[Lessons from the Malaria Vector Control Program Based on Indoors Residual Spraying with DDT or Dieldrin in the Pilot Zone of Bobo-Dioulasso: Failure or Success?] / Les Leçons du Programme de Lutte Contre les Vecteurs du Paludisme Par Aspersions Intradomiciliaires de DDT ou de Dieldrine dans la Zone Pilote de Bobo-Dioulasso: Échec ou Succès?

During five years, from 1953, a village scale indoors residual spraying (IRS) was done in the pilot zone of Bobo-Dioulasso, Burkina Faso, with DDT or dieldrin (DLN) or even HCH with a conceptually both entomological and parasitological evaluation [18].Compared to the control area, DDT induced an approximatively 95% and 67% reduction in the landing rate of Anopheles gambiae, respectively inside and outside human houses but due to its irritant action, DDT greatly increased their exophagic behaviour. However, DLN had no impact on the landing rate of An. gambiae either indoors or outdoors due to the already noticed resistance of this species to this insecticide. The sporozoitic index of An. gambiae was reduced by 96% in the DDT treated areas and by 70% in the DLN treated area.DDT reduced the landing rates of Anopheles funestus by 98% and 91%, inside and outside treated houses respectively. With DLN, these reductions were 98% and 97%, respectively. The sporozoitic index of An. funestus was reduced by 95% in areas treated with DDT.Thus, vector control has reduced malaria transmission due to the two main vectors, An. gambiae and An. funestus, by some 99.8% in DDT treated villages compared to control villages. DLN reduced transmission from An. funestus by 99.9%, but almost not from An. gambiae . Overall, the implementation of vector control based on indoor residual spraying with DDT or DLN reduced by 99.9% the transmission of human Plasmodium in the villages of the pilot zone and therefore the program can be considered as entomologically successful.In children aged 2-9 years (target group for endemicity indices) the splenic index was 84.3% (n = 979) in the control area and 44.4% (n = 8920) in the treated areas (difference -47.3%), the plasmodial prevalence was 60.6% (n = 946) in the control zone and 38.0% (n = 7242) in the treated zones (difference - 37%) but the relatively high level of plasmodic or splenic index in treated villages showed that transmission was maintained at such a level that the program could be considered as a "semi-failure".Besides, the gametocytic indices remained at the same levels (3.28%, n = 946 in the control zone and 3.04%, n = 7242 in the treated zones) indicating the maintenance of the "reservoir of parasites" and the remaining possibilities of transmission.Compared to the control area, the index of new contamination was significantly lower in infants 0-3 months and 4 to 6 months in DDT treated villages but not in infants 7 to 12 months demonstrating that the control vector had some efficacy in the prevention of plasmodial infection but "all newborns were infected within one year" demonstrating that P. falciparum transmission was not completely stopped.In spite of its striking drop, the transmission was not fully stopped, and the programme was considered as a "semi-failure" or even a "failure" and inducing a complete shift in malaria control policy from vector control to mass drug chemotherapy (with several drugs, chloroquine, primaquine, pyriméthamine etc) without complete stop of transmission either. In fact, such vector control operations by DDT may have different analysis; in one side they can be considered an entomological success but, in another side, the actual reduction of plasmodic and splenic indices was not enough to be considered as successful. It was clear that both vector and parasite must be implemented in an integrated programme taking care of insecticide and drug resistance. Nevertheless, such programme, even not as successful as expected, could be considered as encouraging and not "disappointing" as it was. Important lessons can be learned from such large-scale field trial in spite of several methodological and operational issues.

The moth Hylesia metabus and French Guiana lepidopterism: centenary of a public health concern.

The females of the moths Hylesia metabus have their abdomens covered by urticating hairs looking like micro-arrows and causing a puriginous dermatitis to humans known as "papillonite" in French Guiana and also called yellowtail moth dermatitis or Caripito itch. The densities of the moths show great seasonal and annual variations depending on mechanisms mostly unknown. When H. metabus infestations occur, numerous cases of dermatologic manifestations are reported from people living near the mangrove swamps where the moths are developing. One hundred years after the first "papillonite" epidemic reported from French Guiana in 1912, the data presented herein summarize the actual state of knowledge on H. metabus biology and ecology and on the lepidopterism. Some recommendations are proposed for the surveillance and warning systems of H. metabus infestations and to avoid contact with the moths. Research priorities are suggested to improve the control against this problem emerging between nuisance and public health.

Arthropod-borne viruses transmitted by Phlebotomine sandflies in Europe: a review.

Phlebotomine sandflies are known to transmit leishmaniases, bacteria and viruses that affect humans and animals in many countries worldwide. These sandfly-borne viruses are mainly the Phlebovirus, the Vesiculovirus and the Orbivirus. Some of these viruses are associated with outbreaks or human cases in the Mediterranean Europe. In this paper, the viruses transmitted by Phlebotomine sandflies in Europe (Toscana virus, Sicilian virus, sandfly fever Naples virus) are reviewed and their medical importance, geographical distribution, epidemiology and potential spreading discussed. Data on vertebrate reservoirs is sparse for sandfly fever viruses. The factor currently known to limit the spread of diseases is mainly the distribution areas of potential vectors. The distribution areas of the disease may not be restricted to the areas where they have been recorded but could be as wide as those of their vectors, that is to say Larroussius and P. papatasi mainly but not exclusively. Consequently, field work in form of viral isolation from sandflies and possible reservoirs as well as laboratory work to establish vectorial competence of colonised sandflies need to be encouraged in a near future, and epidemiological surveillance should be undertaken throughout the European Union.

Acute and selective regulation of glyceroneogenesis and cytosolic phosphoenolpyruvate carboxykinase in adipose tissue by thiazolidinediones in type 2 diabetes.

AIMS/HYPOTHESIS: Regulation of glyceroneogenesis and its key enzyme cytosolic phosphoenolpyruvate carboxykinase (PEPCK-C) plays a major role in the control of fatty acid release from adipose tissue. Here we investigate the effect of rosiglitazone on the expression of genes involved in fatty acid metabolism and the resulting metabolic consequences. MATERIALS AND METHODS: Rosiglitazone was administered to Zucker fa/fa rats for 4 days and to 24 diabetic patients for 12 weeks, then mRNA expression for the genes encoding PEPCK-C, mitochondrial PEPCK, adipocyte lipid-binding protein, glycerol kinase, lipoprotein lipase and glycerol-3-phosphate dehydrogenase was examined in s.c. adipose tissue by real-time RT-PCR. Glyceroneogenesis was determined using [1-(14)C]pyruvate incorporation into lipids. Cultured adipose tissue explants from overweight women undergoing plastic surgery were incubated with rosiglitazone for various times before mRNA determination and analysis of PEPCK-C protein, activity and glyceroneogenesis. RESULTS: Rosiglitazone administration to rats induced the expression of the gene encoding PEPCK-C mRNA (PCK1) and PEPCK-C activity in adipose tissue with a resulting 2.5-fold increase in glyceroneogenesis. This was accompanied by an improvement in dyslipidaemia as demonstrated by the decrease in plasma NEFAs and triacylglycerol. In rosiglitazone-treated diabetic patients, PCK1 mRNA was raised 2.5-fold in s.c. adipose tissue. Rosiglitazone treatment of adipose tissue explants from overweight women caused a selective augmentation in PCK1 mRNA which reached a maximum of 9-fold at 14 h, while mRNA for other genes remained unaffected. Experiments with inhibitors showed a direct and transcription-only effect, which was followed by an increase in PEPCK-C protein, enzyme activity and glyceroneogenesis. CONCLUSIONS/INTERPRETATION: These results favour adipocyte glyceroneogenesis as the initial thiazolidinedione-responsive pathway leading to improvement in dyslipidaemia.

Horse-, bird-, and human-seeking behavior and seasonal abundance of mosquitoes in a West Nile virus focus of southern France.

After 35 yr of disease absence, West Nile virus (family Flaviviridae, genus Flavivirus, WNV) circulation has been regularly detected in the Camargue region (southern France) since 2000. WNV was isolated from Culex modestus Ficalbi, which was considered the main vector in southern France after horse outbreaks in the 1960s. Recent WNV transmissions outside of the Cx. modestus distribution suggested the existence of other vectors. To study potential WNV vectors, horse- and bird-baited traps and human landing collections of mosquitoes were carried out weekly from May to October 2004 at two Camargue sites: one site in a wet area and the other site in a dry area, both chosen for their past history of WNV transmission. At the wet site, the most abundant species in bird-baited traps were Culex pipiens L. and Cx. modestus; both species also were found in lower proportions on horses and humans. The most abundant species in horse-baited traps and human landing collections were Aedes caspius (Pallas), Aedes vexans (Meigen), and Anopheles hyrcanus (Pallas) sensu lato; some of these species were occasionally collected with avian blood at the end of the summer. Anopheles maculipennis Meigen sensu lato was an abundant horse feeder, but it was rarely collected landing on human bait and never contained avian blood. At the dry site, Cx. pipiens was the most abundant species in bird- and horse-baited traps. The seasonal and circadian dynamics of these species are analyzed, and their potential in WNV transmission in Camargue discussed.

[Local administration of ivermectin for the treatment of Cochliomyia hominivorax's myiasis]. / Traitement des myiases dues à Cochliomyia hominivorax par application locale d'ivermectine.

Myiasis with Cochliomyia homonivorax induce sensitive and nauseating cutaneous ulcerations. The usual treatment is limited to the mechanical extraction of the larvae. The authors have made a report on the treatment of C. hominivorax by local application of ivermectin. The use of this molecule paralyses and then kills the larvae. This allows rapid alleviation of pain and makes the extraction of the larvae easier.

A novel Y243S mutation in the pyruvate dehydrogenase El alpha gene subunit: correlation with thiamine pyrophosphate interaction.

We identified a new Y243S mutation in the X-linked E1 alpha-PDH gene in a patient with pyruvate dehydrogenase complex (PDHc) deficiency. The activity in cultured fibroblasts was very low even in the presence of high thiamine pyrophosphate (TPP) concentrations, indicating that the defect could be due to decreased affinity of PDHc for TPP.

Isoenzyme differentiation of Aedes aegypti populations in French Guiana.

Population genetics of peri-domestic Aedes aegypti (Diptera: Culicidae), vector of dengue and yellow fever, were investigated by gel electrophoresis of 10 enzyme loci in 14 samples of mosquito larvae collected in 1997-1998 from localities separated by distances of 3-275 km in French Guiana. Genetic differentiation between geographical populations was generally high (mean FST = +0.111, P < 10(-5)) even among seven sites <30 km apart (FST = +0.137, P < 0.05), but not positively correlated with distance. Thus, Ae. aegypti comprises a mosaic of genetically differentiated populations in French Guiana. This may be attributed to reinvasion from diverse origins through repeated founder events after this vector species was eliminated during the 1940s to 1960s.

Black flies (Diptera: Simuliidae) of French Guiana: cytotaxonomy and a preliminary list of species.

The objectives of the present study were to broaden the survey of simuliid species in French Guiana and to cytologically analyze the species in the Simulium perflavum species group. Twelve species of Simulium were collected from which S. goeldii, S. quadrifidum, S. trombetense, S. near incrustatum, S. metallicum (s.l.) sp1, S. metallicum (s.l.) sp2 and S. ochraceum (s.l.) are reported for the first time for this region. The only species collected in the S. perflavum group was S. rorotaense; 34 larvae of this species were cytologically analyzed, all of which had the standard sequence. S. metallicum (s.l.), S. ochraceum (s.l.), S. guianense (s.l.) and S. oyapockense (s.l.) are involved with transmission of onchocerciasis in Central and South America, however, in French Guiana these species were not found biting humans during the sampling period. With the few collections made during this study, we increase the number of simulid species known in French Guiana from 6 to 13. It is clear that more simuliid species can be expected to be found when more sampling is done, including collections in other ecoregions in French Guiana.

Hyperinsulinism and hyperammonemia syndrome: report of twelve unrelated patients.

Hyperinsulinism and hyperammonemia syndrome has been reported as a cause of moderately severe hyperinsulinism with diffuse involvement of the pancreas. The disorder is caused by gain of function mutations in the GLUD1 gene, resulting in a decreased inhibitory effect of guanosine triphosphate on the glutamate dehydrogenase (GDH) enzyme. Twelve unrelated patients (six males, six females) with hyperinsulinism and hyperammonemia syndrome have been investigated. The phenotypes were clinically heterogeneous, with neonatal and infancy-onset hypoglycemia and variable responsiveness to medical (diazoxide) and dietary (leucine-restricted diet) treatment. Hyperammonemia (90-200 micromol/L, normal <50 micromol/L) was constant and not influenced by oral protein, by protein- and leucine-restricted diet, or by sodium benzoate or N-carbamylglutamate administration. The patients had mean basal GDH activity (18.3 +/- 0.9 nmol/min/mg protein) not different from controls (17.9 +/- 1.8 nmol/min/mg protein) in cultured lymphoblasts. The sensitivity of GDH activity to inhibition by guanosine triphosphate was reduced in all patient lymphoblast cultures (IC(50), or concentrations required for 50% inhibition of GDH activity, ranging from 140 to 580 nM, compared with control IC(50) value of 83 +/- 1.0 nmol/L). The allosteric effect of ADP was within the normal range. The activating effect of leucine on GDH activity varied among the patients, with a significant decrease of sensitivity that was correlated with the negative clinical response to a leucine-restricted diet in plasma glucose levels in four patients. Molecular studies were performed in 11 patients. Heterozygous mutations were localized in the antenna region (four patients in exon 11, two patients in exon 12) as well as in the guanosine triphosphate binding site (two patients in exon 6, two patients in exon 7) of the GLUD1 gene. No mutation has been found in one patient after sequencing the exons 5-13 of the gene.

Characterization of an in vivo hormonally regulated phosphodiesterase 3 (PDE3) associated with a liver Golgi-endosomal fraction.

The biochemical properties of an in vivo hormonally regulated low Km cAMP phosphodiesterase (PDE) activity associated with a liver Golgi-endosomal (GE) fraction have been characterized. DEAE-Sephacel chromatography of a GE fraction solubilized by a lysosomal extract resulted in the sequential elution of three peaks of activity (numbered I, II, and III), while ion-exchange HPLC resolved five peaks of activity (numbered 1, 2, 3, 4, and 5). Based on the sensitivity of the eluted activity to cGMP and selected phosphodiesterase inhibitors, two phosphodiesterase isoforms were resolved: a cGMP-stimulated and EHNA-inhibited PDE2, eluted in DEAE-Sephacel peak I and HPLC peak 2 and a cGMP-, a cilostamide-, and ICI 118233-inhibited PDE3, eluted in DEAE-Sephacel peak III and HPLC peaks 3, 4, and 5. GE fractions isolated after acute treatments with insulin, tetraiodoglucagon, and growth hormone displayed an increase in phosphodiesterase activity relative to saline-injected controls, as did GE fractions from genetically obese and hyperinsulinemic rats relative to lean littermates. In all experimental rats, an increase in PDE3 activity associated with DEAE-Sephacel peak III and HPLC peaks 4 and 5 was observed relative to control animals. Furthermore, in genetically obese Zucker rats, an increase in the sensitivity of PDE activity to cilostamide and in the amount of PDE activity immunoprecipitated by an antibody to adipose tissue PDE3 was observed relative to lean littermates. These results extend earlier studies on isolated hepatocytes and show that liver PDE3 is the main if not sole PDE isoform activated by insulin, glucagon, and growth hormone in vivo.

New splicing-site mutations in the SURF1 gene in Leigh syndrome patients.

The gene SURF1 encodes a factor involved in the biogenesis of cytochrome c oxidase, the last complex in the respiratory chain. Mutations of the SURF1 gene result in Leigh syndrome and severe cytochrome c oxidase deficiency. Analysis of seven unrelated patients with cytochrome c oxidase deficiency and typical Leigh syndrome revealed different SURF1 mutations in four of them. Only these four cases had associated demyelinating neuropathy. Three mutations were novel splicing-site mutations that lead to the excision of exon 6. Two different novel heterozygous mutations were found at the same guanine residue at the donor splice site of intron 6; one was a deletion, whereas the other was a transition [588+1G>A]. The third novel splicing-site mutation was a homozygous [516-2_516-1delAG] in intron 5. One patient only had a homozygous polymorphism in the middle of the intron 8 [835+25C>T]. Western blot analysis showed that Surf1 protein was absent in all four patients harboring mutations. Our studies confirm that the SURF1 gene is an important nuclear gene involved in the cytochrome c oxidase deficiency. We also show that Surf1 protein is not implicated in the assembly of other respiratory chain complexes or the pyruvate dehydrogenase complex.

Aedes aegypti in French Guiana: susceptibility to a dengue virus.

Twenty-seven samples of Aedes aegypti (F1 generation) from French Guiana were tested for their susceptibility to dengue serotype 2 virus. Very high infection rates were observed by indirect fluorescent antibody (IFA) test. Ae. aegypti samples were pooled according to two groups: the first group (N=10) represented mosquitoes from the urbanized area of Cayenne and surroundings, and the second group (N=17) corresponded to mosquitoes collected in the countryside. Infection rates were found to be similar in these two cases. These findings are discussed in relation with the history of Ae. aegypti in this part of the world.

First prenatal diagnosis of defects in the HsPDX1 gene encoding protein X, an additional lipoyl-containing subunit of the human pyruvate dehydrogenase complex.

We have previously reported a genetic study of a neonatal lactic acidosis linked to a pyruvate dehydrogenase complex deficiency due to the absence of the protein X subunit. This rare autosomal recessive disorder is associated with specific deletions in this polypeptide which is encoded by the HsPDX1 gene, located on chromosome 11p1.3. The pathology of the patient was considered to arise from a large homozygous deletion (78del85) found at the 5' end of the HsPDX1 coding sequence. Her heterozygous mother underwent prenatal diagnosis during a subsequent pregnancy. Chorionic villus samples were used for three independent studies: (1) normal levels of the protein X component of the PDH complex were detected by immunoblotting; (2) RT-PCR analysis showed no deletion at the 5' end of the cDNA but the presence of a distinct heterozygous deletion (965del59) at its 3' end inherited from the father; (3) haplotype analysis revealed the presence of the father's mutated allele and the mother's normal allele. It was concluded that the fetus was heterozygous for this separate 3' deletion, so, it was likely to be not affected. This study permitted us to characterize more precisely the genetic abnormalities of the HsPDX1 cDNA occurring in each family's member.

Residual activity of Bacillus thuringiensis serovars medellin and jegathesan on Culex pipiens and Aedes aegypti larvae.

Bacillus thuringiensis serovar medellin strain 163-131 and Bacillus thuringiensis serovar jegathesan (B.t.jeg.) strain 367 are very toxic to mosquito larvae. However, they are 10 times less toxic than Bacillus thuringiensis var. israelensis (B.t.i.) to mosquito larvae under laboratory conditions. Lyophilized powders were produced from these strains and their toxicities were compared to that of powder produced from the B.t.i. strain. Larvicidal activity was titrated using Aedes aegypti (Bora-Bora strain) larvae, with IPS82 powder as the standard. The efficacy of these powders in the field was determined using Culex pipiens (Montpellier strain) in Paris, France, and Ae. aegypti larvae (French Guiana strain) in Cayenne, French Guiana, in standardized conditions. Residual activity was also assessed in the laboratory, using Cx. pipiens (SLAB strain), in Montpellier, France. Any negative effect of direct sunlight, soil, or polluted water on the residual activity of the 3 powders was recorded. Increasing bacterial concentration by a factor of 8 had little effect on the duration of larvicidal activity, except in the presence of polluted water and when substrate was added. All powders had similar initial efficacies against both types of mosquito larvae, in all conditions except water rich in organic matter. Bacillus thuringiensis serovar medellin had the lowest residual activity, both in the laboratory and in the field, whereas B.t.jeg. remained toxic for as long as B.t.i.

Detection of Bordetella bronchiseptica by the polymerase chain reaction.

Polymerase chain reaction (PCR) assays were developed that enabled not only discriminative detection of three Bordetella species, B. pertussis, B. parapertussis, and B. bronchiseptica (Bspp PCR), but also specific detection of B. bronchiseptica (Bb PCR). An upstream sequence of the flagellin gene was used as a target DNA region. This sequence contained differences in B. pertussis, B. parapertussis, and B. bronchiseptica DNA. These species could then be differentiated using two different sets of primers, Bspp and Bb. When oligonucleotide Bspp primers were used, PCR products were obtained from the three species of Bordetella. A fragment of the expected size (164 bp) was amplified using B. bronchiseptica and B. parapertussis DNA, but a fragment with a distinct molecular weight was amplified with B. pertussis DNA (195 bp). This Bspp PCR was specific and sensitive, but it could not differentiate between B. parapertussis and B. bronchiseptica. When Bb primers were used, a 237-bp PCR product was detected only from B. bronchiseptica DNA. No PCR products were identified after Bb PCR amplification of DNAs either from B. parapertussis isolates or B. pertussis isolates, nor from other respiratory pathogen DNAs tested. This second PCR assay had a sensitivity limit of less than 10 organisms of B. bronchiseptica after detection with a specific probe. The specificity and the sensitivity of the fla PCR assay were evaluated with purified DNA, as was its capacity for detecting the bacteria in human clinical samples and in lungs of infected mice.

Activation of a cGMP-stimulated cAMP phosphodiesterase by protein kinase C in a liver Golgi-endosomal fraction.

The ability of Ca2+/phospholipid-dependent protein kinase (protein kinase C, PKC) to stimulate cAMP phosphodiesterase (PDE) activity in a liver Golgi-endosomal (GE) fraction was examined in vivo and in a cell-free system. Injection into rats of 4 beta-phorbol 12-myristate 13-acetate, a known activator of PKC, caused a rapid and marked increase in PKC activity (+325% at 10 min) in the GE fraction, along with an increase in the abundance of the PKC alpha-isoform as seen on Western immunoblots. Concurrently, 4 beta-phorbol 12-myristate 13-acetate treatment caused a time-dependent increase in cAMP PDE activity in the GE fraction (96% at 30 min). Addition of the catalytic subunit of protein kinase A (PKA) to GE fractions from control and 4 beta-phorbol 12-myristate 13-acetate-treated rats led to a comparable increase (130-150%) in PDE activity, suggesting that PKA is probably not involved in the in-vivo effect of 4 beta-phorbol 12-myristate 13-acetate. In contrast, addition of purified PKC increased (twofold) PDE activity in GE fractions from control rats but affected only slightly the activity in GE fractions from 4 beta-phorbol 12-myristate 13-acetate-treated rats. About 50% of the Triton-X-100-solubilized cAMP PDE activity in the GE fraction was immunoprecipitated with an anti-PDE3 antibody. On DEAE-Sephacel chromatography, three peaks of PDE were sequentially eluted: one early peak, which was stimulated by cGMP and inhibited by erythro-9 (2-hydroxy-3-nonyl) adenine (EHNA); a selective inhibitor of type 2 PDEs; and two retarded peaks of activity, which were potently inhibited by cGMP and cilostamide, an inhibitor of type 3 PDEs. Further characterization of peak I by HPLC resolved a major peak which was activated (threefold) by 5 microM cGMP and inhibited (87%) by 25 microM EHNA, and a minor peak which was insensitive to EHNA and cilostamide. 4 beta-Phorbol 12-myristate 13-acetate treatment caused a selective increase (2.5-fold) in the activity associated with DEAE-Sephacel peak I, without changing the K(m) value. These results suggest that PKC selectively activates a PDE2, cGMP-stimulated isoform in the GE fraction.

Longitudinal study of tissue- and subunit-specific obesity-induced regulation of the pyruvate dehydrogenase complex.

The tissue-specific expression of the mitochondrial pyruvate dehydrogenase complex (PDHc) has been studied in an animal model of obesity with hyperinsulinemia, the obese (fa/fa) Zucker rat. Liver and heart were obtained from 4 and 8 week-old obese rats and age-matched lean animals, and in each tissue the following parameters were analyzed: (1) total activity of the mitochondrial PDHc; (2) abundance of the mitochondrial PDHc subunits on Western blots; and (3) abundance of the E1alpha and E1beta subunit mRNAs on Northern blots and semi-quantitative RT-PCR. Regardless of age, obese rats showed an increase in liver total PDHc activity and a coordinate increase in liver E1alpha and E1beta PDHc subunit abundance. At 4 weeks, obese rats also showed an increase in liver PDH E1alpha mRNA level, but regardless of age E1beta mRNA level was unchanged. In contrast, neither total PDHc activity nor the concentration of its protein subunits were increased in heart of obese rats. Thus, obese Zucker rats display a liver-specific early increase in PDHc which results from a selective up-regulation of the E1alpha gene expression.

Mutations in PDX1, the human lipoyl-containing component X of the pyruvate dehydrogenase-complex gene on chromosome 11p1, in congenital lactic acidosis.

We have identified and sequenced a cDNA that encodes an apparent human orthologue of a yeast protein-X component (ScPDX1) of pyruvate dehydrogenase multienzyme complexes. The new human cDNA that has been referred to as "HsPDX1" cDNA was cloned by use of the "database cloning" strategy and had a 1,506-bp open reading frame. The amino acid sequence of the protein encoded by the cDNA was 20% identical with that encoded by the yeast PDX1 gene and 40% identical with that encoded by the lipoate acetyltransferase component of the pyruvate dehydrogenase and included a lipoyl-bearing domain that is conserved in some dehydrogenase enzyme complexes. Northern blot analysis demonstrated that the major HsPDX1 mRNA was 2.5 kb in length and was expressed mainly in human skeletal and cardiac muscles but was also present, at low levels, in other tissues. FISH analysis performed with a P1-derived artificial chromosome (PAC)-containing HsPDX1 gene sublocalized the gene to 11p1.3. Molecular investigation of PDX1 deficiency in four patients with neonatal lactic acidemias revealed mutations 78del85 and 965del59 in a homozygous state, and one other patient had no PDX1 mRNA expression.

Biochemical and genetic studies of four patients with pyruvate dehydrogenase E1 alpha deficiency.

We report studies of four patients with pyruvate dehydrogenase complex (PDH) deficiency caused by mutations in the E1 alpha subunit. Two unrelated male patients presented with Leigh syndrome and a R263G missense mutation in exon 8. This mutation has previously been described in males with the same phenotype. The two other patients had different novel mutations: (1) an 8-bp deletion at the C-terminus (exon 11) was found in one allele of a young girl suffering from microcephaly and (2) a C88S missense mutation (exon 3) in a boy who only presented with motor neuropathy. These mutations were not found in the mothers of any of the four cases. Immunoblot analysis revealed decreased immunoreactivity for the E1 alpha and E1 beta subunits in three out of the four patients. These findings confirm that: (1) PDH deficiencies are genetically heterogeneous, (2) the R263G mutation is more frequent in male cases than are other mutations and this amino acid is a hot spot for gene mutations, (3) the last eight amino acids may be important for the conformation of the tetrameric E1-PDH enzyme, and (4) the amino acids at positions 88, 263 and 382-387 are essential for the linking of the alpha subunit with the beta subunit and for the activity of the holoenzyme.