'Padhar regime' - a low-dose magnesium sulphate treatment for eclampsia.

AIMS: To determine the recurrent convulsion rate using low-dose magnesium sulphate regime in eclampsia and to identify toxicity and complications with clinical parameters. METHODS: Prospective study with two different magnesium sulphate regimes in two slightly clinically different subgroups. Group A that came directly to our hospital and group B who had already received an injection of diazepam or Phenergan at the referring hospital. Group A received 10 g and group B 6 g loading dose of magnesium sulphate. Both groups received 4 g maintenance dose every 4 h. RESULTS: Out of 95 eclamptic patients, only one woman in group B had recurrent convulsion. All women maintained normal respiratory rates. 39 (41.1%) women had absent knee jerks on at least one occasion when the maintenance dose was omitted. Urinary output was more than 30 ml/h in 92 (96.8%) women. In 5 women, maintenance dose had to be augmented to 5 g as reflexes were exaggerated. CONCLUSION: The low-dose regime appears to control and prevent convulsions effectively in Indian women. Clinical monitoring appears to be sufficient. We hope to be able to reassure health professionals at primary and secondary level hospitals about the safety of magnesium.

Recurrent ovarian malignancy presenting as cutaneous metastasis.

Cutaneous metastasis from ovarian carcinoma is relatively uncommon in clinical practice. We report the case of the woman who presented to us with clitoral nodules and skin nodules. Histopathological examination of nodules confirmed the diagnosis of metastasis of an ovarian carcinoma. Despite poor prognosis, the patient responded and survived well beyond the expected four months survival of similar cases.

Tuberculosis drug resistance and treatment outcomes under DOTS settings in large cities in the Philippines.

SETTING: Two large cities in the Philippines. OBJECTIVES: To describe the problems of drug-resistant tuberculosis (TB) in an urban setting, with special emphasis on their potential impact on the treatment services provided by the National TB Control Programme. DESIGN: Cross-sectional survey and cohort analysis of treatment outcomes. METHODS: All patients with positive sputum smear examination results in Cebu and Mandaue cities during the survey period were included. The survey procedures of the World Health Organization and the International Union Against Tuberculosis and Lung Disease were strictly applied. Treatment outcome data were also collected. RESULTS: Of 306 cases enrolled, 255 were new cases, 28 were previously treated and for 23 treatment history was unknown. Of the new cases, 72.2% were pan-susceptible to all four first-line anti-tuberculosis drugs. Resistance in new cases was 16.9% to isoniazid (INH), 4.7% to rifampicin (RMP), 3.1% to ethambutol, 18.0% to streptomycin, and 3.9% to at least both INH and RMP (multidrug-resistant [MDR]). Over 90% of the new cases, either pan-susceptible or mono-resistant, were successfully treated with the standard regimen, but four of nine MDR new cases could not be cured. CONCLUSION: The drug resistance level was high in this population, but treatment outcome using the standard treatment regimen was not seriously affected unless the patients were MDR.

DOTS expansion: will we reach the 2005 targets?

The global targets for tuberculosis control consist of detecting 70% of estimated infectious cases and curing 85% of these by 2005. Since the introduction of the DOTS strategy, DOTS geographical coverage has increased substantially and treatment success rates under DOTS are approaching the targets, standing at 82% in 2000. However, DOTS case detection, albeit increasing, is still relatively low, at 32% in 2001. This target may not be reached by 2005. The low case detection is unlikely to stem from overestimating the global number of TB cases which has been estimated on several occasions, but from TB cases not being detected or notified for various reasons. The population may have poor access to TB services, cases may not be suspected or correctly diagnosed, cases may not be notified, and/or public health programmes or the private sector may not be adequately linked to the National Tuberculosis Programmes. Since the global TB targets were set, progress has been made. Political commitment has increased, additional financial resources mobilised, access to anti-tuberculosis drugs augmented and planning and coordination improved. Constraints still remain, the most important related to human resource capacity. Although the issue is being tackled, many countries still suffer from a lack of trained health care professionals. Finally, new strategies have been developed to face the current challenges such as public-private mix, community TB care, social mobilisation, TB/HIV collaborative interventions and Practical Approach to Lung Health. The current efforts should be maintained and strengthened in order to approach these targets.

Noncardiogenic pulmonary edema as the chief manifestation of a pheochromocytoma: a case report of MEN 2A with pedigree analysis of the RET proto-oncogene.

Pheochromocytomas are rare neoplasias of the adrenal medulla which generally present with paroxysmal or sustained hypertension. Cardiogenic pulmonary edema is a common feature of these tumors, but few cases have been described with noncardiogenic pulmonary edema. We report a pheochromocytoma with the principle manifestation of noncardiogenic pulmonary edema and characterize a genetic lesion associated with the disorder. A 30-year-old man was admitted with abdominal pain and breathlessness. x-Ray examination of the chest revealed a massive, diffuse infiltration of the left lung without cardiomegaly. No paroxysmal blood pressure fluctuations or heart failure were evident during the entire course, and the infiltrate and dyspnea resolved in three days without inotropic or diuretic agents. Serum norepinephrine and epinephrine levels were elevated twenty and fifty times above normal, respectively. The patient was ultimately diagnosed with multiple endocrine neoplasia type 2A (MEN 2A). Mutations in the RET proto-oncogene have been described recently in patients with MEN 2A. Mutation analysis of selected RET exonic sequences identified a germline mutation at codon 634 in exon 11 of the RET proto-oncogene. The mutation introduces a transition encoding a non-conservative substitution from TGC (Cys) to CGC (Arg) and creates a novel restriction site recognized by HhaI. We further screened for this mutation among four of the proband's relatives by HhaI restriction analysis. One asymptomatic family member was identified who subsequently elected prophylactic total thyroid removal. Histological examination of this specimen confirmed the presence of medullary thyroid carcinoma.

Hereditary orotic aciduria heterozygotes accompanied with neurological symptoms.

We report a family with hereditary orotic aciduria heterozygotes. A 3-year-old boy who had been diagnosed as having cerebral palsy and mental retardation presented himself with an increase in excretion of urinary orotic acid. Enzymatic studies revealed that the boy and his healthy mother were hereditary orotic aciduria heterozygote carriers. We can not prove that this pyrimidine disorder caused his neurological symptoms, but his pyrimidine nucleoside supply may have been insufficient in his neonatal period.

Molecular cloning of the human UMP synthase gene and characterization of point mutations in two hereditary orotic aciduria families.

Uridine monophosphate (UMP) synthase is a bifunctional enzyme catalyzing the last two steps of de novo pyrimidine biosynthesis, orotate phosphoribosyltransferase (OPRT) and orotidine-5'-monophosphate decarboxylase (ODC). Loss of either enzymatic activity results in hereditary orotic aciduria, a rare autosomal recessive disorder characterized by retarded growth, anemia, and excessive urinary excretion of orotic acid. We have isolated the UMP synthase chromosomal gene from a lambdaEMBL-3 human genomic library and report a single-copy gene spanning approximately 15 kb. The UMP synthase genomic structure encodes six exons ranging in size from 115 bp to 672 bp, and all splicing junctions adhere to the canonical GT/AG rule. Cognate promoter elements implicated in glucocorticoid- and cAMP-mediated regulation as well as in liver-, myeloid-, and lymphocyte-specific expression are located within the 5' flanking sequence. Molecular investigation of UMP synthase deficiency in a Japanese orotic aciduria patient revealed mutations R96G (A-to-G transition; nt 286) and G429R (G-to-C transversion; nt 1285) in one allele and V109G (T-to-G transversion; nt 326) in the other allele. Expression of human UMP synthase cDNAs containing these mutations in pyrimidine auxotrophic Escherichia coli and in recombinant baculovirus-infected Sf21 cells demonstrates impaired activity presumably associated with the urinary orotic acid substrate accumulations observed in vivo. We further establish the identity of two polymorphisms, G213A (v = .26) and 440Gpoly (v = .27) located in exons 3 and 6, respectively, which did not significantly compromise either OPRT or ODC function.

Isolation of a rat histidase cDNA sequence and expression in Escherichia coli--evidence of extrahepatic/epidermal distribution.

Histidase (histidine ammonia-lyase) is a cytosolic enzyme responsible for catalyzing the non-oxidative deamination of histidine to urocanic acid. Full-length cDNAs encoding rat histidase have been isolated from a lambdaZAP liver cDNA library using a partial cDNA fragment obtained by PCR. Whereas the initial description of the rat histidase 3' untranslated sequence contained a rare polyadenylation signal sequence, the data presented encompass a more distant 28-bp region, possessing a nucleotide stretch (AATATAAA), identical to that in the mouse histidase cDNA. Dideoxynucleotide chain-termination sequencing of two clones obtained by in vivo excision yielded an additional 376 bp and 105 bp of 5' and 3' untranslated sequences, respectively. A selected rat histidase cDNA clone was introduced into the pET-16b prokaryotic vector and expressed in BL21(DE3)pLysS Escherichia coli. After purification by nickel-chelation chromatography, recombinant histidine-tagged protein was employed to raise anti-(rat histidase) immunoglobulin in a Japanese white rabbit. The polyclonal rabbit antibody recognized and formed immune complexes with rat and recombinant human histidase proteins. Immunoblots of crude rat organ extracts detected a spectrum of histidase expression extending beyond that observed in liver and skin. Among other histidase-positive cells were those of the renal cortex tubular epithelium, fundic mucosal glands of stomach, gastric intramuscular (Auerbach's) plexus, and adrenal cortex. Immunohistochemical studies of histidase in rat liver produced discrete staining of hepatocytes in association with portal triads (Rappaport zone I). Furthermore, in contrast with previous reports of activity confined to epidermal stratum corneum, our findings demonstrate immunoreactive protein within and limited to the adjacent stratum granulosum.

Microsatellite instability in in situ and invasive sporadic breast cancers of Japanese women.

We studied the timing of microsatellite instability (referred to as replication error; RER) presentation during human breast carcinogenesis using tissue microdissected from both in situ and invasive breast cancers of Japanese women. We analyzed 100 breast cancer specimens for RER at nine genomic loci on seven chromosomes. Eight of the 100 cases (8%) were RER-positive at one or more chromosomal loci. Additionally; we obtained genomic DNA from two of four RER-positive patients with an intraductal component, both of which showed microsatellite instability in in situ foci. This finding indicates that microsatellite instability may be an early event during human breast carcinogenesis.

Microsatellite instability in sporadic human breast cancers.

Human breast-cancer specimens from 100 patients were analyzed for microsatellite instability (referred to as replication error; RER) at 12 genomic loci on 7 chromosomes, and results were correlated with clinicopathologic characteristics. In 42 of 100 breast-cancer patients, we investigated whether RER was associated with the amplification of oncogenes and/or suppression of tumor-suppressor genes. Of the 100 patients, 8 (8%) were RER-positive at one or more chromosomal loci. The majority of RER-positive patients had early-stage disease with ER-positive tumors, suggesting that RER occurs early in breast tumorigenesis. However, no significant correlation was observed between RER and oncogenes or tumor-suppressor genes. Thus, the mechanism of RER in sporadic human breast cancer may be independent of the multi-step carcinogenesis caused by the alterations of oncogenes and tumor-suppressor genes.

Identification and expression of a missense mutation (Y446C) in the acid sphingomyelinase gene from a Japanese patient with type A Niemann-Pick disease.

Types A and B Niemann-Pick disease (NPD), an autosomal recessive lysosomal storage disorder, are caused by deficiency of acid sphingomyelinase (ASM). The recent identification of mutations in ASM gene causing types A and B NPD has led to the investigation of the phenotypic heterogeneity and the ethnic distribution of this disease, especially in Ashkenazi Jewish population. To characterize the mutations causing NPD in Japanese population, we analyzed the genomic sequence of ASM from a Japanese patient with type A NPD by PCR amplification and sequencing. A new mutation, Y446C, was identified. The authenticity of this lesion was demonstrated by the expression of the Y446C allele in COS-1 cells. No residual ASM activity was detected from the expression of the Y446C.

Molecular cloning and structural characterization of the human histidase gene (HAL).

Histidase (EC 4.3.1.3) is a cytosolic enzyme that catalyzes the nonoxidative deamination of histidine to urocanic acid. Histidinemia, resulting from reduced histidase activity as reported in Cambridge stock his/his mice and in humans, is the most frequent inborn metabolic error in Japan. The histidase chromosomal gene (HAL) was isolated from a lambda EMBL-3 human genomic library using the human histidase cDNA as a probe. Restriction mapping and Southern blot analysis of the isolated clones reveal a single-copy gene spanning approximately 25 kb and consisting of 21 exons. Exon 1 encodes only 5' untranslated sequence of liver histidase mRNA, with protein coding beginning in exon 2. A rarely observed 5' GC, similar to that reported in the human P-450 (SCC) gene, is present in intron 20. All other splicing junctions adhere to the canonical GT/AG rule. A TATA box sequence is located 25 bp upstream of the liver histidase transcription initiation site determined by S1 nuclease protection analysis. Several liver- and epidermis-specific transcription factor binding sites, including C/EBP, NFIL6, HNF5, AP2/KER1, MNF, and others, are also identified in the 5' flanking region. Consistent with the hepatic and epidermal expression of histidase, this finding suggests that histidase transcription may be regulated by these factors. We further identify a polymorphism (A to G transition) in the histidase coding region of exon 16. The human histidase genomic structure presented here should facilitate the molecular investigation of symptomatic and asymptomatic forms of histidinemia.

Molecular cloning of a cDNA encoding human histidase.

We isolated overlapping cDNA clones encoding human histidase (histidine ammonia-lyase) from a human lambda gt10 library. The cDNA predicted a 657 amino acid protein of 72,651 Da. The human histadase amino acid sequence was 93% conserved with both rat and mouse histidase sequences, including four N-glycosylation consensus sites.

Toward gene therapy for Niemann-Pick disease (NPD): separation of retrovirally corrected and noncorrected NPD fibroblasts using a novel fluorescent sphingomyelin.

The neurologic (type A) and nonneurologic (type B) forms of Niemann-Pick disease (NPD) both result from deficiencies of acid sphingomyelinase (ASM) activity leading to the accumulation of sphingomyelin and other related lipids within lysosomes. Recently, the full-length cDNA and genomic sequences encoding ASM have been isolated and the nature of the molecular lesions causing NPD has been investigated. Although these developments have facilitated diagnosis for this debilitating disease, no effective treatment is currently available. Toward this latter goal, our laboratories recently reported the effectiveness of retroviral-mediated gene transfer for the in vitro correction of the cellular pathology in NPD fibroblasts (Suchi et al., 1992). In addition, novel selection procedures were developed to separate retrovirally corrected and noncorrected NPD fibroblasts based on the receptor-mediated delivery of a fluorescently (pyrene)-labeled sphingomyelin (P12-SPM) to the lysosomes of cells using liposomes coated with apolipoprotein E. In this study, we have used a different, fluorescent derivative of sphingomyelin (lissamine-rhodamine dodecanoyl sphingomyelin; LR12-SPM) to extend and improve this selection system. LR12-SPM offers a number of advantages over P12-SPM, including the facts that apolipoprotein E is not required for its efficient uptake and targeting to lysosomes and that the product of LR12-SPM degradation by ASM is efficiently transported out of cells. Thus, when analyzed in a fluorescence-activated cell sorter (FACS), there was complete separation (i.e., no overlap) of retrovirally corrected and noncorrected NPD cells after the administration of LR12-SPM.(ABSTRACT TRUNCATED AT 250 WORDS)

Identification and expression of five mutations in the human acid sphingomyelinase gene causing types A and B Niemann-Pick disease. Molecular evidence for genetic heterogeneity in the neuronopathic and non-neuronopathic forms.

The deficient activity of the human lysosomal hydrolase, acid sphingomyelinase (ASM, EC 3.1.4.12), results in the neuronopathic (Type A) and non-neuronopathic (Type B) forms of Niemann-Pick disease (NPD). To investigate the genetic basis of the phenotypic heterogeneity in NPD, the molecular lesions in the ASM gene were determined from three unrelated NPD patients and evaluated by transient expression in COS-1 cells. A Type A NPD patient of Asian Indian ancestry (proband 1) was homoallelic for a T to A transversion in exon 2 of the ASM gene which predicted a premature stop at codon 261 of the ASM polypeptide (designated L261X). In contrast, an unrelated Type A patient of European ancestry (proband 2) was heteroallelic for a two-base (TT) deletion in exon 2 which caused a frame-shift mutation at ASM codon 178 (designated fsL178), leading to a premature stop at codon 190, and a G to A transition in exon 3 which caused a methionine to isoleucine substitution at codon 382 (designated M382I). Transient expression of the fsL178, L261X, and M382I mutations in COS-1 cells demonstrated that these lesions did not produce catalytically active ASM, consistent with the severe neuronopathic Type A NPD phenotype. In contrast, an unrelated Type B patient of European descent (proband 3) was heteroallelic for two missense mutations, a G to A transition in exon 2 which predicted a glycine to arginine substitution at ASM codon 242 (designated G242R), and an A to G transition in exon 3 which resulted in an asparagine to serine substitution at codon 383 (designated N383S). Interestingly, the G242R allele produced ASM activity in COS-1 cells at levels about 40% of that expressed by the normal allele, thereby explaining the mild Type B phenotype of proband 3 and the high residual activity (i.e. approximately 15% of normal) in cultured lymphoblasts. In contrast, the N383S allele did not produce catalytically active enzyme. None of these five ASM mutations was detected in over 60 other unrelated NPD patients analyzed, nor were these mutations found in over 100 normal ASM alleles. Thus, small deletions or nonsense mutations which trunctated the ASM polypeptide, or missense mutations that rendered the enzyme noncatalytic, resulted in Type A NPD disease, whereas a missense mutation that produced a defective enzyme with residual catalytic activity caused the milder nonneuronopathic Type B phenotype. These findings have facilitated genotype/phenotype correlations for this lysosomal storage disease and provided insights into the functional organization of the ASM polypeptide.

Retroviral-mediated transfer of the human acid sphingomyelinase cDNA: correction of the metabolic defect in cultured Niemann-Pick disease cells.

Types A and B Niemann-Pick disease (NPD) result from inherited deficiencies of the lysosomal hydrolase, acid sphingomyelinase (ASM; sphingomyelin cholinephosphohydrolase, EC 3.1.4.12). To evaluate the feasibility of somatic gene therapy for the treatment of these disorders, retroviral-mediated gene transfer was used to introduce the full-length ASM cDNA into cultured fibroblasts from two unrelated type A NPD patients. The ASM activities in these cells were less than 4% of mean normal levels, and, consequently, they accumulated approximately 3-fold elevated levels of sphingomyelin. After retroviral-mediated transfer of the ASM cDNA, ASM activities in the NPD cells increased to levels up to 16-fold those found in normal fibroblasts. In addition, the sphingomyelin content was reduced to normal levels, indicating that the vector-encoded enzyme was properly targeted to lysosomes, where it was enzymatically active and able to degrade the accumulated substrate. In situ cell-loading studies also were undertaken to evaluate the effects of retroviral-mediated gene transfer on the pathology of NPD fibroblasts. When a pyrene derivative of sphingomyelin was introduced into the lysosomes of cultured fibroblasts from a type A NPD patient by using apolipoprotein E-mediated endocytosis, only approximately 6% of the delivered substrate was degraded. In contrast, normal cells and NPD cells transduced (i.e., "corrected") by retroviral-mediated gene transfer could degrade approximately 80% of the delivered sphingomyelin. These results provided further evidence that retroviral-mediated gene transfer may be used to correct the pathology of NPD cells. Cell-loading studies were also used to develop a selection system for discriminating between NPD cells and those transduced by retroviral-mediated gene transfer. This selection scheme was based on the fluorescence emission of intact NPD cells, which, when loaded with pyrene-labeled sphingomyelin, was 3- to 5-fold that of normal or transduced cells. As a consequence, the NPD and transduced cells could be efficiently sorted by flow cytometry with a fluorescence-activated cell sorter. In addition, the NPD cells could be selectively killed by photosensitization after irradiation with a long-wavelength UV light. These results should permit direct selection of ASM-expressing cells after retroviral-mediated gene transfer without the need to preselect for a cotransferred marker gene.

Human acid sphingomyelinase. Isolation, nucleotide sequence and expression of the full-length and alternatively spliced cDNAs.

Two types of partial cDNAs encoding human acid sphingomyelinase (EC 3.1.4.12; ASM) were recently isolated from fibroblast and placental cDNA libraries (Quintern, L. E., Schuchman, E.H., Levran, O., Suchi. M., Ferlinz, K., Reinke, H., Sandhoff, K., and Desnick, R. J. (1989) EMBO J. 8, 2469-2473). The cDNA inserts had identical sequences with the exception of an internal region; type 1 cDNAs (representing approximately 90% of the ASM cDNAs isolated) had 172 in-frame base pairs (bp), which were replaced in the type 2 cDNAs by a 40-bp in-frame sequence. Northern hybridization and RNase protection studies indicated that both type 1 and 2 transcripts were approximately 2.5 kilobases; therefore, efforts were directed to isolate full-length type 1 and 2 cDNAs by screening human placental, testis, hepatoma, and retinal cDNA libraries. In addition to type 1 and 2 cDNAs, a new type of ASM cDNA (type 3), which did not contain the type 1- or 2-specific regions, was isolated and sequenced. The full-length type 1 and the reconstructed full-length type 2 and 3 cDNAs were transiently expressed in COS-1 cells. Only the full-length type 1 transcript encoded catalytically active human ASM, demonstrating its functional integrity. The 2347-bp full-length type 1 placental cDNA (pASM-1FL) had an 87-bp 5'-untranslated region, an 1890-bp open reading frame encoding 629 amino acids, and a 370-bp 3'-untranslated sequence. The predicted location of the signal peptide cleavage site was after alanine 46. Two base differences were identified in codons 322 and 506 and shown to be polymorphisms with the common alleles having frequencies of 0.6 and 0.7, respectively. To determine the genomic organization of the type 1, 2, and 3 sequences, a 1665-bp genomic region containing both the unique type 1 (172 bp) and type 2 (40 bp) sequences was amplified by the polymerase chain reaction and sequenced. The 172-bp sequence was exonic, flanked by 5'- and 3'-intronic sequences of 1052 and 229 bp, respectively. The 40-bp type 2 sequence was intronic, occurring at the 5' end of the 1052-bp intron due to the use of a cryptic 5' donor splice site, which deleted the entire 172-bp exon and both flanking intronic sequences. The type 3 cDNA resulted from an alternative splicing event, which excised the 172-bp exon. These studies demonstrate the occurrence of alternatively splicing of the ASM transcript, but the existence of only one functional mRNA.