Leishmania major: histone H1 gene expression from the sw3 locus

Histone H1 in the parasitic protozoan Leishmania is a developmentally regulated protein encoded by the sw3 gene. Here we report that histone H1 variants exist in different Leishmania species and strains of L. major and that they are encoded by polymorphic genes. Amplification of the sw3 gene from the genome of three strains of L. major gave rise to different products in each strain, suggesting the presence of a multicopy gene family. In L. major, these genes were all restricted to a 50-kb Bg/II fragment found on a chromosomal band of 1.3 Mb (chromosome 27). The detection of RFLPs in this locus demonstrated its heterogeneity within several species and strains of Leishmania. Two different copies of sw3 (sw3.0 and sw3.1) were identified after screening a cosmid library containing L. major strain Friedlin genomic DNA. They were identical in their 5' UTRs and open reading frames, but differed in their 3' UTRs. With respect to the originally cloned copy of sw3 from L. major strain LV39, their open reading frames lacked a repeat unit of 9 amino acids. Immunoblots of L. guyanensis parasites transfected with these cosmids revealed that both copies could give rise to the histone H1 protein. The characterization of this locus will now make possible a detailed analysis of the function of histone H1 in Leishmania, as well as permit the dissection of the molecular mechanisms governing the developmental regulation of the sw3 gene.     

Anaerobic expression of Escherichia coli succinate dehydrogenase: functional replacement of fumarate reductase in the respiratory chain during anaerobic growth

Succinate-ubiquinone oxidoreductase (SQR) from Escherichia coli is expressed maximally during aerobic growth, when it catalyzes the oxidation of succinate to fumarate in the tricarboxylic acid cycle and reduces ubiquinone in the membrane. The enzyme is similar in structure and function to fumarate reductase (menaquinol-fumarate oxidoreductase [QFR]), which participates in anaerobic respiration by E. coli. Fumarate reductase, which is proficient in succinate oxidation, is able to functionally replace SQR in aerobic respiration when conditions are used to allow the expression of the frdABCD operon aerobically. SQR has not previously been shown to be capable of supporting anaerobic growth of E. coli because expression of the enzyme complex is largely repressed by anaerobic conditions. In order to obtain expression of SQR anaerobically, plasmids which utilize the PFRD promoter of the frdABCD operon fused to the sdhCDAB genes to drive expression were constructed. It was found that, under anaerobic growth conditions where fumarate is utilized as the terminal electron acceptor, SQR would function to support anaerobic growth of E. coli. The levels of amplification of SQR and QFR were similar under anaerobic growth conditions. The catalytic properties of SQR isolated from anaerobically grown cells were measured and found to be identical to those of enzyme produced aerobically. The anaerobic expression of SQR gave a greater yield of enzyme complex than was found in the membrane from aerobically grown cells under the conditions tested. In addition, it was found that anaerobic expression of SQR could saturate the capacity of the membrane for incorporation of enzyme complex. As has been seen with the amplified QFR complex, E. coli accommodates the excess SQR produced by increasing the amount of membrane. The excess membrane was found in tubular structures that could be seen in thin-section electron micrographs.     

Resistance to Leishmania major is linked to the H2 region on chromosome 17 and to chromosome 9

In Leishmaniasis, as in many infectious diseases, clinical manifestations are determined by the interaction between the genetics of the host and of the parasite. Here we describe studies mapping two loci controlling resistance to murine cutaneous leishmaniasis. Mice infected with L. major show marked genetic differences in disease manifestations: BALB/c mice are susceptible, exhibiting enlarging lesions that progress to systemic disease and death, whereas C57BL/6 are resistant, developing small, self-healing lesions. F2 animals from a C57BL/6 X BALB/c cross showed a continuous distribution of lesion score. Quantitative trait loci (QTL) have been mapped after a non-parametric QTL analysis on a genome-wide scan on 199 animals. QTLs identified were confirmed in a second cross of 271 animals. Linkage was confirmed to a chromosome 9 locus (D9Mit67-D9Mit71) and to a region including the H2 locus on chromosome 17. These have been named Imr2 and Imr1, respectively.     

The mechanism of thioredoxin reductase from human placenta is similar to the mechanisms of lipoamide dehydrogenase and glutathione reductase and is distinct from the mechanism of thioredoxin reductase from Escherichia coli

Thioredoxin reductase, lipoamide dehydrogenase, and glutathione reductase are members of the pyridine nucleotide-disulfide oxidoreductase family of dimeric flavoenzymes. The mechanisms and structures of lipoamide dehydrogenase and glutathione reductase are alike irrespective of the source (subunit M(r) approximately 55,000). Although the mechanism and structure of thioredoxin reductase from Escherichia coli are distinct (M(r) approximately 35,000), this enzyme must be placed in the same family because there are significant amino acid sequence similarities with the other two enzymes, the presence of a redox-active disulfide, and the substrate specificities. Thioredoxin reductase from higher eukaryotes on the other hand has a M(r) of approximately 55,000 [Luthman, M. & Holmgren, A. (1982) Biochemistry 21, 6628-6633; Gasdaska, P. Y., Gasdaska, J. R., Cochran, S. & Powis, G. (1995) FEBS Lett 373, 5-9; Gladyshev, V. N., Jeang, K. T. & Stadtman, T.C. (1996) Proc. Natl. Acad. Sci. USA 93, 6146-6151]. Thus, the evolution of this family is highly unusual. The mechanism of thioredoxin reductase from higher eukaryotes is not known. As reported here, thioredoxin reductase from human placenta reacts with only a single molecule of NADPH, which leads to a stable intermediate similar to that observed in titrations of lipoamide dehydrogenase or glutathione reductase. Titration of thioredoxin reductase from human placenta with dithionite takes place in two spectral phases: formation of a thiolate-flavin charge transfer complex followed by reduction of the flavin, just as with lipoamide dehydrogenase or glutathione reductase. The first phase requires more than one equivalent of dithionite. This suggests that the penultimate selenocysteine [Tamura, T. & Stadtman, T.C. (1996) Proc. Natl. Acad. Sci. USA 93, 1006-1011] is in redox communication with the active site disulfide/dithiol. Nitrosoureas of the carmustine type inhibit only the NADPH reduced form of human thioredoxin reductase. These compounds are widely used as cytostatic agents, so this enzyme should be studied as a target in cancer chemotherapy. In conclusion, three lines of evidence indicate that the mechanism of human thioredoxin reductase is like the mechanisms of lipoamide dehydrogenase and glutathione reductase and differs fundamentally from the mechanism of E. coli thioredoxin reductase.     

Fractal landscapes and molecular evolution: modeling the myosin heavy chain gene family

Mapping nucleotide sequences onto a "DNA walk" produces a novel representation of DNA that can then be studied quantitatively using techniques derived from fractal landscape analysis. We used this method to analyze 11 complete genomic and cDNA myosin heavy chain (MHC) sequences belonging to 8 different species. Our analysis suggests an increase in fractal complexity for MHC genes with evolution with vertebrate > invertebrate > yeast. The increase in complexity is measured by the presence of long-range power-law correlations, which are quantified by the scaling exponent alpha. We develop a simple iterative model, based on known properties of polymeric sequences, that generates long-range nucleotide correlations from an initially noncorrelated coding region. This new model-as well as the DNA walk analysis-both support the intron-late theory of gene evolution.     

Parasite dose determines the Th1/Th2 nature of the response to Leishmania major independently of infection route and strain of host or parasite

Leishmania major causes cutaneous leishmaniasis in mice and man. Infection of mice with relatively low or high numbers of parasites leads respectively to parasite containment, associated with a Th1, cell-mediated response, or progressive disease, associated with a Th2, antibody response in all circumstances studied. These include different parasite strains, different routes of infection, and different hosts previously classified as susceptible, resistant or of intermediate susceptibility. This dose dependency appears to reflect a general rule. We argue that this rule may allow the design of a vaccination strategy that is effective among a genetically diverse population, and that it imposes severe constraints upon proposals for the nature of the "decision criterion" determining whether antigen induces a Th1 or Th2 response.     

GDP-fucose synthetase from Escherichia coli: structure of a unique member of the short-chain dehydrogenase/reductase family that catalyzes two distinct reactions at the same active site

BACKGROUND:. In all species examined, GDP-fucose is synthesized from GDP-mannose in a three-step reaction catalyzed by two enzymes, GDP-mannose 4,6 dehydratase and a dual function 3, 5-epimerase-4-reductase named GDP-fucose synthetase. In this latter aspect fucose biosynthesis differs from that of other deoxy and dideoxy sugars, in which the epimerase and reductase activities are present as separate enzymes. Defects in GDP-fucose biosynthesis have been shown to affect nodulation in bacteria, stem development in plants, and are associated with the immune defect leukocyte adhesion deficiency type II in humans. RESULTS:. We have determined the structure of GDP-fucose synthetase from Escherichia coli at 2.2 A resolution. The structure of GDP-fucose synthetase is closely related to that of UDP-galactose 4-epimerase and more distantly to other members of the short-chain dehydrogenase/reductase family. We have also determined the structures of the binary complexes of GDP-fucose synthetase with its substrate NADPH and its product NADP+. The nicotinamide cofactors bind in the syn and anti conformations, respectively. CONCLUSIONS:. GDP-fucose synthetase binds its substrate, NADPH, in the proper orientation (syn) for transferring the 4-pro-S hydride of the nicotinamide. We have observed a single binding site in GDP-fucose synthetase for the second substrate, GDP-4-keto,6-deoxy-mannose. This implies that both the epimerization and reduction reactions occur at the same site in the enzyme. As is the case for all members of the short-chain family of dehydrogenase/reductases, GDP-fucose synthetase retains the Ser-Tyr-Lys catalytic triad. We propose that this catalytic triad functions in a mechanistically equivalent manner in both the epimerization and reduction reactions. Additionally, the X-ray structure has allowed us to identify other residues that are potentially required for substrate binding and catalysis.     

Pulse radiolysis studies on cytochrome cd1 nitrite reductase from Thiosphaera pantotropha: evidence for a fast intramolecular electron transfer from c-heme to d1-heme

Electron transfer within cytochrome cd1 from Thiosphaera pantotropha was investigated by the technique of pulse radiolysis. The reduction of the heme centers in this nitrite reductase occurred in two phases as judged from kinetic difference spectra. In the faster phase, radiolytically generated N-methylnicotinamide (NMA) radicals selectively reduced the c-heme of the enzyme. From the absorbance increase at 420 nm, a characteristic of formation of the ferrousc-heme, the second-order rate constant for this electron transfer process was estimated to be 3.8 x 10(9) M-1 s-1 at pH 7.0. In the slower phase, a decrease of absorption around 420 and 550 nm, corresponding to a reoxidation of the c-heme, was accompanied by an increase of absorption around 460 and 640 nm, characteristic of formation of the reduced d1-heme. This indicated that an intramolecular electron transfer from the c-heme to the d1-heme occurred. The first-order rate constant of this process was calculated to be 1.4 x 10(3) s-1 at pH 7.0 and was independent of the enzyme concentration. In the presence of nitrite the interheme electron transfer rate was not affected, but on a time scale of seconds a new species associated with the d1-heme, having an absorption maximum at 640 nm, was detected and is proposed to reflect ligand binding to this heme. These results suggest the role of the c-heme as the electron acceptor site in cytochrome cd1 and in mediating the electron transfer to the catalytic site of the enzyme. Moreover, the fast interheme electron transfer rate argues against this process being the rate determining step in catalysis.     

Thermodynamic and kinetic studies on carbon-cobalt bond homolysis by ribonucleoside triphosphate reductase: the importance of entropy in catalysis

In the catalytic mechanism of nucleotide reduction, ribonucleoside triphosphate reductase (RTPR) from Lactobacillus leichmannii catalyzes the homolytic cleavage of the carbon-cobalt bond of adenosylcobalamin (AdoCbl) at a rate approximately 10(11)-fold faster than the uncatalyzed reaction. Model systems have suggested hypotheses for the thermodynamic basis of this reaction, but relevant measurements of the enzymatic reaction have been lacking. To address this question in a system for which the microscopic rate constants can be measured as a function of temperature, we examined the RTPR-catalyzed exchange reaction. RTPR, in the presence of allosteric effector dGTP and in the absence of substrate, catalyzes carbon-cobalt bond homolysis and formation of a thiyl radical from an active-site cysteine in a concerted fashion [Licht, S., Booker, S. , Stubbe, J. (1999) Biochemistry 38, 1221-1233]. Both the kinetics of cob(II)alamin formation and the amounts of cob(II)alamin formed have been studied as a function of AdoCbl concentration and temperature. Analysis of these data has allowed calculation of a DeltaH of 20 kcal/mol, a DeltaS of 70 cal mol-1 K-1, a DeltaH of 46 kcal/mol, and a DeltaS of 96 cal mol-1 K-1 for carbon-cobalt bond homolysis/thiyl radical formation. The results further show that the enzyme perturbs the equilibrium between the reactant (AdoCbl-bound) state and the product (cob(II)alamin/5'-deoxyadenosine (5'-dA)/thiyl radical state, making them approximately equal in energy. The thermodynamic perturbation, in addition to transition-state stabilization, is required for the large rate acceleration observed. Entropic, rather than enthalpic, factors make the largest contribution in both cases.     

Isolation and characterization of a gene expressed mainly in the gastric epithelium, a novel member of the ep37 family that belongs to the betagamma-crystallin superfamily

EP37 family proteins are non-lens members of the betagamma-crystallin superfamily, of which expression is observed in integumental tissues of the Japanese newt, Cynops pyrrhogaster. In the present study, a gene was isolated that has high homology with ep37 and is transcribed mainly in the gastric epithelial cells and hence designated gep. The predicted amino acid sequence of the gep cDNA contains four betagamma-crystallin motifs in the N-terminal half, as is the case in the integumental EP37 proteins. Immunohistochemical analysis showed that GEP protein was mainly localized on the luminal content of the surface mucous cells of the gastric epithelium in both premetamorphic larvae and adults. In addition, GEP protein was also expressed in fundic glands after metamorphosis. Considering the fact that beta- and gamma-crystallins are evolutionarily related to stress-induced proteins, this localization suggests that GEP protein may have an evolutionarily conserved role in protection against physico-chemical stresses, such as physical abrasion and autodigestion, during assimilation.     

NADP-Isocitrate dehydrogenase from Pseudomonas nautica: kinetic constant determination and carbon limitation effects on the pool of intracellular substrates

Variations of intracellular concentrations of isocitrate and NADP+ were measured throughout all growth phases of the marine bacterium Pseudomonas nautica. The intracellular isocitrate concentration tracked the intracellular protein concentration throughout all phases of growth. It rapidly increased in early exponential phase to a maximum and fell to nearly zero in parallel with pyruvate exhaustion in the culture medium. The intracellular NADP+ and protein concentrations increased in parallel during the exponential phase but were poorly correlated. Even after carbon exhaustion, the intracellular NADP+ concentration stayed high, as did protein levels. The results demonstrated that the intracellular isocitrate concentration, but not the intracellular NADP+ concentration, was affected by the carbon availability in the culture. They also suggest that, because of its variability, isocitrate, but not NADP+, plays the larger role in the control of the respiratory CO2 production rate (RCO2). From initial rate studies, bisubstrate Michaelis constants and the dissociation constant were determined for NADP+-specific isocitrate dehydrogenase (IDH) from P. nautica. These studies support the hypothesis that the mechanism of IDH's activity involves the ordered addition of the substrates, D-isocitrate and NADP+. Furthermore, the results support the use of a bisubstrate enzyme kinetic equation to model RCO2 in P. nautica.     

Colon cancer in the elderly: evidence for major improvements in health care and survival

Time trends in therapeutic approaches and in the prognosis of colon cancer for patients aged 75 years and above have been investigated in comparison with corresponding trends for younger patients using a population-based series of 2089 colon cancer patients diagnosed between 1976 and 1990 in the C?te-d'Or area (478,000 inhabitants), Burgundy, France. Significant progress has been achieved in the management of patients with colon cancer in both age groups, but trends have been more noticeable in patients aged 75 years and above. In the elderly, the proportion of cancers limited to the digestive tract wall showed a 3-year average increase of 2.8% (P = 0.02) and the frequency of curative surgery an average increase of 8.6% (P < 0.001), so that it was performed in 80% of cases in the last 3-year period. Operative mortality decreased by 2.5% between 3-year periods (P < 0.004). Crude 5-year survival rates in elderly patients increased from 15% in the 1976-78 period to 29% in the 1985-87 period (P < 0.001), the corresponding figures being 36% and 44% (P > 0.10) in younger patients.     

Pseudosubstrate hydrolysis by the erythrocyte plasma membrane Ca(2+)-ATPase: kinetic evidence for a modified E1 conformation in dimethylsulfoxide

Four different beta-tubulin clones were isolated from carrot genomic and cDNA libraries. Their nucleotide sequences were determined 1 and their predicted amino acids were compared with each other. The predicted amino acid composition of the C-terminal region of three of them (beta-1, 3, 4) resembled one another, but that of one isotype (beta-2) was divergent. The beta-2 tubulin included two hydroxyl amino acids, serine and threonine, and consisted of a lower number of negatively charged amino acids than the others in the C-terminal region. The predicted hydrophobicity profile of the beta-2 tubulin around the residue 200 is less hydrophobic than beta-1, but it is still more hydrophobic than those of animal and fungal beta-tubulins. The beta-2 gene was transcribed in cultured cells and flowers, while the beta-1 gene was ubiquitously transcribed in cultured cells, roots, shoots and flowers. When the predicted amino acids of plant tubulin were compared with those of other organisms, substitutions from non-polar amino acids to those with hydroxyl group were conspicuous in the region corresponding to the third exon in the plant genes.     

Mice defective in Fas are highly susceptible to Leishmania major infection despite elevated IL-12 synthesis, strong Th1 responses, and enhanced nitric oxide production

MRL/MP-lpr/lpr (MRL/lpr) mice have a single mutation (lpr) of the fas apoptosis gene. The mutant mice developed significantly smaller lesions than the wild-type mice at the earlier stage of infection with the intracellular protozoan parasite Leishmania major. However, while all the wild-type mice achieved complete lesion resolution, the disease in the mutant mice progressed inexorably. The mutant mice had more IL-12 and nitrite/nitrate in the serum than wild-type mice following infection. Lymphoid cells from infected MRL/lpr mice produced more IFN-gamma but less IL-4 and IL-5 than cells from MRL-+/+ mice. Peritoneal macrophages from the mutant mice also produced more IL-12 and NO after stimulation with LPS. Thus, Fas expression is essential for resistance against leishmaniasis, and Fas-mediated apoptosis may form an integral part of the Th1-mediated microbicidal function.     

Role of the active-site carboxylate in dihydrofolate reductase: kinetic and spectroscopic studies of the aspartate 26-->asparagine mutant of the Lactobacillus casei enzyme

A mutant of Lactobacillus casei dihydrofolate reductase, D26N, in which the active site aspartic acid residue has been replaced by asparagine by oligonucleotide-directed mutagenesis has been studied by NMR and optical spectroscopy and its kinetic behavior characterized in detail. On the basis of comparisons of a large number of chemical shifts and NOEs, it is clear that there are only very slight structural differences between the methotrexate complexes of the wild-type and mutant enzymes and that these are restricted to the immediate environment of the substitution. The data suggest a slight difference in orientation of the pteridine ring in the binding site in the mutant enzyme. Both NMR and UV spectroscopy show that methotrexate is protonated on N1 when bound to the wild-type enzyme but not when bound to the mutant. Binding constant measurements by fluorescence quenching and steady-state kinetic measurements of dihydrofolate (FH2) and folate reduction show that the substitution has little or no effect on substrate, coenzyme, and inhibitor binding (< 7-fold increase in Kd) and only a modest effect on kcat (up to a factor of 9 for FH2 and 25 for folate) and kcat/KM (up to a factor of 13 for FH2 and 14 for folate). Measurements of deuterium isotope effects and direct measurements of hydride ion transfer and product release by stopped-flow methods revealed that for the mutant enzyme hydride ion transfer is rate-limiting across the pH range 5-8. This allowed a direct comparison of the rate of hydride ion transfer in the wild-type and mutant enzymes; the asparagine substitution was found to decrease this rate by 62-fold at pH 5.5 and 9-fold at pH 7.5. This effect is much smaller than that seen for the corresponding mutant of Escherichia coli dihydrofolate reductase [Howell, E. E., Villafranca, J. E., Warren, M. S., Oatley, S. J., & Kraut, J. (1986) Science 231, 1123-1128], estimated as a 1000-fold decrease in the rate of hydride ion transfer. The change in pH dependence of kcat resulting from the substitution is consistent with, but does not prove, the idea that the group of pK 6.0 which must be protonated for hydride ion transfer to occur is Asp26. For folate reduction, the pH dependence of kcat is determined by two pKs, one of which, pK 5, disappears in the mutant enzyme, suggesting that it may correspond to ionization of Asp26.(ABSTRACT TRUNCATED AT 400 WORDS)     

Degradation of mouse invariant chain: roles of cathepsins S and D and the influence of major histocompatibility complex polymorphism

Antigen-presenting cells (APC) degrade endocytosed antigens into peptides that are bound and presented to T cells by major histocompatibility complex (MHC) class II molecules. Class II molecules are delivered to endocytic compartments by the class II accessory molecule invariant chain (Ii), which itself must be eliminated to allow peptide binding. The cellular location of Ii degradation, as well as the enzymology of this event, are important in determining the sets of antigenic peptides that will bind to class II molecules. Here, we show that the cysteine protease cathepsin S acts in a concerted fashion with other cysteine and noncysteine proteases to degrade mouse Ii in a stepwise fashion. Inactivation of cysteine proteases results in incomplete degradation of Ii, but the extent to which peptide loading is blocked by such treatment varies widely among MHC class II allelic products. These observations suggest that, first, class II molecules associated with larger Ii remnants can be converted efficiently to class II-peptide complexes and, second, that most class II-associated peptides can still be generated in cells treated with inhibitors of cysteine proteases. Surprisingly, maturation of MHC class II in mice deficient in cathepsin D is unaffected, showing that this major aspartyl protease is not involved in degradation of Ii or in generation of the bulk of antigenic peptides.