The molecular biology of lung cancer.
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Genes, Tumor Suppressor↗
Biomedical subjects
M D Holmes
Publications and source records attributed to M D Holmes.
Vaginal douching. Who and why?
Despite the long history of vaginal douching and its estimated practice by more than 67 million U.S. women, little is known about who those women are or why they douche. We addressed these questions by studying 618 women from urban gynecology practices. Fifty-nine percent had douched at some time, with the most frequent reason given as hygiene. The solutions used were evenly divided between commercial and home preparations. Three characteristics stood out among the women who douched: lower socioeconomic status, greater risk of sexually transmitted diseases and symptoms suggestive of vaginal infection. These characteristics were most common among women who douched frequently. The results suggest that douching has a strong cultural component but also raise the possibility that douching may be done in response to a perceived infection, possibly one transmitted through sexual contact.
Adolescent↗
Alpha 1-antitrypsin Wbethesda: molecular basis of an unusual alpha 1-antitrypsin deficiency variant.
Molecular analysis of alpha 1-antitrypsin (alpha 1AT) Wbethesda revealed that it differs from the normal M1 (Ala213) allele by a single base mutation causing an amino acid substitution Ala336 GCT----Thr ACT. Evaluation of alpha 1AT biosynthesis directed by the Wbethesda allele showed that although Wbethesda alpha 1AT mRNA was translated normally in vitro, transfection of the Wbethesda cDNA into COS-I cells was associated with human alpha 1AT secretion of 50% that of cells transfected with a normal alpha 1AT cDNA. The pattern of alpha 1AT biosynthesis was not intracellular accumulation as observed with the common Z alpha 1AT deficiency allele, but reduced intracellular alpha 1AT, suggesting intracellular degradation of the newly synthesized Wbethesda molecule. Together these observations suggest that in heterozygous combination with a Z or Null alpha 1AT allele, the Wbethesda variant causes "alpha 1AT deficiency", thus classifying it as an alpha 1AT "at risk" allele for emphysema.
Alleles↗
Molecular analysis of the heterogeneity among the P-family of alpha-1-antitrypsin alleles.
The rare P-family of alpha 1-antitrypsin (alpha 1AT) variants is defined by the position of migration of the alpha 1AT protein on isoelectric focusing of serum (IEF) between the common M and S variants. To begin to examine the molecular heterogeneity among the P-type alleles, two unrelated subjects and their families identified by IEF to be carrying a P allele were analyzed. The first, Plowell, is a deficiency allele associated with reduced serum alpha 1AT levels, and the second, Psaint albans, is associated with normal serum levels. DNA sequence analysis of Plowell, the more anodal of the two variants on IEF analysis, showed that if differed from the normal M1(Val213) allele by a single base and amino acid substitution Asp256 GAT----Val GTT. In contrast, Psaint albans, a slightly more cathodally positioned variant on IEF analysis, differed from the coding exons of the normal M1(Val213) allele by two mutations, Asp341 GAC----Asn AAC, and a silent substitution in the same codon as the Plowell variant, Asp256 GAT----Asp GAC. Evaluation of Plowell mRNA transcripts by Northern and cytoblot analyses demonstrated they were of normal size and amount, and Plowell mRNA transcripts could be translated normally in vitro. Retroviral insertion of the Plowell cDNA into the genome of 3T3 fibroblasts demonstrated that it directed the synthesis of alpha 1AT, but at levels 24% that of the Psaint albans cDNA or the normal M1 (Val213) cDNA, with a pattern of biosynthesis consistent with the concept that the Plowell alpha 1AT deficiency state results from intracellular degradation of the newly synthesized Plowell protein.(ABSTRACT TRUNCATED AT 250 WORDS)
Aged↗
Characterization of the normal alpha 1-antitrypsin allele Vmunich: a variant associated with a unique protein isoelectric focusing pattern.
alpha 1-Antitrypsin (alpha 1AT), the major serum inhibitor of neutrophil elastase, is a highly polymorphic protein associated with isoelectric focusing (IEF) patterns typical for each variant. alpha 1AT Vmunich, a previously unreported normal alpha 1AT variant, has a unique IEF banding pattern in which the 7 and 8 alpha 1AT protein bands focus with the normal M-type 7 and 8 bands, despite the fact that the major fraction of the Vmunich protein focuses in the "V" region of the IEF gel. To characterize the molecular basis of this variant and its unique IEF pattern, DNA sequence analysis of the coding exons of the Vmunich alpha 1AT gene was carried out using the polymerase chain reaction. The Vmunich allele differed from the common normal M1(Val213) alpha 1AT allele by a single nucleotide substitution of cytosine for adenosine, with the resultant amino acid change Asp2 GAT----Ala GCT. Inheritance of the allele was confirmed by family analysis using allele-specific amplification with the polymerase chain reaction. The Asp2----Ala mutation explains the cathodal position of the Vmunich protein on IEF, as there is a substitution of a negatively charged amino acid by a neutral one.(ABSTRACT TRUNCATED AT 250 WORDS)
Alleles↗
Characterization of the coding sequence of the normal M4 alpha 1-antitrypsin gene.
The nucleotide sequences of the common normal "M" family of alpha 1-antitrypsin (alpha 1AT) variants are known, including M1(Val213), M1(Ala213), M2 and M3. Less common, but also migrating with the "M" family on isoelectric focusing gels, is the normal M4 allele. Being relatively rare, the M4 allele is usually found in heterozygous combination with another alpha 1AT allele making sequence characterization more difficult. To facilitate analysis of the coding exons of the alpha 1AT M4 allele, a method was developed to combine blood monocyte RNA extraction, reverse transcription of the alpha 1AT mRNA, amplification with the polymerase chain reaction and direct sequencing. This analysis demonstrated that the M4 allele differs from the M1(Val213) allele by a single nucleotide substitution G--greater than A, causing the amino acid substitution Arg101 CGT--greater than His101 CAT. This same mutation is also a part of the M2 gene suggesting that this region of the alpha 1AT gene may be one of increased mutational activity.
Alleles↗
Molecular basis of the liver and lung disease associated with the alpha 1-antitrypsin deficiency allele Mmalton.
Alpha 1-Antitrypsin (alpha 1AT) deficiency is characterized by reduced serum levels of alpha 1AT and a risk for the development of emphysema and liver disease. However, whereas there is an increased risk for emphysema associated with at least 10 alpha 1AT deficiency and null alleles, the hepatic disease is observed only in a subset of these alleles, suggesting that it is not the reduced serum levels of alpha 1AT per se which cause the liver disease. The present study characterizes the alpha 1AT deficiency allele Mmalton, an allele that like the common Z deficiency mutation (Glu342----Lys) is associated with both alpha 1AT deficiency and hepatic disease. Capitalizing on the identification of the homozygous inheritance of the rare Mmalton alpha 1AT deficiency allele, it was demonstrated that although caused by a very different mutation, the Mmalton allele shares with the Z allele the association of liver disease with the same type of abnormalities of alpha 1AT biosynthesis. Cloning of the Mmalton gene and sequence analysis demonstrated that it differs from the normal alpha 1AT M2 allele by deletion of the entire codon (TTC) for residue Phe52. Liver biopsy of the Mmalton homozygote revealed inflammation, mild fibrosis, and intrahepatocyte accumulation of alpha 1AT. Evaluation of de novo alpha 1AT biosynthesis in alpha 1AT-synthesizing cells of this individual demonstrated normal levels of alpha 1AT mRNA transcripts but abnormal intracellular accumulation of newly synthesized alpha 1AT at the level of the rough endoplasmic reticulum with consequent reduced alpha 1AT secretion. Finally, retroviral gene transfer of a normal alpha 1AT cDNA and an alpha 1AT cDNA with the Mmalton Phe52 deletion into murine cells demonstrated that the Mmalton cells reproduced the abnormal accumulation of newly synthesized alpha 1AT, thus directly demonstrating that the deletion mutation is responsible for the intracellular accumulation of the newly synthesized alpha 1AT. Thus, not only is the liver disease associated with alpha 1AT deficiency restricted to a subset of alpha 1AT deficiency alleles, it appears to be restricted to those alleles associated with intracellular accumulation of newly synthesized alpha 1AT, suggesting that it is the abnormal intrahepatocyte alpha 1AT accumulation which incites the liver injury.
Alleles↗
Racial inequalities in the use of procedures for ischemic heart disease.
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Coronary Disease↗
Carbamazepine and its epoxide: relation of plasma levels to toxicity and seizure control.
We studied the relation of plasma levels of carbamazepine (CBZ) and carbamazepine 10,11 epoxide (CBZ-E), and their ratio to drug toxicity and seizure control in 7 patients with complex partial seizures. CBZ-E/CBZ increased with increasing CBZ levels and was higher when patients were taking phenytoin or valproic acid. There were weak correlations between CBZ, CBZ-E levels, toxicity scores, and seizure control when patients were taking CBZ alone, but not when other drugs were given as well. There were no significant differences in the correlation with toxicity score or seizure frequency between CBZ, CBZ-E, or their sum. Measurement of CBZ-E levels did not provide additional information useful for monitoring clinical response to CBZ therapy.
Carbamazepine↗
Chlamydia trachomatis cervical infection in women seeking routine gynecologic care: criteria for selective testing.
PURPOSE: Chlamydia trachomatis infection is one of the most common sexually transmitted diseases. Developing methods to identify patients who are at higher risk would enable clinicians to perform selective testing for C. trachomatis infection, which may reduce morbidity, sequelae, and overall costs. Risk factors for C. trachomatis infection of the cervix in women seen for routine gynecologic care were therefore analyzed. PATIENTS AND METHODS: Extensive clinical data and cervical cultures were obtained from 1,141 women between the ages of 18 and 50. Multivariate techniques were used to develop and prospectively test a model to predict infection. RESULTS: In the model development series of 530 patients seen in a hospital-based practice, 24 (4.5%) had a positive culture result. In the validation series of 611 patients seen in private practice, 17 (2.8%) had positive culture results. The majority of infected patients (61%) were asymptomatic. For sexually active women 18 to 35 years of age, the optimal predictive model was based on three factors: level of education (high school or less), a sex partner with other partners during the prior three months, and endocervical bleeding induced by the initial swab. In the validation series, the prevalence of infection for women with one or more of these factors was 5.8% (95% confidence interval, 4.0 to 8.6) compared with 0.9% in patients without these factors (p = 0.005). CONCLUSION: Clinical information can be used in a low-prevalence population to predict a patient's risk for C. trachomatis infection and to select patients for testing.
Adolescent↗
Ribonuclease A cleavage combined with the polymerase chain reaction for detection of the Z mutation of the alpha-1-antitrypsin gene.
Homozygous inheritance of the Z mutation (exon V, Glu342GAG----Lys342AAG), the most common cause of alpha-1-antitrypsin (alpha 1AT) deficiency, is associated with a high risk for emphysema and liver disease. This study presents a rapid and accurate approach to definitive genotypic diagnosis of the Z homozygous state using a combination of polymerase chain reaction amplification of exon V of the alpha 1AT gene and ribonuclease cleavage of an exon V-specific antisense RNA probe. Taking advantage of the concept that ribonuclease A will cleave at points of mismatch of RNA-DNA hybrids, a 0.79 kb antisense RNA probe was designed with complementarity to the sense strand of exon V of the alpha 1AT gene (the site of the Z mutation) along with small regions of the 5' and 3' flanking sequences. After amplification of exon V of the alpha 1AT gene from genomic DNA by the polymerase chain reaction, the amplified DNA was analyzed by hybridization to a 32P-labeled exon V antisense RNA probe followed by digestion with RNase A. Any substitution mutations resulting in DNA-RNA mismatch were detected by evaluation with polyacrylamide gel electrophoresis under denaturing conditions followed by autoradiography (expected fragment lengths: 0.33 kb when the exon V probe hybridized to the normal amplified genomic DNA, 0.25 and 0.08 kb fragments when the exon V probe hybridized to the amplified genomic DNA with the Z mutation). Double-blinded evaluation of genomic DNA of 36 individuals (phenotypes MM n = 14, MZ n = 5, ZZ n = 16, ZNull n = 1; included among the "M" alleles were representatives of all the major normal M alleles) demonstrated definitive diagnosis of the Z mutation with absolute specificity for all 36 specimens, i.e., ZZ homozygotes, MZ heterozygotes, and normals were all detected accurately. This approach should be useful not only for screening for the Z mutation of the alpha 1AT gene, but by this type of analysis, mutational alterations of the alpha 1AT gene can be screened for without prior knowledge of the sequence changes and without complex cloning and sequencing methods.
Alleles↗
The alpha 1-antitrypsin gene and its mutations. Clinical consequences and strategies for therapy.
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Alleles↗
An analytic review of current therapies for obesity.
Weight control is a cornerstone objective for many diseases common in medicine. Substantial, prolonged weight loss is difficult to achieve. Nutrition counseling, very low calorie diets, behavior modification, exercise, intragastric balloon, and gastric restriction surgery are interventions that physicians may recommend for obese patients. This paper analyzes the efficacy of these methods with attention to attrition rates, maximum weight loss, long-term maintenance of weight loss, and morbidity. Strategies for intervention for various classes of obese patients are recommended.
Humans↗
Rapid, nonradioactive detection of mutations in the human genome by allele-specific amplification.
A simple, rapid, nonradioactive method has been developed to facilitate the direct detection of point mutations that cause genetic disease. The method operates on the basis of the specific amplification of a target allele by the polymerase chain reaction with extension primers designed such that their 3' end is placed at the mutation site. When this base is complementary to that of the specific allele, the DNA segment is amplified; when it is not complementary, the polymerase chain reaction cannot proceed. When alpha 1-antitrypsin (alpha 1AT) deficiency was used as a model, the technique of allele-specific amplification was capable of selective detection of five different mutations that cause the alpha 1AT deficiency state, including three different naturally occurring single-base substitution mutations (alleles Z, S, and Nullbellingham), an insertion mutation (Nullmattawa), and a deletion mutation (Nullgranite falls). Double-blind evaluation of 47 samples of genomic DNA demonstrated 100% accuracy of the method. The technique of allele-specific amplification is rapid, simple, and does not require the existence of a convenient restriction endonuclease site or the use of radioactive materials, and thus should have broad applicability for the detection of known genetic diseases in a highly sensitive and specific fashion.
Alleles↗
Structure of the human neutrophil elastase gene.
The gene for human neutrophil elastase (NE), a powerful serine protease carried by blood neutrophils and capable of destroying most connective tissue proteins, was cloned from a genomic DNA library of a normal individual. The NE gene consists of 5 exons and 4 introns included in a single copy 4-kilobase segment of chromosome 11 at q14. The coding exons of the NE gene predict a primary translation product of 267 residues including a 29-residue N-terminal precursor peptide and a 20-residue C-terminal precursor peptide. Analysis of the N-terminal peptide sequence suggests it contains a 27-residue "pre" signal peptide followed by a "proN" dipeptide, similar to that of other blood cell lysosomal proteases. The sequences for the mature 218-residue NE protein are included in exons II-V. The 5'-flanking region of the gene includes typical TATA, CAAT, and GC sequences within 61 base pairs (bp) of the cap site. The sequence 1.5 kilobases 5' to exon I contains several interesting repetitive sequences including six tandem repeats of unique 52- or 53-bp sequences. The 5'-flanking region also contains a 19-bp segment with 90% homology to a segment of the 5'-flanking region of the human myeloperoxidase (MPO) gene, a gene also expressed in bone marrow precursor cells and a protein stored in the same neutrophil granules as NE. In addition, like the MPO gene, the NE 5'-flanking region has several regions with greater than or equal to 75% homology to sequences 5' to c-myc, but there is no overlap between the NE-c-myc and MPO-c-myc homologous sequences.
Amino Acid Sequence↗
Complex partial seizures. Correlation of clinical and metabolic features.
We compared metabolic patterns on 18F-2-deoxyglucose positron emission tomography (PET) with closed circuit television and simultaneous electroencephalographic ictal recordings of complex partial seizures in 48 patients. Closed circuit television and electroencephalographic data and PET scans were scored by "blinded" raters. Of the 48 patients, 26 had unilateral temporal; three, frontal; ten, ipsilateral frontotemporal; one, frontoparietal; and five, temporoparietal hypometabolism; and three had widespread hypometabolism affecting frontal, temporal, and parietal lobes. patients with frontal hypometabolism alone had shorter ictal and postictal durations, but involvement of multiple regions was associated with prolonged seizures. Auras were more likely to be present in patients with temporal hypometabolism alone, but an initial motionless stare did not distinguish this group. However, other metabolic patterns did not predict specific ictal clinical features. Vocalizations (formed or unformed) were not more closely associated with frontal involvement. When hypometabolism is multilobar, it may be difficult to use PET to distinguish between complex partial seizures of frontal and temporal origin.
Brain↗
Excessive daytime somnolence. A series of 100 patients.
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Adolescent↗
Enhancement of healing in osteochondral defects by collagen sponge implants.
Implants of porous, highly cross-linked collagen sponge (CS) were tested for their capacity to enhance the healing of osteochondral defects in rabbits. Comparison was made to the healing of similar defects with polyvinyl alcohol sponge (PVAS) implants and with no implants (CONT). Evaluation was carried out up to 44 weeks following implantation and included observation of host cellular response, biodegradability of implant, gross appearance of restored joint surface, collagenous architecture of repair tissue, and properties of the junctions of implants and host articular cartilage, subchondral bone, and medullary bone. Collagen sponge proved most effective in promoting healing of osteochondral defects with fibrous and fibrocartilaginous tissue over restored subchondral bone. Collagen sponge showed many desirable properties as a potential material for biologic resurfacing of damaged joints. These properties included porosity, biodegradability, biocompatability, ability to mechanically protect cells and matrix while directing cell ingrowth, and an available chemical technology for modifying its biomechanical and biological properties. Comparative analysis of results of healing of CS, PVAS, and CONT osteochondral defects suggest rational design criteria for implant materials to improve their effectiveness in restoration of articular surfaces.
Animals↗