Solution structure and function of a conserved protein SP14.3 encoded by an essential Streptococcus pneumoniae gene.

Streptococcus pneumoniae is a major human pathogen that causes high mortality and morbidity rates and has developed resistance to many antibiotics. The genome of S. pneumoniae has recently been completely sequenced revealing many genes encoding hypothetical proteins of unknown function. We have found that the gene encoding one such conserved protein, SP14.3, is essential for growth of S. pneumonia. Since it is essential, SP14.3 represents a potential target for drug discovery. Here, we describe the three-dimensional solution structure of SP14.3 as determined by NMR spectroscopy. The structure consists of two domains each with an alpha/beta-fold. The N-terminal domain contains two alpha-helices and a three-stranded beta-sheet, while the C-terminal domain is composed of one alpha-helix and a five-stranded beta-sheet. The N-terminal domain of the protein contains a highly negatively charged surface and resembles the fold of the N-terminal domain of Thermus thermophilus ribosomal protein S3. The C-terminal domain has a protein fold similar to human small nuclear ribonucleoprotein Sm D3 and Haloarcula marismortui ribosomal protein L21E. The two domains of the protein tumble in solution overall as a whole with an overall molecular rotational correlation time (tau(m)) of 12.9 ns at 25 degrees C. The relative orientation of the two domains is not defined by the nuclear Overhauser effect data. Indeed, residual dipolar couplings and the structure calculations indicate that the relative orientation of the two domains is not rigidly oriented with respect to one another in solution.

Quantitative immunolocalization of a P29 protein (GRA7), a new antigen of toxoplasma gondii.

Ultrastructural localization of a P29 protein of Toxoplasma gondii was examined on thin sections by an immunogold technique using a P29 antigen-specific monoclonal antibody (5-241-178). Immunolocalization of the P29 protein in extracellular tachyzoites demonstrated that this antigen was present in the dense granules. Thus, we have identified this P29 antigen as the seventh protein (GRA7) to be localized to the dense granules of T. gondii. P29 immunolocalization in intracellular tachyzoites demonstrated association of this antigen with the parasite membrane complex, tubular elements of the intravacuolar network, and with the parasitophorous vacuolar membrane. Our immunolabeling data suggest trafficking of the P29 (GRA7) antigen from the dense granule via the intravacuolar network to the parasitophorous vacuolar membrane on invasion of the tachyzoite into the host cell. (J Histochem Cytochem 46:1411-1421, 1998)

Gemcitabine in locally advanced and metastatic non-small cell lung cancer: the Central European phase II study.

The efficacy and toxicity profile of gemcitabine was evaluated in this phase II study of chemonaive patients with locally advanced and metastatic non-small cell lung cancer (NSCLC). Eighty patients (62 males, 18 females) were entered into this study. The disease stage was IIIA in ten patients, IIIB in 32, and IV in 38 patients. The median age was 61 (range 41 - 78). Karnofsky performance status was > or = 80 in 88% of patients. All patients were chemonaive, but five patients had received prior radiotherapy and 34 patients had undergone prior surgery. Gemcitabine 1250 mg/m2 was given as a 30-min intravenous infusion on days 1, 8, and 15 of a 28-day cycle. Patients received up to nine cycles (median three cycles). Of 872 doses 815 (93%) were administered without dose delay or modification. Of the 80 patients enrolled, 76 were evaluable for efficacy analysis, and 16 patients had a partial response for an overall response rate of 21.1% (95% CI, 11.9-30.3%). A further 47 patients (61.8%) had stable disease. Partial responses were seen in eight of 41 stage III patients (19.5%) and in eight of 35 stage IV patients (22.9%). The median time to progressive disease was 4.6 months. Median survival for all 80 patients was 7.1 months. Haematological toxicity was mild with grade 3 4 neutropenia in 6.3% of patients, grade 3 thrombocytopenia in 3.8% of patients, and grade 3 anaemia in 2.5% of patients. Grade 3 non-laboratory toxicity was: somnolence (1.3% of patients), infection (1.3%), nausea and vomiting (6.4%) and dyspnoea (5.1%). This study confirms that single-agent gemcitabine is active in advanced NSCLC and its well-tolerated safety profile makes it particularly suited to outpatient use.

Evaluation of different inhaled combination therapies (EDICT): a randomised, double-blind comparison of Seretide (50/250 microg bd Diskus vs. formoterol (12 microg bd) and budesonide (800 microg bd) given concurrently (both via Turbuhaler) in patients with moderate-to-severe asthma.

The aim of this study was to compare the efficacy safety and cost of Seretide (salmeterol/fluticasone propionate (Salm/FP), 50/250 microg bd) via Diskus with formoterol (Form; 12 microg bd) and budesonide (Bud; 800 microg bd) given concurrently (Form+Bud) via Turbuhaler in patients with moderate-to-severe asthma who were uncontrolled on existing corticosteroid therapy. The study used a randomised, double-blind, double-dummy, parallel-group design, consisting of a 2-week run-in period on current corticosteroid therapy (1000-1600 microg/day of BDP or equivalent) and a 12-week treatment period. Symptomatic patients (n = 428) with FEV1 of 50-85% predicted and increased symptom scores or reliever use during run-in were randomly allocated to receive either Salm/FP (50/250 microg bd) via a single Diskus inhaleror Form+Bud (12+800 microg bd) via separate Turbuhalers. Clinic, diary card and asthma-related health-care resource utilisation data were collected. Improvement in mean morning peak expiratory flow (PEFam was similar in the Salm/FP and Form+Bud groups. Both PEFam and mean evening PEF (PEFpm) increased by a clinically significant amount (>20 L/min) from baseline in both treatment groups. The mean rate of exacerbations (mild, moderate or severe) was significantly lower in the Salm/FP group (0.472) compared with the Form+Bud group (0.735) (ratio = 0.64; P < 0.001), despite the three-fold lower microgram inhaled corticosteroid dose in the Salm/FP group. Patients in the Salm/FP group also experienced significantly fewer nocturnal symptoms, with a higher median percentage of symptom-free nights (P = 0.04), nights with a symptom score <2 (P = 0.03), and nights with no awakenings (P = 0.02). Total asthma-related health-care costs were significantly lower in the Salm/FP group than the Form+Bud group (P<0.05). Both treatments were well tolerated, with a similar low incidence of adverse events. This study showed that in symptomatic patients with moderate-to-severe asthma, Salm/FP (50/250 microg bd), administered in a single convenient device (Diskus), was at least as effective as an approximately three-fold higher microgram corticosteroid dose of Bud (800 microg bd) given concurrently with Form (12 microg bd) in terms of improvement in PEFam, and superior at reducing exacerbations and nights with symptoms or night-time awakenings. Salm/FP was also the less costly treatment due primarily to lower hospitalisation and drug costs.

Genome scanning in Haemophilus influenzae for identification of essential genes.

We have developed a method for identifying essential genes by using an in vitro transposition system, with a small (975 bp) insertional element containing an antibiotic resistance cassette, and mapping these inserts relative to the deduced open reading frames of Haemophilus influenzae by PCR and Southern analysis. Putative essential genes are identified by two methods: mutation exclusion or zero time analysis. Mutation exclusion consists of growing an insertional library and identifying open reading frames that do not contain insertional elements: in a growing population of bacteria, insertions in essential genes are excluded. Zero time analysis consists of monitoring the fate of individual insertions after transformation in a growing culture: the loss of inserts in essential genes is observed over time. Both methods of analysis permit the identification of genes required for bacterial survival. Details of the mutant library construction and the mapping strategy, examples of mutant exclusion, and zero time analysis are presented.

The DNA-binding protein pUL57 of human cytomegalovirus: comparison of specific immunoglobulin M (IgM) reactivity with IgM reactivity to other major target antigens.

In this work we used PCR to amplify the DNA regions coding for two polypeptides from pUL57 of human cytomegalovirus (amino acids 540 to 601 and 1144 to 1233) and showed that both portions reacted very efficiently with immunoglobulin M in sera of acutely infected subjects. However, pUL57 is not an essential antigen for the replacement of or supplement to a cocktail of recombinant protein antigens containing portions of ppUL32, -44, -83, and -80a in immunoglobulin M serology.

Recombinant antigens to detect Toxoplasma gondii-specific immunoglobulin G and immunoglobulin M in human sera by enzyme immunoassay.

We have evaluated the diagnostic utility of eleven Toxoplasma gondii recombinant antigens (P22 [SAG2], P24 [GRA1], P25, P28 [GRA2], P29 [GRA7], P30 [SAG1], P35, P41 [GRA4], P54 [ROP2], P66 [ROP1], and P68) in immunoglobulin G (IgG) and IgM recombinant enzyme-linked immunosorbent assays (Rec-ELISAs). Following an initial evaluation, six recombinant antigens (P29, P30, P35, P54, P66, and P68) were tested in the IgG and IgM Rec-ELISAs with four groups of samples which span the toxoplasmosis disease spectrum (negative, chronic infection, acute infection, and recent seroconversion). Our results suggest that the combination of P29, P30, and P35 in an IgG Rec-ELISA and the combination of P29, P35, and P66 in an IgM Rec-ELISA can replace the tachyzoite antigen in IgG and IgM serologic tests, respectively. The relative sensitivity, specificity, and agreement for the IgG P29-P30-P35 Rec-ELISA were 98.4, 95.7, and 97.2%, respectively. The resolved sensitivity, specificity, and agreement for the IgM P29-P35-P66 Rec-ELISA were 93.1, 95.0, and 94. 5%, respectively. Relative to the tachyzoite-based immunocapture IgM assay, the IgM P29-P35-P66 Rec-ELISA detects fewer samples that contain IgG antibodies with elevated avidity from individuals with an acute toxoplasmosis.

Development and clinical evaluation of a recombinant-antigen-based cytomegalovirus immunoglobulin M automated immunoassay using the Abbott AxSYM analyzer.

A new microparticle enzyme immunoassay (MEIA), the Cytomegalovirus (CMV) Immunoglobulin M (IgM) test, was developed on the Abbott AxSYM analyzer. This test uses recombinant CMV antigens derived from portions of four structural and nonstructural proteins of CMV: pUL32 (pp150), pUL44 (pp52), pUL83 (pp65), and pUL80a (pp38). A total of 1, 608 specimens from random volunteer blood donors (n = 300), pregnant women (n = 1,118), transplant recipients (n = 6), and patients with various clinical conditions and disease states (n = 184) were tested during development and evaluation of this new assay. In a preliminary clinical evaluation we tested specimens collected prospectively from pregnant women (n = 799) and selected CMV IgM-positive archived specimens from pregnant women (n = 39). The results from the new CMV IgM immunoassay were compared to the results of a consensus interpretation of the results obtained with three commercial CMV IgM immunoassays. The results for specimens with discordant results were resolved by a CMV IgM immunoblot assay. The relative sensitivity, specificity, and agreement for the AxSYM CMV IgM assay were 94.29, 96.28, and 96.19%, respectively, and the resolved sensitivity, specificity, and agreement were 95.83, 97.47, and 97.37%, respectively. We also tested serial specimens from women who experienced seroconversion or a recent CMV infection during gestation (n = 17) and potentially cross-reactive specimens negative for CMV IgM antibody by the consensus tests (n = 184). The AxSYM CMV IgM assay was very sensitive for the detection of CMV IgM during primary CMV infection, as shown by the detection of CMV IgM at the same time as or just prior to the detection of CMV IgG. Specimens from individuals with lupus (n = 16) or parvovirus B19 infection (n = 6) or specimens containing hyper IgM (n = 9), hyper IgG (n = 8), or rheumatoid factor (n = 55) did not cross-react with the AxSYM assay. One specimen each from individuals infected with Epstein-Barr virus (n = 26), measles virus (n = 10), herpes simplex virus (n = 12), or varicella-zoster virus (n = 13) infection, one specimen from an influenza vaccinee (n = 14), and one specimen containing antinuclear antibody cross-reacted with the assay. The overall rate of cross-reactivity of the specimens with the assay was 3.3% (6 of 184). The AxSYM CMV IgM assay is a sensitive and specific assay for the detection of CMV-specific IgM.