Green fluorescent protein as a reporter in rapid screening of antituberculosis compounds in vitro and in macrophages

Trypanosoma cruzi trans-sialidase consists of a C-terminal domain composed essentially of immunodominant amino acid repeat units (SAPA-repeats) and an amino region responsible for the enzymatic activity (catalytic domain). To investigate the possible function(s) of SAPA-repeats, recombinant trans-sialidases either containing or lacking the C-terminal region were tested in mice. The presence of SAPA-repeats in the intravenously injected protein has two consequences. First, they enhance the persistence of the trans-sialidase activity in blood. Second, SAPA-repeats promoted the production of antibodies directed to the catalytic domain that inhibit trans-sialidase activity. These results suggest that SAPA-repeats modulate the trans-sialidase activity in blood.     

beta-Galactosidase reporter system in mycobacteria and its application in rapid antimycobacterial drug screening

Osteopontin (OPN) is a ubiquitous multiphosphorylated secretory glycoprotein. Twenty-seven phosphorylated serines have been identified in bovine milk OPN (E. S. Sorensen et al. (1995) Protein Sci. 4, 2040-2049). Nineteen of these phosphoacceptor sites are fully conserved in rat OPN, all displaying the consensus for the Golgi apparatus casein kinase, G-CK (S-x-E/Sp). Here we show that rat OPN is indeed phosphorylated more readily than casein itself by G-CK from either rat mammary gland or liver. OPN is also phosphorylated by casein kinases-1 and -2 (CK1, CK2), though less readily than casein. If OPN kinase activities are normalized in terms of casein phosphorylation, OPN phosphorylation rate by G-CK is 78-fold and 19-fold higher than those measured with CK2 and CK1, respectively. These data, in conjunction with the specific location of G-CK to the Golgi apparatus, where CK2 and CK1 are hardly detectable, support the view that G-CK is the main if not the only physiological agent committed to the phosphorylation of OPN.     

The amplification refractory mutation system (ARMS): a rapid and direct prenatal diagnostic technique for beta-thalassaemia in Singapore

beta-Thalassaemia major patients have chronic anaemia and since 3-4 per cent of Singaporeans carry the beta-gene, prenatal diagnosis is essential. We evaluated the amplification refractory mutation system (ARMS) technique as a routine test for prenatal diagnosis of beta-major. Six mutations along the beta-gene were studied--41-42 (-TCTT), IVSII #654 (C-T), 17 beta (A-T), -28 TATA (A-G), IVSI #5 (G-C), and IVSI #1 (G-T). Our results indicate that prenatal diagnosis using these mutations can be offered to 90 per cent (35/39) of our Chinese couples and 54.6 per cent (12/22) of our Malay couples at risk. Confirmation of ARMS results was carried out using allele-specific oligonucleotide hybridization. Prenatal diagnosis using ARMS was successfully carried out in nine cases which included a set of triplets and twins. The triplets were diagnosed with the beta-trait carrying the 41-42 mutation. The couple with twins possessed the #654 mutation and one twin was diagnosed with the beta-trait and the other with #654 homozygosity. Genomic sequencing of the undefined mutations in the Chinese couples revealed rarer mutations at -29 and an ATG-AGG base substitution at the initiation codon for translation. In the Malay couples, genomic sequencing detected mutations at codon 15 (TGG-TAG) and codon 26 (GAG-AAG). We conclude that ARMS with its direct detection of amplified products by gel electrophoresis provides an accurate, rapid, and simpler method for our beta-thalassaemia prenatal diagnosis programme in Singapore.     

Detection of blotted antigen using a fusion protein between protein A and pepsinogen C

Human pepsinogen (PG) A and C were fused with protein A and expressed in Escherichia coli. Although the fusion proteins (PA-PGA and PA-PGC) were not expressed at high levels and were almost totally recovered from the insoluble fraction, the renaturation and purification procedures were easy and simple. PA-PGA and PA-PGC possessed proteolytic activity equivalent to the gastric mucosal PGA and PGC, respectively. However, the activity of PA-PGC was about 3-fold higher than that of PA-PGA. Therefore, PA-PGC was applied to the subsequent immunoblotting studies. The proteolytic activity of PA-PGC was used for digesting the blocking reagent around the target antigen (in situ digestion method) or casein-clotting in the agarose plate containing skimmed milk (caseogram print method). Although the sensitivity of these methods was lower than that of the conventional color detection, the caseogram print method was superior in that the reaction was linear over a wide range. On the other hand, the in situ digestion method possessed a unique property on Western blotting, and it was very easy to identify the relative position of the target, which could be recognized as a clear band. For PA-PGC detection, no special chemicals are required, and so the procedure is simple, rapid, and inexpensive.     

CCR: a rapid and simple approach for mutation detection

We describe a simple approach for detecting known mutations in genomic DNA. The strategy entails a DNA amplification reaction that combines the use of thermostable DNA polymerase and ligase, and that has been designated the Combined Chain Reaction (CCR). CCR consists of four phases: denaturation, annealing, elongation and ligation. Unlike most PCR-based mutation detection systems it relies on mismatch between primer and template at the primer 5'ends. It is rapid and simple, and requires neither the use of radioactivity, nor polyacrylamide gel electrophoresis, nor autoradiography for mutation detection at the single base-pair level.     

Synthesis and use of a biotinylated 3-azidophenothiazine to photolabel both amino- and carboxyl-terminal sites in calmodulin

The biotinylated probe, 3-azido-10-(4-(4-biotinyl-1-piperazinyl)butyl)phenothiazine, was used to examine the phenothiazine binding domains in calmodulin (CaM) by photolabeling. This phenothiazine, synthesized from 3-azido-10-(4-(1-piperazinyl)butyl)phenothiazine and d-biotinyl tosylate, inhibited the CaM-mediated activation of phosphodiesterase (PDE) with an I50 of 12.5 (+/- 2.8) microM. Photolabeling of CaM produced covalent adducts in excellent yield (32%) in a light- and Ca2+-dependent manner. Studies performed over a range of drug concentrations suggested a 2:1 stoichiometry for the binding of the phenothiazine probe to CaM. Limited trypsin digestion and purification of the resulting fragments by either SDS-PAGE or HPLC provided two principal phenothiazine-containing peptides. Amino acid composition and sequence analyses performed on these two peptides established that both the N- and C-terminal domains in CaM, particularly the regions amino terminal to Ca2+-binding loops 1 and 3, were modified by the photoactivated phenothiazine derivative. These data, particularly for the C-terminal domain, are in excellent agreement with the X-ray structure analysis of a 1:1 CaM-trifluoperazine complex.     

A new method for measuring menstrual blood loss and its use in screening women before endometrial ablation

OBJECTIVE: 1. To develop and validate a method for measuring menstrual blood loss in a routine setting, and 2. To assess the value of measuring menstrual blood loss before endometrial ablation. DESIGN: A prospective, observational study. SETTING: Four Yorkshire hospitals: The General Infirmary at Leeds, St. James's University Hospital, Leeds, St. Luke's Hospital, Bradford and The Friarage Hospital, Northallerton. PARTICIPANTS: Three hundred and seventy-two women who had been offered endometrial ablation for menorrhagia. MEASUREMENT: Sanitary material was washed with a nonionic detergent in a known volume of water. The haemoglobin in a sample of solution was measured by mixing with sodium carbonate for spectrophotometric analysis. INTERVENTIONS: The menstrual blood loss result was revealed to each women. Electrosurgical endometrial ablation was performed for those who decided to have surgery. MAIN OUTCOME MEASURES: Proportion of women with normal menstrual blood loss (< or = 80 mL) who avoided surgery. Comparison of endometrial ablation outcome in women with and without genuine menorrhagia. RESULTS: Thirty-six women (10%) with normal menstrual blood loss who declined surgery continued to avoid surgery after a mean of 27 months. Two hundred and ninety-two women were followed up for one year after endometrial ablation. Those with genuine menorrhagia (n = 122) were less likely to be dissatisfied (9% vs 18%) (OR 2.5, 95% CI 1.1-4.7) or to require hysterectomy (4% vs 7%) (OR 1.8, 95% CI 0.6-5.2) than women with normal menstrual blood loss (n = 170). CONCLUSIONS: The objective diagnosis of menorrhagia can be undertaken in a routine setting and may provide some women, who have a normal menstrual blood loss, sufficient reassurance to refrain from surgery. Women with genuine menorrhagia have a better outcome after endometrial ablation than those with normal menstrual blood loss.     

A fusion protein designed for noncovalent immobilization: stability, enzymatic activity, and use in an enzyme reactor

We have designed a new method for enzyme immobilization using a fusion protein of yeast alpha-glucosidase containing at its C-terminus a polycationic hexa-arginine fusion peptide. This fusion protein can be directly adsorbed from crude cell extracts on polyanionic matrices in a specific, oriented fashion. Upon noncovalent immobilization by polyionic interactions, the stability of the fusion protein is not affected by pH-, urea-, or thermal-denaturation. Furthermore, the enzymatic properties (specific activity at increasing enzyme concentration, Michaelis constant, or activation energy of the enzymatic reaction) are not influenced by this noncovalent coupling. The operational stability of the coupled enzyme under conditions of continuous substrate conversion is, however, increased significantly compared to the soluble form. Fusion proteins containing polyionic peptide sequences are proposed as versatile tools for the production of immobilized enzyme catalysts.     

MutS and MutL activate DNA helicase II in a mismatch-dependent manner

MutS, MutL, and DNA helicase II are required for the mismatch-provoked excision step that occurs during Escherichia coli methyl-directed mismatch repair. In this study MutL is shown to enhance the unwinding activity of DNA helicase II more than 10-fold on a conventional helicase substrate in which a 35-residue oligonucleotide is annealed to a M13 circular single-stranded phage DNA under conditions where the two proteins are present at approximately molar stoichiometry with respect to the substrate. MutS- and MutL-dependent activation of DNA helicase II has also been demonstrated with a model substrate in which a 138-residue oligonucleotide was hybridized to a 138-nucleotide gap in an otherwise duplex 7,100-base pair circular DNA. Displacement of the oligonucleotide requires MutS, MutL, DNA helicase II, and ATP and is dependent on the presence of a mismatch within the hybrid region. Although DNA helicase II and Rep helicase share substantial sequence homology and features of mechanism, Rep helicase is inactive in this reaction.     

Characterization of a natural mutation in an antigenic site on the fusion protein of measles virus that is involved in neutralization

Although measles virus is an antigenically monotypic virus, nucleotide sequence analysis of the hemagglutinin and nucleoprotein genes has permitted the differentiation of a number of genotypes. In contrast, the fusion (F) protein is highly conserved; only three amino acid changes have been reported over a 40-year period. We have isolated a measles virus strain which did not react with an anti-F monoclonal antibody (MAb) which we had previously shown to be directed against a dominant antigenic site. This virus strain, Lys-1, had seven amino acid changes compared with the Edmonston strain. We have shown that a single amino acid at position 73 is responsible for its nonreactivity with the anti-F MAb. With the same MAb, antibody-resistant mutants were prepared from the vaccine strain. A single amino acid change at position 73 (R-->W) was observed. The possibility of selecting measles virus variants in vaccinated populations is discussed.     

Quantitation of induced mutation in Dictyostelium discoideum: characterization and use of a methanol-resistance mutation assay

An assay based on forward mutation of Dictyostelium discoideum to 3% methanol resistance allows quantitation of induced mutation following treatment with physical and chemical agents. Properties of this assay are: uniform recovery of methanol-resistant (MeOHr) cells over a wide range of plated cell densities, equal growth rates of methanol-sensitive and methanol-resistant cells in the absence of methanol during the expression period and the attainment of plateau in the number of mutants as a function of expression time. When 4 mutagens were tested with this system and compared at the 10% survival level, N-methyl-N'-nitrosoguanidine was the most effective for mutation induction, followed by 60Co-gamma-rays, ultraviolet light, and methyl methanesulfonate.     

HRX leukemic fusion proteins form a heterocomplex with the leukemia-associated protein SET and protein phosphatase 2A

One of the most common chromosomal abnormalities in acute leukemia is a reciprocal translocation involving the HRX gene at chromosome locus 11q23, resulting in HRX fusion proteins. Using the yeast two-hybrid system, in vitro binding studies, and human cell culture coimmunoprecipitation experiments, we show here that a region of the HRX protein that is consistently retained in HRX leukemic fusion proteins interacts directly with SET, another protein implicated in leukemia. We have identified the binding sites on HRX for SET and show that these sequences are clustered near the A.T hooks that have been shown to bind DNA. We also show that carboxyl-terminal SET sequences, possibly the acidic tail of SET, bind to HRX. We have also found serine/threonine-specific protein phosphatase activity in anti-HRX coimmunoprecipitates. Using the phosphatase inhibitor okadaic acid and Western blotting, the phosphatase was identified as protein phosphatase 2A (PP2A). Mutation of a single amino acid in one of the SET binding sites of HRX resulted in lower amounts of both coimmunoprecipitated SET protein and coimmunoprecipitated PP2A. These results suggest that the leukemogenic effects of HRX fusion proteins may be related to interactions with SET and PP2A.     

Large-scale screening for factor V Leiden mutation in a north-eastern German population

A 37-year-old man was referred with thoracic pain after a deceleration trauma. He also had a cerebral contusion and a wrist fracture. There were no sings of hypovolemic shock. Computerized tomography (CT) of the chest and transoesophageal echocardiography (TEE) demonstrated a type B aortic dissection originating just distal to the left subclavian artery. There was a patent false lumen without rupture or distal ischaemia. Conservative treatment was given. A paralytic ileus developed and abdominal complaints persisted for several months. Angiography showed normal patency of mesenteric vessels. On follow-up, 3 years after the accident a slight aortic dilation was found on CT thorax without development of a post-dissection aneurysm. Blunt thoracic injury to the aorta usually gives rise to aortic rupture in the region of the isthmus, which can be complete or partial. In the latter case a false aneurysm may develop. An intimal tear after blunt trauma leading to type B aortic dissection rarely occurs. General principles regarding treatment of type B dissection also apply to this particular condition.     

A RANTES-antibody fusion protein retains antigen specificity and chemokine function

The successful eradication of cancer cells in the setting of minimal residual disease may require targeting of metastatic tumor deposits that evade the immune system. We combined the targeting flexibility and specificity of mAbs with the immune effector function of the chemokine RANTES to target established tumor deposits. We describe the construction of an Ab fusion molecule with variable domains directed against the tumor-associated Ag HER2/neu, linked to sequences encoding the chemokine RANTES (RANTES.her2.IgG3). RANTES is a potent chemoattractant of T cells, NK cells, monocytes, and dendritic cells, and expression of RANTES has been shown to enhance immune responses against tumors in murine models. RANTES.her2.IgG3 fusion protein bound specifically to HER2/neu Ag expressed on EL4 cells and on SKBR3 breast cancer cells as assayed by flow cytometry. RANTES.her2.IgG3 could elicit actin polymerization of THP-1 cells and transendothelial migration of primary T lymphocytes. RANTES.her2.IgG3 prebound to SKBR3 cells also facilitated migration of T cells. RANTES.her2.IgG3 bound specifically to the CCR5 chemokine receptor, as demonstrated by flow cytometry, and inhibited HIV-1 infection via the CCR5 coreceptor. RANTES.her2.IgG3, alone or in combination with other chemokine or cytokine fusion Abs, may be a suitable reagent for recruitment and activation of an expanded repertoire of effector cells to tumor deposits.     

A rapid silver staining and destaining technique for the nucleolus organizer region

Silver staining of nucleolar organizing regions (NOR) is common, but a standard protocol is lacking. A modification of a rapid silver nitrate staining technique for NORs is presented here. Advantages of the modified technique include reliability, speed, cost and the fact that it can be carried out in the light.