Rapid methods for the analysis of immunoglobulin gene hypermutation: application to transgenic and gene targeted mice

Hypermutation of immunoglobulin genes is a key process in antibody diversification. Little is known about the mechanism, but the availability of rapid facile assays for monitoring immunoglobulin hypermutation would greatly aid the development of culture systems for hypermutating B cells as well as the screening for individuals deficient in the process. Here we describe two such assays. The first exploits the non-randomness of hypermutation. The existence of a mutational hotspot in the Ser31 codon of a transgenic immunoglobulin V gene allowed us to use PCR to detect transgene hypermutation and identify cell populations in which this mutation had occurred. For animals that do not carry immunoglobulin transgenes, we exploited the fact that hypermutation extends into the region flanking the 3'-side of the rearranged J segments. We show that PCR amplification of the 3'-flank of VDJH rearrangements that involve members of the abundantly-used VHJ558 family provides a large database of mutations where the germline counterpart is unequivocally known. This assay was particularly useful for analysing endogenous immunoglobulin gene hypermutation in several mouse strains. As a rapid assay for monitoring mutation in the JH flanking region, we show that one can exploit the fact that, following denaturation/renaturation, the PCR amplified JH flanking region DNA from germinal centre B cells yields mismatched heteroduplexes which can be quantified in a filter binding assay using the bacterial mismatch repair protein MutS -Wagner et al. (1995) Nucleic Acids Res. 23, 3944-3948-. Such assays enabled us, by example, to show that antibody hypermutation proceeds in the absence of the p53 tumour suppressor gene product.     

Fast and accurate numerical method for principal components analysis dealing with large image data sets

The principal components analysis has been applied to various imaging studies in nuclear medicine. This technical report describes a fast and accurate numerical method of calculating eigenvalues and eigenvectors in the principal components analysis dealing with larger image data sets. The method employs both data transformation and matrix transpose of original data sets to calculate a variance-covariance or correlation matrix. The method was tested on actual image data sets using a common workstation, confirming faster execution time and efficient accuracy in comparison to a standard method.     

Molecular biological access to the chemistry of unknown soil microbes: a new frontier for natural products

Cultured soil microorganisms have provided a rich source of natural-product chemistry. Because only a tiny fraction of soil microbes from soil are readily cultured, soil might be the greatest untapped resource for novel chemistry. The concept of cloning the metagenome to access the collective genomes and the biosynthetic machinery of soil microflora is explored here.     

Cognitive representations of bodily parts and products: implications for health behavior

This study investigated the unexplored hypothesis that people may have cognitive representations of the body's parts and products (body schema) and that these may be relevant to illness behavior. Factor and MANOVA analyses revealed that body parts are viewed as differing in the Stigma attached to them and in the extent to which they are seen as Important, Vulnerable, Sexual, and Private, with these five dimensions found to be independent of age and health history. Seeking help for dysfunction varied across body parts, with subjects reluctant to seek help for highly stigmatized and private parts but likely to seek help for parts viewed as important and vulnerable. Significant gender and ethnic differences in representations of body parts also emerged.     

Patients'' and relatives'' opinion and knowledge about electroconvulsive therapy: implications for nursing

This study examines sources of motivation to seek treatment. Participants were 105 male alcoholics and their non-alcoholic female partners who participated in a study of three different approaches to the conjoint treatment of alcoholism. Participants' sources of motivation were coded from responses to questions at the initial clinical screening interview. Sources of motivation were classified as "internal" or "external." More participants had internal sources of motivation (74%) than external sources. Participants with internal sources of motivation scored higher on the Michigan Alcoholism Screening Test than participants with external sources of motivation, but did not differ on other measures of pretreatment severity of alcohol problems. About half of the participants (53%) cited their partner as a primary source of motivation to seek treatment. Other sources of motivation cited were: increasing problems with alcohol, mental health problems, and physical health problems. There was greater variability among internal sources of motivation than among external sources of motivation. Participants' partners but not the male participants themselves, experienced an increase in marital satisfaction from pre- to within-treatment when the participant was motivated to come to treatment by his partner.     

Apoptosis by retrovirus- and adenovirus-mediated gene transfer of Fas ligand to glioma cells: implications for gene therapy

Astrocytic tumors frequently express Fas/APO-1 (Fas), in sharp contrast to surrounding normal brain cells, providing a potential window through which selective killing of tumor cells could be pursued. To assess this possibility, we transduced Fas into U251, a glioma cell line resistant to anti-Fas antibody-mediated apoptosis, and obtained transfectants with high levels of Fas expression. Anti-Fas antibody showed significantly enhanced cytotoxicity for the transfectants, suggesting that U251 cells maintained an intercellular cascade of Fas-mediated apoptosis. When U251 transfectants with high-level Fas expression were transduced with Fas ligand-encoding gene via retrovirus, they were unaffected by exposure to anti-Fas antibody or Fas ligand adenovirus (Adeno-FL). Thus, retroviral induction of Fas ligand into the glioma cells with high levels of Fas led to the selection of cells that were resistant to Fas-dependent apoptosis. These resistant U251 transfectants were susceptible to FADD adenovirus (Adeno-FADD)-induced apoptosis, indicating that a cascade of death signals was blocked at the steps between Fas ligand and FADD. As for adenoviral transduction of Fas ligand into gliomas, gliomas with a relatively high level of expression of Fas were remarkably sensitive to Adeno-FL-induced apoptosis. Besides, Adeno-FADD induced pronounced apoptosis in all glioma cells. Our data suggest the possibility of using adenovirus-mediated transduction of Fas ligand and FADD genes as a therapeutic approach to target gliomas.     

Strategies for mutational analysis of the large multiexon ATM gene using high-density oligonucleotide arrays

Mutational analysis of large genes with complex genomic structures plays an important role in medical genetics. Technical limitations associated with current mutation screening protocols have placed increased emphasis on the development of new technologies to simplify these procedures. High-density arrays of >90,000-oligonucleotide probes, 25 nucleotides in length, were designed to screen for all possible heterozygous germ-line mutations in the 9.17-kb coding region of the ATM gene. A strategy for rapidly developing multiexon PCR amplification protocols in DNA chip-based hybridization analysis was devised and implemented in preparing target for the 62 ATM coding exons. Improved algorithms for interpreting data from two-color experiments, where reference and test samples are cohybridized to the arrays, were developed. In a blinded study, 17 of 18 distinct heterozygous and 8 of 8 distinct homozygous sequence variants in the assayed region were detected accurately along with five false-positive calls while scanning >200 kb in 22 genomic DNA samples. Of eight heterozygous sequence changes found in more than one sample, six were detected in all cases. Five previously unreported sequence changes, not found by other mutational scanning methodologies on these same samples, were detected that led to either amino acid changes or premature truncation of the ATM protein. DNA chip-based assays should play a valuable role in high throughput sequence analysis of complex genes.     

Two dogmas of conceptual empiricism: implications for hybrid models of the structure of knowledge

Concepts seem to consist of both an associative component based on tabulations of feature typicality and similarity judgments and an explanatory component based on rules and causal principles. However, there is much controversy about how each component functions in concept acquisition and use. Here we consider two assumptions, or dogmas, that embody this controversy and underlie much of the current cognitive science research on concepts. Dogma 1: Novel information is first processed via similarity judgments and only later is influenced by explanatory components. Dogma 2: Children initially have only a similarity-based component for learning concepts; the explanatory component develops on the foundation of this earlier component. We present both empirical and theoretical arguments that these dogmas are unfounded, particularly with respect to real world concepts; we contend that the dogmas arise from a particular species of empiricism that inhibits progress in the study of conceptual structure; and finally, we advocate the retention of a hybrid model of the structure of knowledge despite our rejection of these dogmas.     

Changes in the norms governing practices for the manufacture of pharmaceutical products: implications for the MERCOSUR

It is done a comparative study between the "Recommended rules for drug products manufacturing and inspection", approved in 1975 by the World Health Organization (and still in force in the MERCOSUR); and the standards published in 1992 by the WHO Expert Committee on Specifications for Pharmaceutical Preparations 32nd Report, named "Good Manufacturing Practices for pharmaceutical products". The correspondence between the regulation in force in the MERCOSUR and the Good Manufacturing Practices Inspection Guide for pharmaceutical industry, used by Health Authorities in the Common Market Member States, is analysed. It is noticed a disagreement between the rule in force and the instrument for verifying its fulfillment. The proposal of this article is the adoption by the Common Market Group, of the rules published by the WHO in 1992, and the establishment of an inspection guide which absolute agrees with it.     

Two-dimensional electrophoretic analysis of human milk-fat-globule membrane proteins with attention to apolipoprotein E patterns

Two-dimensional (2-D; Iso Dalt method) electrophoresis was done on lipid membrane fractions from human milk samples collected in polypropylene tubes at home by the mother. The samples represent milk from two weeks to ten months post partum. Identification was made by immunoassay of Western blots. Apolipoprotein E was not easily seen on silver-stained gels because concentration is small and spots overlap another protein. Blot assays show a more complex pattern than 34 kDa plasma apo E with molecular size isoforms at 36 kDa, 34 kDa, 20 kDa and 15 kDa. One sample was negative for apo E. Apo E plays an important role in lipid transport in tissue and in the brain. It is synthesized in a variety of tissues by specific cells. Milk may reflect several sources.     

Chemiluminescent reporter gene assays: sensitive detection of the GUS and SEAP gene products

Chemiluminescent assays are described for the secreted alkaline phosphatase (SEAP) and beta-glucuronidase (GUS) reporter gene products. These assays provide simple, sensitive, non-isotopic alternatives to existing detection methods and are performed in microplate or tube luminometers or in a scintillation counter. The SEAP reporter gene product is secreted from mammalian cells and is thus easily detected in a sample of culture medium. Sensitive detection of secreted placental alkaline phosphatase is performed with CSPD chemiluminescent alkaline phosphatase substrate, and approximately 3 fg of enzyme can be detected. GUS has become the major reporter gene used for the analysis of plant gene expression. Sensitive chemiluminescent detection of GUS activity can be performed with an assay system we have developed using Glucuron, a beta-glucuronidase substrate. This chemiluminescent assay detects 60 fg of GUS and is linear over six orders of magnitude of enzyme concentration. CSPD and Glucuron substrates have been incorporated into two new chemiluminescent reporter gene assay kits for SEAP and GUS.     

Clinical economics review: the health-care economic implications of minimal access gastrointestinal surgery

Sporadic progressive external ophthalmoplegia and Kearns-Sayre syndrome are usually associated with single large-scale mitochondrial DNA deletions in muscle. In progressive external ophthalmoplegia with autosomal dominant inheritance, multiple mitochondrial DNA deletions have been reported. We studied several members of a Swedish family with autosomal dominant progressive external ophthalmoplegia and multiple mitochondrial DNA deletions by polymerase chain reaction analysis of single muscle fibers and by in situ hybridization, combined with enzyme histochemical analysis. Muscle fiber segments with deficiency of cytochrome c oxidase, which is partially encoded by mitochondrial DNA, had accumulated mitochondrial DNA with deletions and showed reduced levels of wild-type mitochondrial DNA. The deletions varied between individual muscle fibers. There was one predominant deletion in each cytochrome c oxidase-deficient muscle fiber segment. Sequencing of the deletion breakpoints showed that most but not all of the deletions were flanked by direct repeats. Young, clinically affected individuals of this family without limb muscle symptoms did not show mitochondrial DNA deletions or cytochrome c oxidase-deficient muscle fibers. Our results indicate that a nuclear factor predisposes to the development of somatic multiple mitochondrial DNA deletions. Mitochondrial DNA with multiple different deletions shows clonal expansion, which leads to mitochondrial myopathy with ragged-red fibers and muscle weakness.     

Baculovirus systems for the expression of human gene products

Significant advances in basic and applied biology have resulted from the use of baculovirus vectors for the expression of heterologous proteins in cultured insect cells and in insect larvae. The development of improved vectors has greatly facilitated the construction of recombinant baculoviruses, both by increasing the efficiency of identifying recombinant viruses and by reducing or eliminating the tedious steps used to purify the desired recombinant virus from its non-recombinant parent virus.     

Advanced glycation end products in vitreous: Structural and functional implications for diabetic vitreopathy

PURPOSE: Advanced glycation end products (AGEs) form irreversible cross-links with many macromolecules and have been shown to accumulate in tissues at an accelerated rate in diabetes. In the present study, AGE formation in vitreous was examined in patients of various ages and in patients with diabetes. Ex vivo investigations were performed on bovine vitreous incubated in glucose to determine AGE formation and cross-linking of vitreous collagen. METHODS: By means of an AGE-specific enzyme-linked immunosorbent assay (ELISA), AGE formation was investigated in vitreous samples obtained after pars plana vitrectomy in patients with and without diabetes. In addition, vitreous AGEs were investigated in bovine vitreous collagen after incubation in high glucose, high glucose with aminoguanidine, or normal saline for as long as 8 weeks. AGEs and AGE cross-linking was subsequently determined by quantitative and qualitative assays. RESULTS: There was a significant correlation between AGEs and increasing age in patients without diabetes (r = 0.74). Furthermore, a comparison between age-matched diabetic and nondiabetic vitreous showed a significantly higher level of AGEs in the patients with diabetes (P < 0.005). Collagen purified from bovine vitreous incubated in 0.5 M glucose showed an increase in AGE formation when observed in dot blot analysis, immunogold labeling, and AGE ELISA. Furthermore, there was increased cross-linking of collagen in the glucose-incubated vitreous, when observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and protein separation. This cross-linking was effectively inhibited by coincubation with 10 mM aminoguanidine. CONCLUSIONS: This study suggests that AGEs may form in vitreous with increasing age. This process seems to be accelerated in the presence of diabetes and as a consequence of exposure to high glucose. Advanced glycation and AGE cross-linking of the vitreous collagen network may help to explain the vitreous abnormalities characteristic of diabetes.