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S Krishna

Publications and source records attributed to S Krishna.

At least 37 records · Page 2Linked to original sources
PubMed2000

A brief illustrated guide to the ultrastructure of Plasmodium falciparum asexual blood stages.

Interpretation of the new information arising from the Plasmodium falciparum Genome Project requires a good working knowledge of the ultrastructure of the parasite; however many aspects of the morphology of this species remain obscure. Lawrence Bannister, John Hopkins and colleagues here give an illustrated overview of the three-dimensional (3-D) organization of the merozoite, ring, trophozoite and schizont stages of the parasite, based on available data that include 3-D reconstruc-tion from serial electron microscope sections. The review describes the chief organelles present in these stages, emphasizing the continuity of structure in addition to specialized, stage-specific features developed during the asexual erythrocytic cycle.

Animals↗
PubMed2000

Hexose transport in asexual stages of Plasmodium falciparum and kinetoplastidae.

The hexose sugar, glucose, is a vital energy source for most organisms and an essential nutrient for asexual stages of Plasmodium falciparum. Kinetoplastid organisms (e.g. Trypanosoma and Leishmania spp) also require glucose at certain critical stages of their life cycles. Although phylogenetically unrelated, these organisms share many common challenges during the mammalian stages of a parasitic life cycle, and possess hexose uptake mechanisms that are amenable to study using similar methods. Defining hexose permeation pathways into parasites might expose an Achilles' heel at which both antidisease and antiparasite measures can be aimed. Understanding the mode of entry of glucose also presents a good general model for substrate acquisition in multicompartment systems. In this review, Sanjeev Krishna and colleagues summarize current understanding of hexose transport processes in P. falciparum and provide a comparison with data obtained from kinetoplastids.

Animals↗
PubMed2000

The Trypanosoma cruzi genome contains ion motive ATPase genes which closely resemble Leishmania proton pumps.

DNA fragments homologous to members of the family of P-type ion-motive ATPases were identified in Trypanosoma cruzi by polymerase chain reaction (PCR) amplification. The sequence of one fragment, which closely resembled (87% identity) the tandemly linked proton pumps in Leishmania, was used to characterize the H(+)-ATPase genes in T. cruzi. The T. cruzi proton pump locus contains four tandemly repeated genes (TCH1-4) separated by 1.1 kb intergenic regions. The nucleotide sequence of one cloned gene of the tandem array contains a 2775 nt open reading frame encoding a predicted 101908-Da protein of 925 amino acids. The TCH genes are expressed as 3.8 and 4.9 kb polyadenylated transcripts in the epimastigote stage; expression of both transcripts is reduced in metacyclic trypomastigotes. Results of 5' and 3' RACE transcript mapping indicate that the 3.8 kb message is generated from within the tandemly repeated locus. The 3.8 kb TCH transcript has the T. cruzi mini-exon appended to a short (40 nt) 5' untranslated region (UTR) and has a 927 nt 3' UTR. The full peptide sequence of the T. cruzi proton pump is 80% identical to the Leishmania pump but lacks the extended carboxyl tail present in the Leishmania ATPase. An antibody that recognizes the 110-kDa Leishmania donovani proton pump cross-reacts with a 100-kDa protein in lysates of T. cruzi epimastigotes.

Amino Acid Sequence↗
PubMed2000

Ultrastructure of rhoptry development in Plasmodium falciparum erythrocytic schizonts.

Prior to the separation of merozoites from the Plasmodium falciparum schizont, various stage-specific organelles are synthesized and assembled within each merozoite bud. The apical ends of the merozoites are initiated close to the ends of endomitotic spindles. At each of these sites, the nuclear membrane forms coated vesicles, and a single discoidal or cup-like Golgi cisterna appears. Reconstruction from serial sections indicates that this structure receives vesicles from the nuclear envelope and in turn gives off coated vesicles to generate the apical secretory organelles. Rhoptries first form as spheroidal structures and grow by progressive fusion of small vesicles around their margins. As each rhoptry develops, 2 distinctive regions separate within it, an apical reticular zone with electron-lucent areas separated by cords of granular material, and a more homogenously granular basal region. The apical part elongates into the duct, with evidence for further vesicular fusion at the duct apex. The rounded rhoptry base becomes progressively more densely packed to form a spheroidal mass, and compaction also occurs in the duct. Typically, one rhoptry matures before the other. Cryofractured rhoptry membranes show asymmetry in the sizes and numbers of intramembranous particles at the internally- and externally-directed fracture faces.

Animals↗
PubMed2000

Glucose and lactate kinetics in children with severe malaria.

Children with severe malaria often present with lactic acidosis and hypoglycemia. Although both complications independently predict mortality, mechanisms underlying their development are poorly understood. To study these metabolic derangements we sequentially allocated 21 children with falciparum malaria and capillary lactate concentrations of 5 mmol/L or more to receive either quinine or artesunate as antimalarial therapy, and dichloroacetate or saline placebo for lactic acidosis. We then administered a primed infusion (90 min) of L-[3-13C1]sodium lactate and D-[6,6-D2]glucose to determine the kinetics of these substrates. The mean (SD) glucose disposal rate in all patients was 56 (16) micromol/kg x min, and the geometric mean (range) lactate disposal rate was 100 (66-177) micromol/kg x min. Glucose and lactate disposal rates were positively correlated (r = 0.62; P = 0.005). Artesunate was associated with faster parasite clearance, lower insulin/glucose ratios, and higher glucose disposal rates than quinine. Lactate disposal was positively correlated with plasma lactate concentrations (r = 0.66; P = 0.002) and time to recovery from coma (r = 0.82; P < 0.001; n = 15). Basal lactate disposal rates increased with dichloroacetate treatment. Elevated glucose turnover in severe malaria mainly results from enhanced anaerobic glycolysis. Quinine differs from artesunate in its effects on glucose kinetics. Increased lactate production is the most important determinant of lactic acidosis.

Acidosis, Lactic↗
PubMed1999

Mycobacterium tuberculosis expresses a novel pH-dependent divalent cation transporter belonging to the Nramp family.

Mammalian natural resistance-associated macrophage protein (Nramp) homologues are important determinants of susceptibility to infection by diverse intracellular pathogens including mycobacteria. Eukaryotic Nramp homologues transport divalent cations such as Fe(2+), Mn(2+), Zn(2+), and Cu(2+). Mycobacterium tuberculosis and Mycobacterium bovis (bacillus Calmette-Guérin [BCG]) also encode an Nramp homologue (Mramp). RNA encoding Mramp induces approximately 20-fold increases in (65)Zn(2+) and (55)Fe(2+) uptake when injected into Xenopus laevis oocytes. Transport is dependent on acidic extracellular pH and is maximal between pH 5.5 and 6.5. Mramp-mediated (65)Zn(2+) and (55)Fe(2+) transport is abolished by an excess of Mn(2+) and Cu(2+), confirming that Mramp interacts with a broad range of divalent transition metal cations. Using semiquantitative reverse transcription PCR, we show that Mramp mRNA levels in M. tuberculosis are upregulated in response to increases in ambient Fe(2+) and Cu(2+) between <1 and 5 microM concentrations and that this upregulation occurs in parallel with mRNA for y39, a putative metal-transporting P-type ATPase. Using a quantitative ratiometric PCR technique, we demonstrate a fourfold decrease in Mramp/y39 mRNA ratios from organisms grown in 5-70 microM Cu(2+). M. bovis BCG cultured axenically and within THP-1 cells also expresses mRNA encoding Mramp. Mramp exemplifies a novel prokaryotic class of metal ion transporter. Within phagosomes, Mramp and Nramp1 may compete for the same divalent cations, with implications for intracellular survival of mycobacteria.

Amino Acid Sequence↗
PubMed1999

Human papillomavirus type 16 E6-enhanced susceptibility of L929 cells to tumor necrosis factor alpha correlates with increased accumulation of reactive oxygen species.

Human papillomavirus type 16 (HPV-16) E6 has been shown to prevent or enhance apoptosis depending on the stimulus and cell type. Here we present evidence that HPV-16 E6 sensitized murine fibrosarcoma L929 cells to tumor necrosis factor alpha (TNF)-induced cytolysis. The E6-enhanced cytolysis correlated with a precedent increase in reactive oxygen species (ROS) level and antioxidant treatment could completely block the E6-dependent sensitization. These findings represent the first demonstration of a link between a viral oncogene-sensitized cytolysis and ROS. Previous studies have shown conflicting results regarding whether TNF-induced cytolysis of L929 cells is through necrosis or apoptosis. Here we report that, although L929 cells underwent DNA fragmentation after exposure to TNF, they retained the morphology of intact nuclei while gaining permeability to propidium iodide, features characteristic of necrosis rather than apoptosis. We confirmed that the broad spectrum caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethylketone markedly increased the susceptibility of L929 cells to TNF, and further demonstrated that E6 enhanced this susceptibility, which again correlated with increased ROS accumulation. We showed that the expression of E6 in L929 cells did not alter the stability of p53, and the cells retained a p53 response to actinomycin D. Furthermore, two E6 mutants defective for p53 degradation in other systems exhibited differential effects on TNF sensitization. These results suggest that the enhancement of TNF-induced L929 cytolysis by E6 is independent of p53 degradation. We also found that TNF-induced activation of NF-kappaB did not account for the enhanced TNF susceptibility by E6.

Amino Acid Chloromethyl Ketones↗
PubMed1999

Maternally inherited hearing loss in a large kindred with a novel T7511C mutation in the mitochondrial DNA tRNA(Ser(UCN)) gene.

Thirty-six of 43 maternally related members of a large African American family experienced hearing loss. A muscle biopsy specimen from the proband showed cytochrome c oxidase (COX)-deficient fibers but no ragged-red fibers; biochemical analysis showed marked reduction of COX activity. A novel T7511C point mutation in the tRNA(Ser(UCN)) gene was present in almost homoplasmic levels (>95%) in the blood of 18 of 20 family members, and was also found in lower abundance in the other two. Single-fiber PCR showed that the mutational load was greater in COX-deficient muscle fibers. The tRNA(ser(UCN)) gene may be a "hot spot" for mutations associated with maternally transmitted hearing loss.

Adult↗
PubMed1999

Intraerythrocytic Plasmodium falciparum expresses a high affinity facilitative hexose transporter.

Asexual stages of Plasmodium falciparum cause severe malaria and are dependent upon host glucose for energy. We have identified a glucose transporter of P. falciparum (PfHT1) and studied its function and expression during parasite development in vitro. PfHT1 is a saturable, sodium-independent, and stereospecific transporter, which is inhibited by cytochalasin B, and has a relatively high affinity for glucose (Km = 0.48 mM) when expressed in Xenopus laevis oocytes. Competition experiments with glucose analogues show that hydroxyl groups at positions C-3 and C-4 are important for ligand binding. mRNA levels for PfHT1, assessed by the quantitative technique of tandem competitive polymerase chain reaction, are highest during the small ring stages of infection and lowest in gametocytes. Confocal immunofluorescence microscopy localizes PfHT1 to the region of the parasite plasma membrane and not to host structures. These findings have implications for development of new drug targets in malaria as well as for understanding of the pathophysiology of severe infection. When hypoglycemia complicates malaria, modeling studies suggest that the high affinity of PfHT1 is likely to increase the relative proportion of glucose taken up by parasites and thereby worsen the clinical condition.

Amino Acid Sequence↗
PubMed1999

Thiamine deficiency and malaria in adults from southeast Asia.

BACKGROUND: Thiamine deficiency (beriberi) is common in some parts of southeast Asia. Acute thiamine deficiency can mimic many complications of malaria, such as encephalopathy and lactic acidosis. We examined the incidence of thiamine deficiency in adults admitted to hospital with malaria in Thailand. METHODS: For this prospective study, we recruited consecutive patients with malaria or other febrile illness who presented to Paholpolpayuhasena Hospital, Kanchanaburi, Thailand, between May and July, 1992. We used the activation coefficient (alpha) for transketolase activity in erythrocytes to measure thiamine deficiency (defined as alpha>1.31) in patients with severe and uncomplicated malaria and in controls (patients' relatives and healthy volunteers). To exclude the possibility of interference in the assays, transketolase activity was also measured in erythrocytes used to culture parasites. FINDINGS: 12 (52%) of 23 patients with severe malaria and ten (19%) of 54 patients with uncomplicated malaria had alpha values above the normal range (p<0.0001 and p=0.0014, respectively, compared with controls), which indicated severe thiamine deficiency. Thiamine deficiency was more severe in patients with cerebral malaria than in those with uncomplicated malaria and the controls (p=0.008). INTERPRETATION: In adults admitted to hospital in Thailand, thiamine deficiency commonly complicates acute falciparum malaria, particularly in severe infections, and could contribute to dysfunction of the central nervous system.

Acidosis, Lactic↗
PubMed1999

A nonsense mutation (G15059A) in the cytochrome b gene in a patient with exercise intolerance and myoglobinuria.

We describe a new mitochondrial DNA mutation in the cytochrome b gene in a patient presenting with progressive exercise intolerance and myoglobinuria associated with complex III deficiency in muscle. The point mutation results in the replacement of a glycine at amino acid position 190 with a stop codon. This change predicts premature termination of translation, leading to a truncated protein missing 244 amino acids at the C-terminus of cytochrome b. The mutation fulfills all the accepted criteria for pathogenicity, suggesting that this is the primary cause of the myopathy in the patient.

Adult↗
PubMed1999

Expression of parasite transporters in Xenopus oocytes.

The Xenopus laevis oocyte heterologous expression system is particularly useful for the study of transporter proteins. We demonstrated the utility of this expression system for studies on Plasmodium falciparum transporters by inducing increased uptake of metabolites or their analogues (nucleosides, nucleobases, lactate and glucose) into oocytes after microinjection of mRNA obtained from asexual stages of P. falciparum. We identified a hexose transporter of P. falciparum (PfHT1) and studied its function. Higher levels of functional activity are obtained when 5' and 3' untranslated Xenopus globin gene sequences and a strong Kozak consensus are included in RNA used for microinjection studies. PfHT1 is a saturable, sodium-independent and stereospecific transporter with a relatively high affinity for glucose (K(m) = 0.48 mM). Competition experiments with glucose analogues show that hydroxyl groups at positions C3 and C4 are important for ligand binding. mRNA levels for PfHT1 are highest during the small ring stages of infection and lowest in gametocytes. Confocal immunofluorescence microscopy localizes PfHT1 to the region of the parasite plasma membrane and not to host structures. When hypoglycaemia complicates cerebral malaria, modelling studies using data obtained from oocyte experiments suggest that the high affinity of PfHT1 may increase the proportion of glucose taken up by parasites compared with that transported across the blood-brain barrier.

Animals↗
PubMed1999

The plastid in Plasmodium falciparum asexual blood stages: a three-dimensional ultrastructural analysis.

The plastid in Plasmodium falciparum asexual stages is a tubular structure measuring about 0.5 micron x 0.15 micron in the merozoite, and 1.6 x 0.35 microns in trophozoites. Each parasite contains a single plastid until this organelle replicates in late schizonts. The plastid always adheres to the (single) mitochondrion, along its whole length in merozoites and early rings, but only at one end in later stages. Regions of the plastid are also closely related to the pigment vacuole, nuclear membrane and endoplasmic reticulum. In merozoites the plastid is anchored to a band of 2-3 subpellicular microtubules. Reconstructions show the plastid wall is characteristically three membranes thick, with regions of additional, complex membranes. These include inner and outer membrane complexes. The inner complex in the interior lumen is probably a rolled invagination of the plastid's inner membrane. The outer complex lies between the outer and middle wall membranes. The interior matrix contains ribosome-like granules and a network of fine branched filaments. Merozoites of P. berghei and P. knowlesi possess plastids similar in structure to those of P. falciparum. A model is proposed for the transfer of membrane lipid from the plastid to other organelles in the parasite.

Animals↗
PubMed1999

Fatal infantile cardioencephalomyopathy with COX deficiency and mutations in SCO2, a COX assembly gene.

Mammalian cytochrome c oxidase (COX) catalyses the transfer of reducing equivalents from cytochrome c to molecular oxygen and pumps protons across the inner mitochondrial membrane. Mitochondrial DNA (mtDNA) encodes three COX subunits (I-III) and nuclear DNA (nDNA) encodes ten. In addition, ancillary proteins are required for the correct assembly and function of COX (refs 2, 3, 4, 5, 6). Although pathogenic mutations in mtDNA-encoded COX subunits have been described, no mutations in the nDNA-encoded subunits have been uncovered in any mendelian-inherited COX deficiency disorder. In yeast, two related COX assembly genes, SCO1 and SCO2 (for synthesis of cytochrome c oxidase), enable subunits I and II to be incorporated into the holoprotein. Here we have identified mutations in the human homologue, SCO2, in three unrelated infants with a newly recognized fatal cardioencephalomyopathy and COX deficiency. Immunohistochemical studies implied that the enzymatic deficiency, which was most severe in cardiac and skeletal muscle, was due to the loss of mtDNA-encoded COX subunits. The clinical phenotype caused by mutations in human SCO2 differs from that caused by mutations in SURF1, the only other known COX assembly gene associated with a human disease, Leigh syndrome.

Amino Acid Sequence↗
PubMed1999

Pleiotropic effects of human papillomavirus type 16 E6 oncogene expression in human epithelial cell lines.

Two human papillomavirus (HPV)-negative epithelial cell lines, HaCaT and C33A, were transfected with HPV-16 E6 and analysed for functional consequences which are relevant to invasive tumour progression. After transfection with E6, both cell lines invaded collagen matrices, in contrast to vector-transfected control cells. The E6-expressing cells showed a marked increase in expression of the beta1 integrin subunit, with no or relatively minor alterations in the levels of a range of integrin subunits. In addition, the epithelial cell lines expressing E6 displayed resistance to apoptosis generated by serum starvation. This resistance is comparable to that generated by ras and is not generated by HPV-11 E6 or HPV-16 E7. Both C33A and HaCaT cells have mutations in the p53 loci and hence these functional consequences of E6 are probably independent of wild-type p53 function.

Cell Line↗
PubMed1999

The pfmdr1 gene is associated with a multidrug-resistant phenotype in Plasmodium falciparum from the western border of Thailand.

On the western border of Thailand, Plasmodium falciparum has become resistant to almost all antimalarial agents. The molecular basis of resistance in these parasite populations has not been well characterized. This study assessed genetic polymorphisms in the pfmdr1 gene in 54 parasites collected from the western border of Thailand to determine the relationship of pfmdr1 copy number and codon mutations with parasite sensitivities to mefloquine, chloroquine, halofantrine, quinine, and artesunate assessed in vitro. A point mutation at codon 86 (resulting in a change of Asn to Tyr) was associated with a significantly lower 50% inhibitory concentration (IC(50)) of mefloquine (median, 9 ng/ml versus 52.4 ng/ml; P = 0.003). Overall 35% of the isolates (19 of 54) had an increase in pfmdr1 copy number, and all 19 carried the wild-type allele at codon 86. Increased pfmdr1 copy number was associated with higher IC(50)s of mefloquine (P = 0.04) and artesunate (P = 0.005), independent of polymorphism at codon 86. The relationship between pfmdr1 and resistance to structurally distinct antimalarial agents confirms the presence of a true multidrug-resistant phenotype.

ATP-Binding Cassette Transporters↗