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M Pritchard

Publications and source records attributed to M Pritchard.

At least 37 records · Page 2Linked to original sources
PubMed1999

Alu-splice cloning of human Intersectin (ITSN), a putative multivalent binding protein expressed in proliferating and differentiating neurons and overexpressed in Down syndrome.

By Alu-splice PCR we have trapped two exons and subsequently identified the full length cDNA of a human gene, Intersectin (ITSN), which maps to chromosome 21q22.1 between markers D21S320 and D21S325. The gene has the potential to code for at least two different protein isoforms by alternative splicing (ITSN-L and ITSN-S). Intersectin exists with a high degree of similarity in flies, frogs and mammals, suggesting a conserved role in higher eukaryotes. Analysis of the expression pattern of human and mouse Intersectin detected mRNAs in all adult and foetal tissues tested, with the longer isoform present in brain. In situ hybridisation studies in the developing mouse brain showed ITSN expression in both proliferating and differentiating neurons. The genomic structure of ITSN was determined using the chromosome 21 sequences deposited in the public databases. The protein contains several known motifs which implicate ITSN in clathrin mediated endocytosis and synaptic vesicle recycling. The expression pattern of Intersectin in mouse brain, its presumed function and its overexpression in brains from Down syndrome patients, suggest that Intersectin may contribute in a gene dosage-dependent manner to some of the abnormalities of Down syndrome.

Adaptor Proteins, Vesicular Transport↗
PubMed1999

Characterization of mild whole-body hyperthermia protocols using human breast, ovarian, and colon tumors grown in severe combined immunodeficient mice.

OBJECTIVE: We have shown that one treatment of fever-like whole body hyperthermia (WBH) on mice bearing human breast tumors results in a tumor growth delay. Our goal was to repeat this study in mice bearing human ovarian or colon tumors. We further evaluated this WBH protocol by performing multiple and interrupted WBH treatments. METHODS: Human tumors were grown in severe combined immunodeficient (SCID) mice. For WBH, core body temperatures were maintained at 39.8+/-0.2 degrees C for 6-8 hours. Multiple treatments were given 6-7 days apart. Interrupted WBH consisted of three 2-hour heatings, 15 minutes apart. Tumor growth time (TGT) was the number of days to grow 1.5 or 2 times in volume. RESULTS: For WBH-treated ovarian tumors, TGT was 12+/-1.2d, compared with 5.0+/-0.1d for untreated mice (P < 0.05). For colon tumors with one WBH treatment TGT was 4.4+/-1.1d. Two and three treatments had TGTs of 9+/-2.3d and 8+/-1.6d. For the untreated tumors, TGT was 2+/-0.7d (P < 0.01 for one, two, and three treatments). Histological examination indicated that one and two treatments were associated with cellular damage within the tumors. With a slower growing colon tumor, the TGT was 24+/-3.3d with three WBH treatments, compared with 14+/-1.8d for controls (P < 0.01). The TGT of breast tumors treated with interrupted WBH was not significantly different than the noninterrupted, with TGT of 7.3+/-0.8d and 6.2+/-1.0d, respectively. CONCLUSIONS: These data illustrate that WBH causes a tumor growth delay in mice bearing human ovarian and colon tumors. This response is enhanced with a second treatment of WBH. Interrupted and noninterrupted WBH give comparable anti-tumor results. We will continue to evaluate WBH in various animal models to optimize its potential for clinical administration and maximize the anti-tumor response.

Animals↗
PubMed1999

Treatment of mammalian cells with the endoplasmic reticulum-proliferator compactin strongly induces recombinant and endogenous xenobiotic metabolizing enzymes and 3-hydroxy-3-methylglutaryl-CoA reductase in vitro.

Some xenobiotics induce membrane-bound drug metabolizing enzymes (Xme) and a profound proliferation of the endoplasmic reticulum (ER) in vivo. However these effects are much weaker in vitro, possibly due to absence of certain transcription factors. We tested the possibility that ER proliferation can affect the level of ER-resident enzymes even in the absence of transcriptional activation. For this purpose we analysed the effects of compactin, which has been shown to induce ER proliferation in vitro, on recombinant Xme, which were expressed from a constitutive viral promoter. High levels of recombinant UDP-glucuronosyltransferase UGT1A6 were achieved by amplification of the UGT1A6 cDNA using the dihydrofolate reductase cDNA as selectable marker in DHFR- CHO cells. Treatment of the resulting cell lines with lipoprotein-deficient serum in the absence and presence of compactin for 5 days resulted in a 1.3- and 2.3-fold, respectively, increase of the UGT enzyme activity towards 4-methylumbelliferone, paralleled by an induction of immunoreactive UGT1A6 protein. Similarly, treatment with this 3-hydroxy-3-methylglutaryl-CoA reductase inhibitor increased the endogenous P450 reductase activity 2.6-fold, concomitant with an increase of immunodetectable protein. As expected compactin induced the level of 3-hydroxy-3-methylglutaryl-CoA reductase. Increased levels of this protein have been associated with a proliferation of the ER. Compactin treatment of a separate cell line that expressed recombinant human P450 reductase increased this enzyme activity fivefold. Pulse-chase experiments revealed that the induction of the recombinant Xme by compactin was most likely due to decreased protein degradation. Our results show that enzyme systems unrelated to those involved in cholesterol biosynthesis are affected by compounds known to affect membrane biogenesis. Since this effect extends to heterologously expressed enzymes, it also provides an efficient means by which to increase the levels of recombinant ER proteins.

Animals↗
PubMed1999

Cancer vaccines with emphasis on a viral oncolysate melanoma vaccine.

Biotherapy of malignant diseases has become the fourth treatment modality besides surgery, chemo- and radiotherapy. Whole cell melanoma vaccines with or without BCG and other adjuvants, purified ganglioside and shed antigens, recombinant viruses carrying tumor antigens, dendritic cells pulsed with antigenic peptides etc. are in clinical trials. Efficacious viral oncolysate vaccines induce the host to mount tumor-specific cytotoxic T-cell response and prevention of relapses is supported by clinical trials. The use of "polyvalent" whole cell vaccines vs. purified or genetically engineered single antigen vaccines is justified as i. only very few single tumor antigens are present in all tumors of a given histological type; and ii. antigen modulation occurs in tumors rendering them resistant to immune attack generated by vaccine against a single antigen. Thus polyvalent vaccines immunize against several antigens vs. against a selected antigen.

Adjuvants, Immunologic↗
PubMed1998

Cloning, chromosome mapping and expression analysis of the HIRA gene from Drosophila melanogaster.

The human HIRA gene was identified as a putative transcriptional regulator mapping within the DiGeorge syndrome critical region at 22q11. HIRA-related proteins have been described in a number of species, but functional information concerning family members is only available in Saccharomyces cerevisiae, where the Hir1p and Hir2p proteins are known to be transcriptional corepressors. In order to analyse conservation of HIRA-related genes and to provide resources for functional studies in another model organism we have isolated the HIRA gene from Drosophila melanogaster (dhira). The 3374 nucleotide cDNA encodes a protein of 1047 aa, showing 42% identity with the human protein. Alignment with the predicted HIRA proteins from human, mouse, chick and pufferfish reveals strong conservation within the N-terminal region which contains seven WD domains, with less conservation of C-terminal sequences. In situ hybridisation to salivary gland chromosomes indicates that the gene resides in region 7B2-3 of the X chromosome. Dhira is expressed through embryonic development and at lower levels during larval and pupal development. The expression of dhira is dramatically increased in early embryos and in females, suggesting that the dhira mRNA could be maternally deposited in the embryos.

Amino Acid Sequence↗
PubMed1998

Isolation and genomic characterization of the TUPLE1/HIRA gene of the pufferfish Fugu rubripes.

In an effort to obtain a small genomic construct for the generation of a HIRA transgenic mouse, we have isolated and sequenced the Fugu TUPLE1/HIRA gene. We have compared the gene organization and the proteins encoded in pufferfish and human and also searched for conserved DNA sequences that might be important in gene regulation. The pufferfish gene spans approx. 9 kb, which is approx. 11 times smaller than the human gene, owing to the reduced size of the introns. Like its human counterpart, it is organized into 25 exons. The majority of the splice sites are in identical positions to those found in the human gene, however, for three internal exons the positions of the splice sites are not directly comparable. The coding regions are almost identical in size and show a high degree of similarity, especially at the amino and carboxy termini. Comparisons of 5' and 3' sequences failed to detect similarities or sequences involved in regulation.

Amino Acid Sequence↗
PubMed1998

Cognitive-behavioral treatment of school-refusing children: a controlled evaluation.

OBJECTIVE: To evaluate the efficacy of a 4-week cognitive-behavioral treatment program for children who refuse to go to school. METHOD: Thirty-four school-refusing children (aged 5 to 15 years) were randomly assigned to a cognitive-behavioral treatment condition or a waiting-list control condition. Treatment consisted of individual child cognitive-behavioral therapy plus parent/teacher training in child behavior management skills. Measures taken before and after treatment included school attendance, child self-report of emotional distress and coping, caregiver reports on emotional and behavioral problems, and clinician ratings of global functioning. RESULTS: Relative to waiting-list controls, children who received cognitive-behavioral therapy exhibited a significant improvement in school attendance. These children also improved on self-reports of fear, anxiety, depression, and coping. Significant improvements also occurred in relation to caregiver reports and clinician ratings. Maintenance of therapeutic gains was demonstrated at a 3-month follow-up assessment. CONCLUSIONS: Cognitive-behavioral treatment of school refusal was efficacious and acceptable. The relative contributions of child therapy and parent/teacher training require further study.

Adolescent↗
PubMed1997

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.

Animals↗
PubMed1997

Alu-splice PCR: a simple method to isolate exon-containing fragments from cloned human genomic DNA.

We have developed a simple, straightforward procedure to isolate exons from cloned human genomic DNA. The method is PCR based and relies upon the conservation of splice-site sequences and the frequency of Alu repeat elements in the genome to capture coding sequences. We designed two different sets of primers: a primer from each end of the Alu element and primers with the 5' or 3' splice-site consensus sequences. Putative exons were amplified by PCR using YAC DNA as starting material. We applied Alu-splice PCR to two overlapping YACs, 72H9 and 860G11, from human chromosome 21. Sequence and northern analysis of 37 initial clones resulted in the identification of five novel exons.

Amino Acid Sequence↗
PubMed1997

Confirmation of linkage of oculopharyngeal muscular dystrophy to chromosome 14q11.2-q13 in American families suggests the existence of a second causal mutation.

Oculopharyngeal muscular dystrophy (OPMD) is a late-onset, autosomal dominant disorder characterized by progressive ptosis, dysphagia, and extremity weakness. Linkage of OPMD to 14q11.2-q13 has been reported in a series of French-Canadian families. Tightly linked markers have been defined and haplotype analysis in these data show a single segregating disease chromosome throughout the OPMD French-Canadian families. We have ascertained and sampled five multigenerational outbred American OPMD families. Four of the five families have known French-Canadian ancestry while the fifth is of English/Scottish origin. Linkage analysis was performed using standard likelihood methods. A peak multipoint lod score of 6.30 was obtained for the marker MYH7.1 in the OPMD families. The English/ Scottish family exhibited a different chromosomal haplotype for the OPMD alleles than the families of French-Canadian origin. These data suggest this family may represent a second, possibly independent mutation in this disorder. Linkage was confirmed to chromosome 14q11.2-q13 with no evidence of genetic heterogeneity.

Adult↗
PubMed1997

Down syndrome: characterisation of a case with partial trisomy of chromosome 21 owing to a paternal balanced translocation (15;21) (q26;q22.1) by FISH.

A patient with a typical Down syndrome (DS) phenotype and a normal karyotype was studied by FISH. Using painting probes, we found that the patient had partial trisomy of chromosome 21 owing to an unbalanced translocation t(15;21) (q26; q22.1) of paternal origin. To correlate genotype with phenotype as accurately as possible, we localised the breakpoint using a contig of YACs from the long arm of chromosome 21 as probes and performed FISH. We ended up with two YACs, the most telomeric giving signal on the der (15) in addition to signal on the normal chromosome 21 and the most centromeric giving signal only on both normal chromosomes 21. From these results we could conclude that the breakpoint must be located within the region encompassing YACs 280B1 and 814C1, most likely near one end of either YAC or between them, since neither YAC814C1 nor 280B1 crossed the breakpoint (most likely between marker D21S304 and marker D21S302) onband 21q22.1. The same study was performed on the chromosomes of the father and of a sister and a brother of the patient; all three carried a balanced translocation between chromosomes 15 and 21 and had a normal phenotype. We also performed a prenatal study using FISH for the sister. The fetus was also a carrier of the balanced translocation.

Adult↗
PubMed1997

Minibrain (MNBH) is a single copy gene mapping to human chromosome 21q22.2.

We have identified a human homologue of the Drosophila mnb gene (MNBH) on chromosome 21, while another study mapped an EST clone (R38268) with similarity to minibrain to human chromosome 1. This report describes the mapping of MNBH to a single locus on human chromosome 21q22.2 by FISH and Southern blotting. Comparison of the similarities between the two sequences and mnb demonstrates that MNBH on chromosome 21 is the true human homologue of Drosophila mnb.

Amino Acid Sequence↗
PubMed1997

A program to immunize hospitalized preschool-aged children: evaluation and impact.

OBJECTIVE: The Standards for Pediatric Immunization Practices suggest that hospitalization be viewed as an opportunity to vaccinate children. The purpose of the present study is 1) to determine the immunization status of an urban population of hospitalized preschool-aged children, 2) to study the impact of an immunization program designed to vaccinate hospitalized 0 to 2-year-old children who are underimmunized at admission, and 3) to make immunization a routine part of care for the hospitalized child. METHODS: Prospective evaluation of the immunization status of hospitalized 0 to 2-year-old residents of Philadelphia admitted to an urban children's hospital was performed. With verification of the child's immunization record through the primary care provider (PCP), needed immunizations were given and records were forwarded to notify the PCP. Educational information was provided to families and health care providers. MAIN OUTCOME MEASURE: The percentage of children fully immunized on admission compared with the percentage at the time of discharge. Results. Two thousand three hundred twenty-nine children from 0 to 2 years of age were hospitalized during the 22-month study period. Immunization records were verified in 86% (2006), requiring an average of 1.5 phone calls to the PCP. The mean patient age was 10 months. Average hospital length of stay was 4 days. On admission, 49% (980) of the 2006 study patients were fully immunized. The remaining 51% (1026) were eligible for vaccination. Immunizations were delayed greater than or equal to 2 months in 18% (355) of the children. Neither type of health care insurance nor site of primary care affected the immunization status of those evaluated at the time of admission. Sixty-six percent (N = 674) of eligible patients received at least one vaccination before hospital discharge. Medical contraindications accounted for only 4% of the reasons eligible patients were not immunized. Of the 2006 children evaluated, the percentage of those fully vaccinated for age increased significantly from 44% on admission to 70% on discharge. CONCLUSION: As a result of this program, there was a significant improvement in vaccination percentage at the time of hospital discharge in this group of urban preschool-aged children. The development of an immunization program to vaccinate hospitalized preschool children is an opportunity to immunize in the urban setting where there is a high prevalence of underimmunization. In addition, it provides an opening for educational programs for families, nurses, and housestaff and linkage to the community PCPs.

Child, Preschool↗
PubMed1997

Heterologous expression of human drug-metabolizing enzymes.

This article is a report on a symposium held at the March 1997 meeting of the American Society for Pharmacology and Experimental Therapeutics in San Diego. Current developments in the heterologous expression of cytochrome P450, NADPH-cytochrome P450 reductase, glutathione transferase, and UDP-glucuronosyltransferase enzymes are described. Systems include bacteria, insect cells, and transient and stable mammalian cells. Uses of the products are described for discernment of which enzymes are involved in metabolism of drugs, genotoxicity assays, mutagenesis (for structure-activity relationships), large scale production of enzyme products, antibody production, and production of proteins for biophysical studies.

Enzymes↗
PubMed1996

Coexpression of a human P450 (CYP3A4) and P450 reductase generates a highly functional monooxygenase system in Escherichia coli.

The catalytic activities of recombinant cytochrome P450s expressed in E. coli have been impeded by the absence of endogenous P450 reductase. To solve this problem, we coexpressed P450 reductase with CYP3A4. Membranes from this strain contained 215 pmol P450/mg protein and a reductase activity of 1315 nmol cytochrome c reduced/min per mg. We detected 6beta-hydroxylation of testosterone and oxidation of nifedipine in vivo with turnover numbers of 15.2 and 17.3 min(-1), respectively. These values compare favourably with those obtained using an optimally reconstituted system. Our data demonstrate that a catalytically efficient human P450 system can be generated in E. coli.

Base Sequence↗