Crystallization and preliminary X-ray diffraction studies of the N-terminal domain of the phosphorylating subunit of mannose permease from Escherichia coli.

Mannose permease is a constitutive component of the phosphotransferase system in Escherichia coli. This complex consists of two transmembrane subunits (II-PMan, Mr = 28,000 and II-MMan, Mr = 31,000) and a hydrophilic subunit (IIIMan). IIIMan functions as a phosphorylating enzyme and exists as a soluble homo-dimer of Mr = 70,000 in the cytosol. The N-terminal domain (P13) of IIIMan contains a phosphorylation site and the interface for dimerization. P13 has been crystallized in two different forms: type I, orthorhombic, space group C222 with a = 98.7 A, b = 106.5 A and c = 57.4 A, and type II, monoclinic, space group P2(1), with a = 54.4 A, b = 100.5 A, c = 58.1 A and beta = 90.5 degrees. Both types of crystal are suitable for X-ray diffraction studies.

Crystallization and preliminary X-ray diffraction studies of the spinach-chloroplast thioredoxin f.

Thioredoxins are low-molecular-mass proteins that function as hydrogen carriers in DNA synthesis and in the transformation of sulfur metabolites. They also act as regulatory proteins in the light-dependent enzyme activation during photosynthesis. F-type thioredoxin from spinach chloroplasts, a monomeric protein of 113 amino acid residues, has been found to specifically activate fructose-1,6-bisphosphatase and other key enzymes of CO2 assimilation. It has been crystallized in the monoclinic system, space group P2(1) with a = 30.6 A, b = 63.1 A, c = 31.6 A and beta = 110.7 degrees. The crystals are suitable for X-ray diffraction studies.

Structure of the IIA domain of the mannose transporter from Escherichia coli at 1.7 angstroms resolution.

The mannose transporter from Escherichia coli is a member of the phosphoenolpyruvate-dependent phosphotransferase system. The multi-subunit complex couples translocation across the bacterial inner membrane with phosphorylation of the solute. A functional fragment (IIA(Man), residues 2 to 133) of the membrane-associated IIAB(Man) subunit of the mannose transporter was expressed as a selenomethionine protein, and the unphosphorylated molecule was crystallized and its structure solved by X-ray crystallography. The protein consists of a central five-stranded beta-sheet covered by helices on either face. The order of the secondary structure elements is (beta alpha)4, alpha beta. Four beta-strands are arranged in a parallel manner with strand order 2134 and are linked by helices forming right-handed cross-over connections. The fifth strand that forms one edge of the sheet and runs antiparallel to the others is swapped between the subunits of the dimeric structure. Helices D and E form a helical hairpin. Histidine 10, which is transiently phosphorylated during catalysis, is located at the topological switch-point of the structure, close to the subunit interface. Its imidazole ring is hydrogen bonded to the buried side-chain of Asp67. It is likely that Asp67 acts as a general base and thus increases the nucleophilicity of the histidine. Modeling suggests that the covalently bound phosphoryl group would be stabilized by the macrodipole of helix C. Putative interactions between IIA(Man) and the histidine-containing phosphocarrier protein are discussed.

Predicted topology of the N-terminal domain of the hydrophilic subunit of the mannose transporter of Escherichia coli.

A folding topology for the homodimeric N-terminal domain (IIA, 2 x 14 kDa) of the hydrophilic subunit (IIABman) of the mannose transporter of E. coli is proposed. The prediction is based on (i) tertiary structure prediction methods, and (ii) functional properties of site-directed mutants in correlation with NMR-derived alpha/beta secondary structure data. The 3D structure profile suggested that the overall fold of IIA is similar to that of the unrelated protein, flavodoxin, which is an open-stranded parallel beta-sheet with a strand order of 5 4 3 1 2. The 3D model of IIA, constructed using the known atomic structure of flavodoxin, is consistent with the results from site-directed mutagenesis. Recently NMR results confirmed the open parallel beta-sheet with a strand order of 4 3 1 2 (residues 1-120) of our model whereas beta-strand 5 (residues 127-130) was shown to be antiparallel to beta-strand 4. The correctly predicted fold includes 90% of the monomeric subunit sequence and contains all functional sites of the IIA domain.

Macrophages as effector cells in immunity to malaria.

Experiments with malaria in mice suggest that protective immunity depends not only on antibody but also on activation of macrophages. Activated macrophages may cause intra-erythrocytic death of parasites by releasing reactive oxygen intermediates and/or tumour necrosis factor. Macrophage activation for both types of product correlates well with the timing of recovery in a range of different malaria infections and in mice protected by vaccination.

Malaria exoantigens induce TNF, are toxic and are blocked by T-independent antibody.

The production of cytokines, including tumour necrosis factor (TNF), may be involved in the pathology of malaria, as well as in protection against the parasite. We have shown that parasite exoantigens induce the secretion of TNF in vitro and in vivo and kill mice made hypersensitive to TNF. They elicit T-independent antibody that inhibits their capacity to stimulate TNF production and protects against toxicity in vivo, and those of human and rodent parasites are serologically related. Their active component does not appear to be protein. Here we review their properties and consider the epidemiological significance of our findings and their possible contribution to the development of an "anti-disease" vaccine.

[Bone histology and 25-OH vitamin D plasma levels in alcoholics without cirrhosis (author's transl)]. / Etude histologique de la minéralisation osseuse et taux de 25-hydroxycholecalciférol dans l'éthylisme chronique.

Bone mineralization is impaired in alcoholic non cirrhotics admitted to hospital for withdrawal. The defect is more marked in women than in men. Low 25-OH vitamin D plasma values may be observed in these patients and seem to be related to malnutrition. The degree of bone demineralization is not statistically correlated to 25-OH vitamin D plasma level.

[Should elevated beta-HCG levels be an exclusion criteria in clinical trials? A case report of paraneoplastic secretion associated with lung adenocarcinoma]. / L'élévation du taux de bêta-HCG doit-elle être un critère d'exclusion des essais thérapeutiques ? À propos d'un cas de sécrétion paranéoplasique associée à un adénocarcinome bronchique.

We report the case of a 55-year-old woman with pulmonary adenocarcinoma and bone metastases who was diagnosed with paraneoplastic secretion of the beta subunit of human chorionic gonadotropin (beta-HCG) while being screened for inclusion in a clinical trial. Immunohistochemistry analysis of a bone biopsy revealed strong staining of cancer cells with anti-beta HCG antibodies. Serial measurements of circulating Beta HCG seemed to be influenced by antineoplastic treatments, although they were not strictly associated with tumour evolution assessed by CT scans. Little is known about paraneoplastic secretion of beta HCG, although it has been found in 12% to 24% of non-small cell lung cancers. Usefulness of serial measurements of beta HCG for monitoring NSCLC has yet to be demonstrated, but its use as a criterion for inclusion in clinical trials needs to be questioned.

Transgenic mice and the study of cytokine function in infection.

Most information about the involvement of the different cytokines in immunity to infection has been obtained by the administration to infected animals of recombinant molecules or of antibodies against them. Now another approach to the study of cytokine function, in vivo, is available in the form of transgenic mice that express a transgene encoding a particular cytokine, and of 'knockout' animals, in which a cytokine gene, or a gene for its receptor (which usually comes to the same thing), have been rendered inactive by targeted disruption. While a few of these lines of mice have been analysed for their response to infection by protozoan parasites or worms, more have been tested for their ability to withstand intracellular infections by bacteria or viruses. In this review, Janice Taverne outlines those described to date in which the immune (or immunopathological mechanisms concerned may be relevant to parasitic diseases.

ParaSite: The Pick of Parasites on the Web.

While a steady flow of questions were put to the general parasitology newsgroup during November, none provoked discussion. Other discussion lists came to life:

The internet for scientists.

by Kevin O'Donnell and Larry Winger, Harwood Academic Publishers, 1997. pound14.50 (pbk)/ pound30.00 (hbk) (xi+309 pages) ISBN 90 5702 221 4.

Some consequences of the multiple infection of cell cultures by TRIC organisms.

In BHK 21 cells infected with more than one TRIC organism the inclusions coalesce so that 30 hr. after infection only one inclusion per cell remains. Six hours after infection the cells are as susceptible to further infection as uninfected cells.

Cultivation of TRIC agents: a comparison between the use of BHK-21 and irradiated McCoy cells.

BHK-21 cells and HeLa cells were as sensitive as irradiated McCoy cells for titrating both fast- and slow-growing strains of TRIC agents. BHK-21 cells were as efficient as irradiated McCoy cells for isolating TRIC agents from trachoma and from the genital tract of patients with non-gonococcal urethritis. More inclusions were formed in BHK-21 than in irradiated McCoy cells.

Neutralization of TRIC organisms by antibody: enhancement by antisera prepared against immunoglobulins.

The neutralizing activities of fowl, rabbit and mouse antisera prepared against TRIC agents were enhanced, in some cases over a 100-fold, by the addition of antiserum against the appropriate IgG. Significant non-specific inactivation of the organisms was observed in the absence of antiserum against TRIC agents, in reaction mixtures containing normal serum and antiserum against IgG. Since the extent of this inactivation showed a prozone, control tests with dilutions of normal serum equivalent to the full range of dilutions of the antiserum tested are necessary to show that enhancement of neutralization is specific.