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Biomedical subjects

E Shapira

Publications and source records attributed to E Shapira.

At least 19 recordsLinked to original sources
PubMed2003

Effective expression of the green fluorescent fusion proteins in cultured Aplysia neurons.

The green fluorescent fusion protein and its isoforms are extensively used to monitor gene expression, protein localisation and their dynamics in relations to fundamental cellular processes. However, it has not yet been effectively applied to Aplysia neurons that serve as a powerful model to study the mechanisms underlying neuroplasticity. We report here the development of a procedure combining in vitro transcription of mRNA encoding fluorescent-tagged proteins and its subsequent injection into the cytoplasm to image, in real-time, protein dynamics in cultured Aplysia neurones. To illustrate the efficiency of the procedure we report here the visualisation of actin, microtubules and vesicle trafficking. The results presented here introduce a reliable and effective method to express green fluorescent protein (GFP) fusion proteins in cultured Aplysia neurons.

Animals↗
PubMed2000

The Xvex-1 antimorph reveals the temporal competence for organizer formation and an early role for ventral homeobox genes.

The organizer in vertebrate embryos has been shown to play a central role in their development by antagonizing ventralizing signals and promoting dorsal development. The ventral homeobox gene, Xvex-1, is capable of fulfilling some of the functions of BMP-4. By fusion to activation and repression domains, Xvex-1 was shown to function as a repressor of transcription. The activator version of Xvex-1, the antimorph, was made inducible by fusion to the ligand binding domain of the glucocorticoid receptor. The organizer genes, gsc and Otx-2, were identified as direct targets of Xvex-1. The XVEX-1 antimorph can induce the formation of secondary axes. Temporal analysis of secondary axis induction revealed that the competence to induce a secondary organizer ends with the onset of gastrulation. The same temporal competence window was exhibited by an inducible gsc construct. Partial loss of Xvex-1 activity was able to improve the efficiency of secondary axis induction by the dominant negative BMP receptor or Smad6. These observations together with the early widespread expression of Xvex-1 throughout the embryo prior to gastrulation encoding a homeodomain repressor protein, suggest that elements of the ventral signaling pathway play an important role during late blastula in restricting the formation of Spemann's organizer.

Animals↗
PubMed1999

Metachromatic leukodystrophy: subtype genotype/phenotype correlations and identification of novel missense mutations (P148L and P191T) causing the juvenile-onset disease.

Metachromatic leukodystrophy (MLD) is a lysosomal storage disease resulting from the deficient activity of arylsulfatase A (ASA) and the accumulation of sulfatides. The disease is characterized by several subtypes, designated by age at onset: the late-infantile-, juvenile-, and adult-onset variants. Mutation analysis of genomic DNA from a proband with each variant was performed to identify and characterize their causative ASA mutations. Two sisters with the infantile-onset disease were homoallelic for the missense mutation D335V, a juvenile-onset proband was heteroallelic for two novel missense mutations, P148L and P191T, and an adult-onset patient was heteroallelic for the H397Y and P426L mutations. The novel mutations were not identified in 108 normal alleles indicating that these base substitutions were not common polymorphisms. To further characterize the mutant gene products, the mutant enzymes were partially purified from cultured fibroblasts and their molecular weights and charges were compared by immunoblotting following SDS-PAGE or isoelectric focusing (IEF). Normal fibroblast ASA had a single, broad band at 54 kDa. The enzyme from the late-infantile-onset patient had distinct bands of 36 and 78 kDa, but lacked the normal 54-kDa species. The juvenile- and adult-onset patients each had a faint band of 54 kDa and several other bands ranging from 29 to 64 kDa. IEF revealed several bands for the partially purified normal enzyme with a relatively narrow pH range around 4.0, whereas numerous bands with a wider range of isoelectric points were observed with the enzymes from the juvenile- and adult-onset fibroblasts. In contrast, the enzyme from the late-infantile-onset proband had four bands with more acidic isoelectric points, none corresponding to those of the normal enzyme. These results document changes in both size and charge of the mutant enzymes from patients with different mutations and MLD subtypes.

Adolescent↗
PubMed1999

A role for the homeobox gene Xvex-1 as part of the BMP-4 ventral signaling pathway.

BMP-4 is believed to play a central role in the patterning of the mesoderm by providing a strong ventral signal. As part of this ventral patterning signal, BMP-4 has to activate a number of transcription factors to fulfill this role. Among the transcription factors regulated by BMP-4 are the Xvent and the GATA genes. A novel homeobox gene has been isolated termed Xvex-1 which represents a new class of homeobox genes. Transcription of Xvex-1 initiates soon after the midblastula transition. Xvex-1 transcripts undergo spatial restriction from the onset of gastrulation to the ventral marginal zone, and the transcripts will remain in this localization including at the tailbud stage in the proctodeum. Expression of Xvex-1 during gastrula stages requires normal BMP-4 activity as evidenced from the injection of BMP-4, Smad1, Smad5 and Smad6 mRNA and antisense BMP-4 RNA. Xvex-1 overexpression ventralizes the Xenopus embryo in a dose dependent manner. Partial loss of Xvex-1 activity induced by antisense RNA injection results in the dorsalization of embryos and the induction of secondary axis formation. Xvex-1 can rescue the effects of overexpressing the dominant negative BMP receptor. These results place Xvex-1 downstream of BMP-4 during gastrulation and suggest that it represents a novel homeobox family in Xenopus which is part of the ventral signaling pathway.

Amino Acid Sequence↗
PubMed1998

Biochemical and clinical response to hydroxocobalamin versus cyanocobalamin treatment in patients with methylmalonic acidemia and homocystinuria (cblC).

OBJECTIVE: To compare the therapeutic effectiveness of hydroxocobalamin and cyanocobalamin in patients with combined methylmalonic acidemia and homocystinuria. STUDY DESIGN: Analysis of urine methylmalonic acid, plasma homocystine, and growth of two unrelated patients with cobalamin C disease who were initially receiving cyanocobalamin and were subsequently switched to hydroxocobalamin. RESULTS: Each patient had a significant decrease in urine methylmalonic acid excretion while receiving cyanocobalamin, but levels remained at least 10 times normal. Cyanocobalamin treatment resulted in a decrease of plasma homocystine to near normal in one patient but had no effect on plasma homocystine in the second patient. Each patient was switched to hydroxocobalamin and urine methylmalonic acid levels decreased to the limit of detection. Plasma homocystine values while taking hydroxocobalamin remained < 5 nmol/ml in both patients. In patient 1, who continued to receive cyanocobalamin therapy for more than 1 year, growth rates (height, weight, and head circumference) were very poor. After initiation of hydroxocobalamin, growth parameters normalized with growth rates above normal. CONCLUSION: Intramuscular cyanocobalamin treatment is inadequate in the treatment of patients with cobalamin C disease. Appropriate management of cobalamin C disease should include only the hydroxocobalamin form of cobalamin.

Child↗
PubMed1997

The chicken caudal genes establish an anterior-posterior gradient by partially overlapping temporal and spatial patterns of expression.

The caudal genes in vertebrates as in invertebrates assume a posterior position along the anterior-posterior axis and they appear to regulate the expression of the Hox genes. The third chicken caudal gene, Cdx-C, was cloned. Extensive comparisons of the sequence of this protein to the other known members of this homeobox family has lead to the suggestion that vertebrate genomes contain three members of the caudal homeobox family. A comparative study of the chicken Cdx-A and Cdx-C genes during gastrulation and neurulation revealed the differences between the genes. The caudal genes exhibit sequential activation in the newly formed neural plate and sequential extinction in axial midline structures during the primitive streak regression along the anterior-posterior axis. This pattern of expression suggests that the number and identity of caudal genes expressed along the anterior-posterior axis changes dynamically.

Amino Acid Sequence↗
PubMed1996

Guidelines for the retention, storage, and use of residual dried blood spot samples after newborn screening analysis: statement of the Council of Regional Networks for Genetic Services.

These guidelines provide scientific information for policy development by state health departments considering appropriate use of newborn screening specimens after screening tests are finished. Information was collected, debated, and formulated into a policy statement by the Newborn Screening Committee of the Council of Regional Networks for Genetic Services (CORN), a federally funded national consortium of representatives from 10 regional genetics networks. Newborn screening programs vary widely in approaches and policies concerning residual dried blood spot samples (DBS) collected for newborn screening. Recognition of the epidemiological utility of DBS samples for HIV seroprevalence surveys and a growing interest in DBSs for DNA analysis has intensified consideration of issues regarding retention, storage, and use of residual DBS samples. Potentially these samples provide a genetic material "bank" for all newborns nationwide. Their values as a resource for other uses has already been recognized by scientists, administrators, and judicial officials. Programs should promulgate rules for retention and use of residual newborn screening DBS samples based on scientifically valid information. Banking of newborn samples as sources of genetic material should be considered in light of potential benefit or harm to society.

Blood Specimen Collection↗
PubMed1995

Goldenhar sequence and mosaic trisomy 22.

We describe a term infant with facioauriculo-vertebral "dysplasia" (Goldenhar sequence), hypertelorism, and mosaic trisomy 22: peripheral blood, 46, XY/47, XY, + 22 (72%/28%); skin fibroblasts, 47, XY, + 22 (100%). This is the second report of Goldenhar anomaly with epibulbar dermoids in a liveborn infant with aneuploidy. Hypertelorism is rare in Goldenhar sequence, but typical of trisomy 22. We recommend chromosome analysis in all patients with Goldenhar sequence. Those with hypertelorism may be more likely to have aneuploidy as well.

Chromosomes, Human, Pair 22↗
PubMed1995

The awareness of chromosomal disarrangements in the evaluation of child psychiatry patients.

This case report reviews genetics consultations on psychiatrically hospitalized school-age children. Six of 100 patients were noted to have positive genetic findings: four with congenital anomalies and/or biochemical/metabolic abnormalities and two with chromosomal abnormalities. Three of these patients are used to illustrate the occurrence of genetic abnormalities in psychiatrically hospitalized children. The indications for chromosomal analysis are discussed.

Child↗
PubMed1993

Isolation and characterization of target sequences of the chicken CdxA homeobox gene.

The DNA binding specificity of the chicken homeodomain protein CDXA was studied. Using a CDXA-glutathione-S-transferase fusion protein, DNA fragments containing the binding site for this protein were isolated. The sources of DNA were oligonucleotides with random sequence and chicken genomic DNA. The DNA fragments isolated were sequenced and tested in DNA binding assays. Sequencing revealed that most DNA fragments are AT rich which is a common feature of homeodomain binding sites. By electrophoretic mobility shift assays it was shown that the different target sequences isolated bind to the CDXA protein with different affinities. The specific sequences bound by the CDXA protein in the genomic fragments isolated, were determined by DNase I footprinting. From the footprinted sequences, the CDXA consensus binding site was determined. The CDXA protein binds the consensus sequence A, A/T, T, A/T, A, T, A/G. The CAUDAL binding site in the ftz promoter is also included in this consensus sequence. When tested, some of the genomic target sequences were capable of enhancing the transcriptional activity of reporter plasmids when introduced into CDXA expressing cells. This study determined the DNA sequence specificity of the CDXA protein and it also shows that this protein can further activate transcription in cells in culture.

Amino Acid Sequence↗
PubMed1993

The prevalence and nosocomial acquisition of Clostridium difficile in elderly hospitalized patients.

Rectal swabs obtained from 10 of 49 chronic-care geriatric patients were positive for Clostridium difficile, for a prevalence rate of 20.4%. Simultaneous sampling of 29 patients in an acute geriatric ward revealed four colonized patients, for a prevalence rate of 13.7%. A prospective study of C. difficile colonization in 100 consecutive patients admitted to an acute geriatric ward was carried out. All patients were sampled upon admission and biweekly during hospitalization. Two patients (2%) were positive on admission, and 12 of the 98 initially negative patients became colonized, representing a nosocomial acquisition rate of 12.2%. A major determinant for C. difficile colonization in asymptomatic patients appears to be length of hospitalization. Previous antibiotic administration was not found to be a significant factor.

Aged↗
PubMed1993

The chicken CdxA homeobox gene and axial positioning during gastrulation.

The chicken homebox containing gene, CdxA (formerly CHox-cad), was previously shown to be expressed during gastrulation. Localization of CdxA transcripts by in situ hybridization to tissue sections revealed that, during gastrulation, expression of this gene exhibits a posterior localization along the primitive streak. The transcripts are localized to epiblast cells in the vicinity of the primitive streak, to cells of the primitive streak itself and in the definitive endoderm as it replaces the hypoblast. In order to study in greater detail the pattern of expression of the CdxA gene during gastrulation, we expressed the full-length CdxA protein as a fusion protein in E. coli and generated monoclonal antibodies against it. Chicken embryos at different stages of gastrulation were processed for whole-mount immunohistochemical localization of the protein using anti-CdxA antibodies. Once the pattern of expression in the whole embryo was determined, the same embryos were sectioned to determine the identity of the cells expressing the CdxA protein. Detailed analysis of the CdxA protein in embryos, from the onset of primitive streak formation to the beginning of the tail bud stage (stages 2 to 10), has shown different patterns of expression during primitive streak elongation and regression. The CdxA protein is initially detected at the posterior marginal zone and the expression moves rostrally into the primitive streak during mid-streak stages. As the primitive streak elongates, the CdxA stripe of expression moves anteriorly. By definitive streak stages, the CdxA stripe of expression delineates a position along the anterior-posterior axis in the primitive streak. CdxA, like its Drosophila homologue cad, is expressed during gastrulation in a stripe localized to the posterior region of the embryo. These observations suggest that CdxA as a homebox gene may be part of a regulatory network coupled to axial determination during gastrulation in the early chick embryo.

Animals↗
PubMed1992

Deletion of the proximal short arm of chromosome 8.

We report on a 5-month-old boy with a de novo interstitial deletion of the proximal short arm of chromosome 8 (p21p11.2). He manifested bilateral cleft lip and palate, and apparent hypogonadism. Four previous case reports with similar deletions (p11.1p21) were associated with hypogonadotropic hypogonadism [Beighle et al., Hum Genet 38:113-121, 1977] and hereditary spherocytosis (HS) [Chilcote et al., Blood 6:156-159, 1987; Kitatani et al., Hum Genet 78:94-95, 1988; Lux et al., Nature 345:736-739, 1990]. Our patient has no demonstrable red blood cell abnormality, suggesting that the gene for HS is located in the region 8p11.1 to 8p11.2.

Chromosome Deletion↗