Isolation of endothelial-like stromal cells that express Kit ligand and support in vitro hematopoiesis.

Although macrophages account for 70-90% of the adherent cells in mouse long-term bone marrow cultures (LTBMC) and CFU-F colonies, the predominant nonhematopoietic stromal cell is endothelial-like (EL), expressing cytoplasmic collagen IV, laminin, and an antigen recognized by the monoclonal antibody MECA-10. We report the isolation of this stromal cell lineage from primary LTBMC by immunomagnetic cell selection using MECA-10. More than 95% of the cells in the MECA-10-positive fraction are EL cells as judged by morphology, surface staining for MECA-10, cytoplasmic staining for collagen IV, and electrophoretic analysis of MECA-10-positive cells isolated from radiation chimeras. When plated under LTBMC conditions, EL cell monolayers recharged with either wild-type or Sl/Sld marrow support an increased density and number of clonogenic and mature hematopoietic cells in short-term cultures. In accord with this finding, Northern blots of mRNA from unstimulated EL cells demonstrate constitutive expression of Kit ligand (KL). Moreover, in situ two-color immunofluorescence staining for cytoplasmic collagen IV and surface KL suggests that EL cells are the exclusive source of membrane-bound KL in mouse cultures. The ability to isolate EL cells from primary cultures without the need for repeated cell passage or immortalization provides a novel approach to dissecting the molecular basis of stem cell-stromal cell interactions.

Mutations in the ligand-binding domain of the kit receptor: an uncommon site in human piebaldism.

Heterozygous mutations in the gene for the Kit transmembrane receptor have been identified recently in human piebaldism and mouse "dominant spotting." Interestingly, all of the 14 known missense mutations that cause depigmentation in these species map to the tyrosine kinase domain of the receptor, whereas none have involved the extracellular ligand-binding domain. In an attempt to detect these uncommon mutations, we screened the nine exons encoding the extracellular portion of Kit for single-strand conformation polymorphisms (SSCP) in eight piebald subjects previously reported to be negative for kinase mutations. Four of these eight kindreds proved to carry novel mutations. The first mutation, found in two apparently unrelated probands with mild piebaldism and English ancestry, substitutes an arginine for a highly conserved cysteine at codon 136. This substitution disrupts a putative disulfide bond required for formation of the second Ig-like (D2) loop of the Kit ligand-binding domain. The second mutation, detected in a piebald kindred characterized by unusually limited depigmentation, substitutes a threonine for an alanine at codon 178, a site just proximal to conserved cysteines at codons 183 and 186. The third mutation, occurring in a kindred with more extensive depigmentation, is a novel four-base insertion in exon 2 that results in a proximal frameshift and premature termination. The data strongly suggest that piebaldism can result from missense mutations in the Kit ligand-binding domain, although the resulting phenotype may be milder than that observed for null or kinase mutations. The apparent clustering of these uncommon mutations at or near the conserved cysteines for the D2 Ig-like loop further suggests a critical role for this region in Kit receptor function.

Semisynthesis of antitumoral acetogenins: SAR of functionalized alkyl-chain bis-tetrahydrofuranic acetogenins, specific inhibitors of mitochondrial complex I.

The acetogenins of Annonaceae are known by their potent cytotoxic activity. In fact, they are promising candidates as a new future generation of antitumoral drugs to fight against the current chemiotherapic resistant tumors. The main target enzyme of these compounds is complex I (NADH:ubiquinone oxidoreductase) of the mitochondrial respiratory chain, a key enzymatic complex of energy metabolism. In an attempt to characterize the relevant structural factor of the acetogenins that determines the inhibitory potency against this enzyme, we have prepared a series of bis-tetrahydrofuranic acetogenins with different functional groups along the alkyl chain. They comprise several oxo, hydroxylimino, mesylated, triazido, and acetylated derivatives from the head series compounds rolliniastatin-1, guanacone, and squamocin. Our results suggest a double binding point of acetogenins to the enzyme involving the alpha,alpha'-dihydroxylated tetrahydrofuranic system as well as the alkyl chain that links the terminal alpha, beta-unsaturated-gamma-methyl-gamma-lactone. The former mimics and competes with the ubiquinone substrate. The latter modulates the inhibitory potency following a complex outline in which multiple structural factors probably contribute to an appropriate conformation of the compound to penetrate inside complex I.

MarC-V: a spreadsheet-based tool for analysis, normalization, and visualization of single cDNA microarray experiments.

The comprehensive analysis and visualization of data extracted from cDNA microarrays can be a time-consuming and error-prone process that becomes increasingly tedious with increased number of gene elements on a particular microarray. With the increasingly large number of gene elements on today's microarrays, analysis tools must be developed to meet this challenge. Here, we present MarC-V, a Microsoft Excel spreadsheet tool with Visual Basic macros to automate much of the visualization and calculation involved in the analysis process while providing the familiarity and flexibility of Excel. Automated features of this tool include (i) lower-bound thresholding, (ii) data normalization, (iii) generation of ratio frequency distribution plots, (iv) generation of scatter plots color-coded by expression level, (v) ratio scoring based on intensity measurements, (vi) filtering of data based on expression level or specific gene interests, and (vii) exporting data for subsequent multi-array analysis. MarC-V also has an importing function included for GenePix results (GPR) raw data files.

Specific interactions of monotetrahydrofuranic annonaceous acetogenins as inhibitors of mitochondrial complex I.

Annonaceous acetogenins (ACG) are a wide group of cytotoxic compounds isolated from plants of the Annonaceae family. Some of them are promising candidates to be a future new generation of antitumor drugs due to the ability to inhibit the NADH:ubiquinone oxidoreductase of the respiratory chain (mitochondrial complex I), main gate of the energy production in the cell. ACG are currently being tested on standard antitumor trials although little is known about the structure activity relationship at the molecular level. On recent studies, the relevance of several parts of the molecule for the inhibitory potency has been evaluated. Due to the great diversity of skeletons included in this family of natural products, previous studies on the presence and distribution of oxygenated groups along the alkyl chain only covered the compounds with different bis-tetrahydrofuranic (bis-THF) relative configurations. Therefore, we have investigated the inhibitory action of all the mono-tetrahydrofuranic (mono-THF) acetogenins available, which differ in the oxygenated arrangements along the molecule. Our results show that the hydroxyl and carbonyl groups, placed in the aliphatic chain that links the initial gamma-lactone moiety with the dihydroxylated tetrahydrofuranic ring system, significantly contribute for modulating the inhibitory potency of the ACG through specific effects.

[Hemolytic-uremic syndrome in Chile: clinical features, evolution and prognostic factors]. / Síndrome hemolítico urémico en Chile: presentación clínica, evolución y factores pronósticos.

BACKGROUND: Hemolytic-uremic syndrome (HUS) is characterized by acute renal failure, microangiopathic hemolytic anemia and thrombocytopenia. AIM: To describe the characteristics of patients with the diagnosis of HUS in Chile, and to identify the most reliable early predictors of morbidity and mortality. MATERIAL AND METHODS: The clinical records of patients with HUS aged less than 15 years, attended between January 1990 and December 2003 in 15 hospitals, were reviewed. Demographic, clinical, biochemical, hematological parameters, morbidity and mortality were analyzed. RESULTS: A cohort of 587 patients aged 2 to 8 years, 48% males, was analyzed. Ninety two percent had diarrhea. At the moment of diagnosis, anuria was observed in 39% of the patients, hypertension in 45% and seizures in 17%. Forty two percent required renal replacement therapy (RRT) and peritoneal dialysis was used in the majority of cases (78%). The most frequently isolated etiological agent was Escherichia coli. Mortality rate was 2.9% in the acute phase of the disease and there was a positive correlation between mortality and anuria, seizures, white blood cell count (WCC)>20.000/mm3 and requirements of renal replacement therapy (p<0.05). Twelve percent of patients evolved to chronic renal failure and the risk factors during the acute phase were the need for renal replacement therapy, anuria, WCC>20.000/mm3, seizures and hypertension. CONCLUSIONS: The present study emphasizes important clinical and epidemiological aspects of HUS in a Chilean pediatric population.

[Fasting glucose versus oral glucose tolerance test for detection of glucose intolerance in obese children]. / Glicemia de ayuno versus prueba de tolerancia oral a la glucosa en la detección de intolerancia a la glucosa en niños y adolescentes obesos.

BACKGROUND: Recently, the cut-off point for normal fasting glucose (FG) level, was decreased to 100 mg/dl. AIM: To determine the frequency of abnormal carbohydrate abnormalities in children with obesity and evaluate if the fasting glucose level is a useful tool for the screening of glucose intolerance (GI). PATIENTS AND METHODS: Children and adolescents, referred for evaluation of obesity were evaluated with an oral glucose tolerance test (OGTT) and FG. The sensitivity of FG for detection of GI, using the 100 and 110 mg/dl cut-off point, was evaluated. RESULTS: We studied 186 patients (125 females) aged 12.1 (range: 5.4-19.3) years with a body mass index (BMI) of 29.9 (18.3-44.6) kg/mt2 and a BMI Z score of 2.1 (1.7-3.2). Seven patients (3.8%) had abnormalities in the carbohydrate metabolism. The sensitivity of FG for the detection of GI using the 100 and 110 mg/dl cut-off values was 42.9 and 14.3%, respectively. Receiver operating characteristic (ROC) curves showed that the optimal diagnostic level for FG corresponds to 80 mg/dl (sensitivity: 85.7% and specificity of 74.9%). CONCLUSIONS: An abnormal carbohydrate metabolism was detected in 3.8% of the obese children and adolescents in this sample. FG of 100 mg/dl does not detect 57.1% of the patients with glucose intolerance. These data suggest that FG is not a useful screening tool for glucose intolerance in young patients.

[Clinical diagnosis of childhood psychoses]. / Diagnóstico clínico de las psicósis infantiles.

I have tried to summarize the different points of view in regard to clinical diagnosis of child psychosis. The main purpose is to reach a more universal agreement to base a diagnosis that allows us not only to facilitate an early diagnosis but also its treatment and above all better bases for research. Infantile psychosis varies at different levels of growth, according to age, however it is considered that psychosis in children is basically a disturbance in ego-functions. This is clearly evident in the thinking process, in affect, perception, motility, language, individualization, disturbance of object relations, and lost of contact with reality. The basic points for the diagnosis of child psychosis proposed by the "Group for the Advancement of Psychiatry" and initially studied by the English working party headed by Goldberg and col. are discussed: disturbances of their interpersonal relationships, indifference or preoccupation with inanimate objects, lack or failure in speech development, alteration in sensorial perception, bizarre or stereotyped behavior, resistance to change routines or change of environment, poor personal identification, crises of anger or panic which are not predictable and finally an uneven intellectual development.

Five novel genes from the cri-du-chat critical region isolated by direct selection.

Cri-du-chat is a well described partial aneusomy resulting from deletion of the short arm of chromosome 5. The hallmark clinical feature of cri-du-chat, a high-pitched monochromatic cry, has recently been localized to 5p15.3, separate from the remaining clinical features of the syndrome, which have been localized to 5p15.2. Five chromosome 5-specific probes from the latter region, designated the cri-du-chat critical region (CDCCR), were used to isolate 30 cosmids from the LANL chromosome 5 specific cosmid library. The 30 framework cosmids were used in a direct selection with three cDNA sources to isolate an initial set of expressed sequences. Nine unique cDNAs were found that hybridized to four discrete sets of cosmids in the CDCCR. The nine cDNAs are novel by sequence database comparisons, and conservatively represent four transcription units. More recently, we have also constructed a YAC contig of the CDCCR which spans approximately 2 Mb. As expected, ESTs derived from the nine novel cDNAs map back to the contig. Limited expression profiles of these cDNAs have been obtained. Two cDNAs that map to one discrete set of cosmids have different expression patterns, suggesting that they represent two different genes and increasing the number of putative genes to five. Further characterization of these genes and the estimated 100 additional genes deleted in cri-du-chat should lead to better diagnostic markers and an understanding of the molecular mechanisms of the disease.

Indoor respirable particulate matter concentrations from an open fire, improved cookstove, and LPG/open fire combination in a rural Guatemalan community.

Improved biomass cookstoves have the potential to reduce pollutant emissions and thereby reduce pollution exposure among populations in developing countries who cook daily with biomass fuels. However, evaluation of such interventions has been very limited. This article presents results from a study carried out in 30 households in rural Guatemala. Twenty-four hour PM3.5 concentrations were compared over 8 months for three fuel/cookstove conditions (n = 10 households for each condition): a traditional open fire cookstove, an improved cookstove called the plancha mejorada, and a liquefied petroleum gas (LPG) stove/open fire combination. Twenty-four hour geometric mean PM3.5 concentrations were 1560 micrograms/m3 (n = 58; 95% C.I. 1310, 1850), 280 micrograms/m3 (n = 59; 95% C.I. 240-320), and 850 micrograms/m3 (n = 60; 95% C.I. 680-1050) for the open fire, plancha, and LPG/open fire combination, respectively. A generalized estimating equation model showed a 45% reduction in PM3.5 concentrations for the LPG/open fire combination as compared to the open fire alone. The difference approached significance (p < 0.0737). The plancha showed an 85% reduction in PM3.5 concentrations as compared to the open fire (p < 0.0001). An analysis of the interaction of time with stove type showed that the temporal trend in pollution did not significantly differ among the three stove types. The reduced PM3.5 concentrations were maintained over time. Season did not affect pollutant concentrations. Of the two interventions, the plancha appears to offer the best prospects for achieving substantial reductions in indoor air pollution levels, although issues of cost and stove maintenance remain to be addressed.

10-Oximeguanacone, the first nitrogenated acetogenin derivative found to be a potent inhibitor of mitochondrial complex I.

A new 10-keto bis-tetrahydrofuran acetogenin, guanacone (1), has been isolated from a cytotoxic extract of Annona aff. spraguei seeds. The 10-oximeguanacone derivative 1f is the first bioactive nitrogenated acetogenin found to be a very potent inhibitor of complex I. In addition, a SAR study of guanacone analogues is reported based on the titration of the NADH oxidase and NADH:ubiquinone oxidoreductase activities.

Direct selection of expressed sequences within a 1-Mb region flanking BRCA1 on human chromosome 17q21.

Direct selection of genes within the interval of chromosome 17q21 containing BRCA1 was performed. YAC and cosmid contigs spanning the BRCA1 region were used to select cDNA clones from pools of cDNAs derived from human placenta, HeLa cells, activated T cells, and fetal head. A minimum set of 48 fragments of nonoverlapping cDNAs that unequivocally mapped within a 1-Mb region was identified, although it is not yet known how many of these are derived from the same transcript. DNA sequence analyses revealed that 4 of these cDNAs were derived from known genes (EDH17B2, glucose-6-phosphatase, IAI.3B, and E1AF), 1 is a member of a previously described gene family (HMG-17), and 7 share substantial identity with previously described genes from human or other species. The remainder showed no significant homology to known genes. Limited PCR-based expression profiles of a set of 13 of the genes were performed, and all gave positive results with at least some cDNA sources supporting the contention that they truly represent transcribed sequences. A comparison between genes obtained from this region by direct selection with those obtained by direct screening or exon trapping (see accompanying papers, this issue) revealed that over 90% of the genes identified by exon trapping were represented in the selected material and that at least two additional genes that appear to represent low abundance transcripts with restricted expression profiles were identified by selection but not by other means.

Cosmid contig and transcriptional map of three regions of human chromosome 21q22: identification of 37 novel transcripts by direct selection.

Human chromosome 21 is associated with many disorders, including Down syndrome (DS). In an effort to identify genes involved in brain development or function and therefore implicated in the mental retardation associated with DS, we chose YACs from three regions of chromosome 21: a region within the so-called "Down syndrome critical region," a region proximal to it, and one distal to it. We made cosmid libraries from these YACs and generated high-resolution physical maps by constructing cosmid contigs. These are the first cosmid contigs on chromosome 21 outside the critical region. The cosmids were used for direct selection of cDNAs to isolate chromosome 21 expressed sequences. We have isolated 45 nonredundant partial cDNAs and mapped these back to the cosmid contigs. We isolated 3 nonoverlapping portions of DSCR1 and a part of GIRK2 and identified 3 nonoverlapping partial cDNAs with similarity to the rat Dyrk gene, which turned out to be the human homologue (MNB) of the Drosophila minibrain gene. Twelve sequences had matches with either STS or EST entries in the databases, including a chromosome 21 EST, a chromosome 21 STS, and 6 unmapped expressed sequence entries. Only 1 sequence resulted in a match with a protein entry. The remaining 25 sequences revealed no similarity to any database entry. All of these partial cDNAs are expressed as determined by Northern blotting or by RT-PCR.

Gamma-lactone-Functionalized antitumoral acetogenins are the most potent inhibitors of mitochondrial complex I.

To study the relevance of the terminal alpha,beta-unsaturated gamma-methyl-gamma-lactone moiety of the antitumoral acetogenins of Annonaceae for potent mitochondrial complex I inhibition, we have prepared a series of semisynthetic acetogenins with modifications only in this part of the molecule, from the natural rolliniastatin-1 (1) and cherimolin-1 (2). Some of the hydroxylated derivatives (1b, 1d and 1e) in addition to two infrequent natural beta-hydroxy gamma-methyl gamma-lactone acetogenins, laherradurin (3) and itrabin (4), are more potent complex I inhibitors than any other known compounds.

Human chromosome-specific cDNA libraries: new tools for gene identification and genome annotation.

To date, only a small percentage of human genes have been cloned and mapped. To facilitate more rapid gene mapping and disease gene isolation, chromosome 5-specific cDNA libraries have been constructed from five sources. DNA sequencing and regional mapping of 205 unique cDNAs indicates that 25 are from known chromosome 5 genes and 138 are from new chromosome 5 genes (a frequency of 79.5%). Sequence complexity estimates indicate that each library contains -20% of the approximately 5000 genes that are believed to reside on chromosome 5. This study more than doubles the number of genes mapped to chromosome 5 and describes an important new tool for disease gene isolation.