCK-like immunoreactivity (CCK-LI) in the plasma of 12 healthy subjects was determined to be 12.9 +/- 2.1 pM CCK-4 equivalents. Concentrations of each individual peptide in plasma were determined to be 1.0 +/- 0.2 pM, 3.4 +/- 0.8 pM and 1.9 +/- 0.4 pM for CCK-4, CCK-8s and CCK-8ns respectively.     

An enzyme linked immunosorbent assay (ELISA) for detection of seminal fluid using a monoclonal antibody to prostatic acid phosphatase

A microtitre plate format enzyme linked immunosorbent assay (ELISA), employing commercially available PASE/4LJ mouse monoclonal hybridoma antibody is described. The technique is a solid phase indirect ELISA for prostatic acid phosphatase, applicable to specific detection of semen. Maximal detectability was found to be one hundred thousand fold dilution of pooled seminal plasma. No cross reactivities with human vaginal fluid, blood, saliva, female urine, nasal discharge, earwax, sweat or faeces have been found.     

Detection of glomerular basement membrane antibodies using the ELISA enzyme assay

BACKGROUND: Hamartoma of the optic disc is a rare disease but a very important one for differential diagnosis with choroidal melanoma. METHODS: A case of a boy, 17, referred to the clinic with suspicion of intrabulbar tumor is presented. Basing on the clinical picture ultrasonography and fluorescein angiography studies, diagnosis of retinal and pigment epithelium hamartoma of the optic disc was established. Laser treatment was applied. RESULTS: During 4-year follow-up visual acuity and ophthalmoscopic picture of the lesion has not deteriorated.     

Rapid assay processing by integration of dual-color fluorescence cross-correlation spectroscopy: high throughput screening for enzyme activity

Dual-color fluorescence cross-correlation spectroscopy (dual-color FCS) has previously been shown to be a suitable tool not only for binding but also for catalytic rate studies. In this work, its application as a rapid method for high-throughput screening (HTS) and evolutionary biotechnology is described. This application is called RAPID FCS (rapid assay processing by integration of dual-color FCS) and does not depend on the characterization of diffusion parameters that is the prerequisite for conventional fluorescence correlation spectroscopy. Dual-color FCS parameters were optimized to achieve the shortest analysis times. A simulated HTS with homogeneous assays for different restriction endonucleases (EcoRI, BamHI, SspI, and HindIII) achieved precise yes-or-no decisions within analysis times of about 1 s per sample. RAPID FCS combines these short analysis times with the development of fast and flexible assays resulting in sensitive, homogeneous fluorescence-based assays, where a chemical linkage between different fluorophores is either cleaved or formed, or where differently labeled molecules interact by noncovalent binding. In principle, assay volumes can be reduced to submicroliters without decreasing the signal strength, making RAPID FCS an ideal tool for ultra-HTS when combined with nanotechnology. RAPID FCS can accurately probe 10(4) to 10(5) samples per day, and possibly more. In addition, this method has the potential to be an efficient tool for selection strategies in evolutionary biotechnology, where rare and specific binding or catalytic properties have to be screened in large numbers of samples.     

Induction of nephrotoxic nephritis with purified antibody: preparation, properties and assay of antibody

The effects of Vinca alkaloids on the vas deferens have been examined after in vitro and in vivo administration. In the organ bath, vinblastine (1 X 10-4M for 4 hr) caused an increase in sensitivity to noradrenaline and biochemical, histochemical and ulstrastructural signs of noradrenaline depletion from the nerves supplying the outer longitudinal muscle layer. After intravenous injection of Vinca alkaloids the nerve fibres seemed less affected. It is concluded that the apparent resistance to the drugs in vivo is largely due to low tissue perfusion.     

A continuous colorimetric assay for rhinovirus-14 3C protease using peptide p-nitroanilides as substrates

Primary salivary gland lymphomas are almost always of B lineage, with most being represented by low grade B-cell lymphoma of mucosa-associated lymphoid tissue. This study characterizes the rare non-B-cell lymphomas of the salivary gland based on an analysis of six cases. All patients were men, with a mean age of 53.5 years. They presented with submandibular or parotid mass, which on histological examination showed extensive interstitial infiltration by small, medium-sized, or large lymphoid cells. There was prominent invasion and expansion of the ducts and acini in five cases. Angioinvasion was evident in two cases. Three cases were of T lineage and were CD56 negative; one of these cases expressed CD30. Three cases showed an immunophenotype of CD2+ CD3(f)- CD3(p)+ CD56+, consistent with T/natural killer (NK) cell lymphoma. In situ hybridization for Epstein-Barr virus (EBV)-encoded early nuclear RNA (EBER) showed positive reaction exclusively in the three CD56+ cases. Clonal T-cell populations were shown in two CD56-negative cases by polymerase chain reaction on paraffin sections using primers for the T-cell-receptor (TCR) gamma-chain gene, but not in the other four cases (the three CD56+ cases and one CD56- case). Four patients (two CD56+ and two CD56-) died within 3 years, and two were disease free at 4 and 1.5 years, respectively. This study shows that salivary gland T- or T/NK-cell lymphomas cannot be reliably distinguished from B-cell lymphomas on morphological grounds alone, because both can show prominent lymphoepithelial lesions. It appears that T/NK-cell lymphomas, which are often extranodal in localization and strongly associated with Epstein-Barr virus (EBV), show a predilection to involve the salivary glands as well.     

应用酶联免疫吸附试验检测血清桥粒芯蛋白抗体水平监测寻常型天疱疮患者病情变化

目的:研究较大样本寻常型天疱疮(pemphigus vulgaris,PV)患者,血清抗桥粒芯蛋白(desmoglein,Dsg)1和抗Dsg3特异性抗体酶联免疫吸附试验(enzyme-linked immunosorbent assay,ELISA)指数长期的变化情况,观察其与病情变化的相关性,探讨其用于病情监测、预测疾病复发和指导治疗的可行性.方法:对20例确诊PV的患者进行长期随访,收集患者在各随访时间点的血清,记录每次随访时的病情并进行评分(autoimmune bullous skin disorder intensity score,ABSIS);检测患者血清的间接免疫荧光(indirect immunofluorescence,IIF)滴度、Dsg3 ELISA指数和Dsg1 ELISA指数;利用统计学方法和做图方法分析病情评分与Dsg3 ELISA指数、Dsg1 ELISA指数和IIF滴度之间的关系.结果:PV患者皮肤和口腔的病情评分,分别与Dsg3 ELISA指数、Dsg1 ELISA指数和IIF滴度均具有显著性关联(P＜0.01),患者疾病活动期和临床缓解期两组间Dsg3 ELISA指数、Dsg1 ELISA指数和IIF滴度差异均有统计学意义(P＜0.01).各组患者相关性分析发现,Dsg3 ELISA指数、Dsg1 ELISA指数和IIF滴度几乎均与病情平行波动变化,ELISA指数优于IIF的平行性,并且ELISA指数可以预测病情是否会反复,从而指导治疗.结论:PV患者血清抗Dsg3抗体ELISA指数与病情变化平行波动,可以反映疾病的活动程度,可用于病情监测,并可为临床上预测疾病复发,指导临床调整治疗提供有利的实验室证据.