Role of molecular diagnostic testing in familial adenomatous polyposis and hereditary nonpolyposis colorectal cancer families.

PURPOSE: Genetic tests are available for familial adenomatous polyposis and hereditary nonpolyposis colorectal cancer. The goal of this review was to develop an algorithm for application of molecular diagnostic techniques to the management of hereditary colorectal carcinoma and to familiarize the clinician with the vocabulary of molecular genetic testing for hereditary colorectal carcinoma. METHODS: Studies examining the clinical use of genetic testing for hereditary colorectal carcinoma syndromes are evaluated. Recent advances in molecular genetic technology are reviewed, and clinical management as practiced here and elsewhere is outlined. RESULTS: This review is a guide to the most reliable molecular diagnostic techniques. Three key questions are answered: who, when, and how to test. CONCLUSIONS: When integrated with existing testing protocols for colorectal carcinoma and when applied with appropriate caveats, particularly regarding interpretation of negative results, genetic testing can result in improved management of patients and families.

Genetic counseling and interpretation of genetic tests in familial adenomatous polyposis and hereditary nonpolyposis colorectal cancer.

PURPOSE: Genetic counseling and interpreting genetic test results can be complex. Moreover, without knowing the limitations of the methods used and the lifetime probability of developing cancer in individuals who carry a gene that predisposes to cancer, misinterpretation may lead to false assurance. The purpose of this review is to discuss how genetic counseling will benefit patients and their family, the genetic tests available for hereditary colorectal cancer syndromes, and the interpretation of results. METHODS: Current literature was reviewed and our clinical and research experiences were incorporated. RESULTS: This review serves as a guide to enable various health care providers to better counsel patients in their quest for advice on prevention, early detection, and surveillance for colorectal cancer. Notable topics of discussion are who should undergo genetic counseling and consider testing and how the interpretation of test results can be misleading; for example, understanding the difference between a no mutation detected vs. a negative test result. CONCLUSIONS: Genetic counseling is of paramount importance for patients to fully understand the limitations of genetic testing and will aid in the management of patients who are susceptible to colorectal cancer.

XRCC1 stimulates human polynucleotide kinase activity at damaged DNA termini and accelerates DNA single-strand break repair.

XRCC1 protein is required for DNA single-strand break repair and genetic stability but its biochemical role is unknown. Here, we report that XRCC1 interacts with human polynucleotide kinase in addition to its established interactions with DNA polymerase-beta and DNA ligase III. Moreover, these four proteins are coassociated in multiprotein complexes in human cell extract and together they repair single-strand breaks typical of those induced by reactive oxygen species and ionizing radiation. Strikingly, XRCC1 stimulates the DNA kinase and DNA phosphatase activities of polynucleotide kinase at damaged DNA termini and thereby accelerates the overall repair reaction. These data identify a novel pathway for mammalian single-strand break repair and demonstrate a concerted role for XRCC1 and PNK in the initial step of processing damaged DNA ends.

Bacterial response to acetate challenge: a comparison of tolerance among species.

Although acetate formation and tolerance are important criteria for various aspects of biotechnological process development, available studies on acetate tolerance in different species are disparate. We evaluate the response of eight bacterial strains, including two variants of Escherichia coli, two variants of Staphylococcus capitis, and one each of Acetobacter aceti, Gluconobacter suboxydans, Lactobacillus acetotolerans, and L. bulgaricus, to acetate challenges under identical conditions. Our findings were: (1) wild-type organisms of species that are considered tolerant of acetate perform only slightly better than E. coli in unadapted shaker cultures; (2) the ability to tolerate acetate is strongly dependent on the carbon source, and is, especially for E. coli, much greater on glycerol than on glucose; (3) respiration is not as important to acetate tolerance in E. coli and S. capitis as has been reported for the acetic acid bacteria; (4) S. capitis was the least affected by acetate under all conditions and grew at up to 44 g/l acetate without any preconditioning; and (5) qualitative high-throughput screening of growth characteristics can be achieved with relatively inexpensive multiwell plate readers.

Metabolic flux ratio analysis of genetic and environmental modulations of Escherichia coli central carbon metabolism.

The response of Escherichia coli central carbon metabolism to genetic and environmental manipulation has been studied by use of a recently developed methodology for metabolic flux ratio (METAFoR) analysis; this methodology can also directly reveal active metabolic pathways. Generation of fluxome data arrays by use of the METAFoR approach is based on two-dimensional (13)C-(1)H correlation nuclear magnetic resonance spectroscopy with fractionally labeled biomass and, in contrast to metabolic flux analysis, does not require measurements of extracellular substrate and metabolite concentrations. METAFoR analyses of E. coli strains that moderately overexpress phosphofructokinase, pyruvate kinase, pyruvate decarboxylase, or alcohol dehydrogenase revealed that only a few flux ratios change in concert with the overexpression of these enzymes. Disruption of both pyruvate kinase isoenzymes resulted in altered flux ratios for reactions connecting the phosphoenolpyruvate (PEP) and pyruvate pools but did not significantly alter central metabolism. These data indicate remarkable robustness and rigidity in central carbon metabolism in the presence of genetic variation. More significant physiological changes and flux ratio differences were seen in response to altered environmental conditions. For example, in ammonia-limited chemostat cultures, compared to glucose-limited chemostat cultures, a reduced fraction of PEP molecules was derived through at least one transketolase reaction, and there was a higher relative contribution of anaplerotic PEP carboxylation than of the tricarboxylic acid (TCA) cycle for oxaloacetate synthesis. These two parameters also showed significant variation between aerobic and anaerobic batch cultures. Finally, two reactions catalyzed by PEP carboxykinase and malic enzyme were identified by METAFoR analysis; these had previously been considered absent in E. coli cells grown in glucose-containing media. Backward flux from the TCA cycle to glycolysis, as indicated by significant activity of PEP carboxykinase, was found only in glucose-limited chemostat culture, demonstrating that control of this futile cycle activity is relaxed under severe glucose limitation.

Molecular cloning of the human gene, PNKP, encoding a polynucleotide kinase 3'-phosphatase and evidence for its role in repair of DNA strand breaks caused by oxidative damage.

Mammalian polynucleotide kinases catalyze the 5'-phosphorylation of nucleic acids and can have associated 3'-phosphatase activity, predictive of an important function in DNA repair following ionizing radiation or oxidative damage. The sequences of three tryptic peptides from a bovine 60-kDa polypeptide that correlated with 5'-DNA kinase and 3'-phosphatase activities identified human and murine dbEST clones. The 57.1-kDa conceptual translation product of this gene, polynucleotide kinase 3'-phosphatase (PNKP), contained a putative ATP binding site and a potential 3'-phosphatase domain with similarity to L-2-haloacid dehalogenases. BLAST searches identified possible homologs in Caenorhabditis elegans, Schizosaccharomyces pombe, and Drosophila melanogaster. The gene was localized to chromosome 19q13.3-13.4. Northern analysis indicated a 2-kilobase mRNA in eight human tissues. A glutathione S-transferase-PNKP fusion protein displayed 5'-DNA kinase and 3'-phosphatase activities. PNKP is the first gene for a DNA-specific kinase from any organism. PNKP expression partially rescued the sensitivity to oxidative damaging agents of the Escherichia coli DNA repair-deficient xth nfo double mutant. PNKP gene function restored termini suitable for DNA polymerase, consistent with in vivo removal of 3'-phosphate groups, facilitating DNA repair.

Purification of a polynucleotide kinase from calf thymus, comparison of its 3'-phosphatase domain with T4 polynucleotide kinase, and investigation of its effect on DNA replication in vitro.

Mammalian polynucleotide kinases (PNKs) carry out 5'-phosphorylation of nucleic acids. Although the cellular function(s) of these enzymes remain to be delineated, important suggestions have included a role in DNA repair and, more recently, in DNA replication. Like T4 PNK, some preparations of mammalian PNKs have been reported to have an associated 3'-phosphatase activity. Previously, we have identified in calf thymus glands an apparently novel PNK with a neutral to alkaline pH optimum that lacked 3'-phosphatase activity. In this report, we describe purification of another bovine PNK, SNQI-PNK, with a slightly acidic pH optimum that copurifies with a 3'-phosphatase activity. The enzyme appears to be a monomer of 60 kDa. Mammalian DNA replication reactions were supplemented with T4 PNK or SNQI-PNK, and no significant effect on DNA replication in vitro was observed. Database searches support the earlier mapping of the 3'-phosphatase activity of T4 PNK to the C-terminus and suggest that the 3'-phosphatase domain of T4 PNK is related to the protein superfamily of L-2-haloacid dehalogenases. Exopeptidase digestion experiments were carried out to compare the SNQI-PNK enzyme with T4 PNK and led to the inference that the domain organization of the bovine polypeptide may differ from that of the T4 enzyme.

Feelings and attitudes of gifted students.

Differences between the self-perceptions of gifted high school freshmen (n = 62) and nongifted peers (n = 162) were assessed regarding intimacy with family and peers, social support, family responsibilities, self-esteem, depression, and risk-taking behavior. Gifted students perceived themselves as being more intimate with friends, assuming fewer family responsibilities, and taking more risks (both sports- and danger-related risks). Contrary to the literature suggesting delays in the social development of gifted students, these data indicate that gifted students may be socially precocious when compared with their nongifted peers. Gifted students and their teachers were also administered the Perceptions about Giftedness Scale. Gifted students reported feeling the same as, or better than, their peers about their academic and social skills, and their teachers closely agreed. However, the teachers rated the gifted students as being less happy than the students rated themselves.

Acetate-specific stress response in acetate-resistant bacteria: an analysis of protein patterns.

Many metabolic byproducts have toxic effects on bacteria, and acetic acid is an excellent model for such molecules. The negative effects of acetate, which include decreased growth rates and specific productivities, appear for Escherichia coli at acetate concentrations lower than 5 g/L. Acetic acid bacteria, however, are naturally resistant to the detrimental effects of acetate in their surroundings; they remain active at acetate levels well over 40 g/L. This study investigated the response to acetate challenges by the naturally acetate-resistant bacteria Acetobacter aceti and Gluconobacter suboxydans to learn more about possible mechanisms of tolerance to otherwise toxic low molecular weight metabolites. Growth studies showed that the resistant bacteria grow more slowly in the presence of acetate but are not slowed nearly so much as is E. coli. In addition, two-dimensional gel electrophoresis (2DE) was applied to study the relative protein patterns of acetate-resistant bacteria during growth in the presence and absence of acetate. In each organism, growth in acetate-containing medium led to elevated levels of many stress response proteins. 2DE analysis of heat-shocked cultures was used to determine which were nonspecific. Elimination of those proteins that were also amplified following heat shock left only eight proteins, here designated acetate-specific stress proteins (Asps), which are overexpressed specifically in response to acetate. Three of these, AspA, AspB, and AspC, appear to be analogous in the two bacterial strains studied, based on their apparent pIs and molecular weights.

Adolescents' perceptions of family responsibility-taking.

A scale was developed to solicit adolescents' perceptions of their family responsibility-taking (defined as helping out and being supportive). Four hundred adolescents were administered this scale together with self-report measures of intimacy with parents and peers as well as other psychological variables (self-esteem, depression, risk-taking, and drug use). The results revealed that adolescents who felt they assumed more family responsibility reported less depression, more intimate relationships with their parents, and higher self-esteem.

Adolescent psychiatric patients' interactions with their mothers.

Thirty-eight adolescent psychiatric patients and their mothers engaged in two dyadic interactions. The participants rated themselves and each other on four behavioral dimensions (calmness, friendliness, involvement, and bossiness) during a videotaped playback. An independent observer rated the dyads on the same dimensions. Analyses were conducted based on classification of adolescents as internalizers/externalizers, depressed/nondepressed, and socially anxious/nonanxious. Internalizing adolescent dyads were significantly calmer, friendlier, and more involved than were externalizing adolescent dyads. The dyads in which the adolescents scored lower on the depression scale were calmer, friendlier, and more involved than were the dyads with adolescents who had higher depression scores. No differences were noted between high and low socially anxious dyads. The findings indicated that the videotaping procedure could help familiarize clinical staff with the dynamics of parent-adolescent interactions.

Specificity of mutagenesis by 4-aminobiphenyl: mutations at G residues in bacteriophage M13 DNA and G-->C transversions at a unique dG(8-ABP) lesion in single-stranded DNA.

Mutagenesis by the human bladder carcinogen 4-aminobiphenyl (ABP) was studied in single-stranded DNA from a bacteriophage M13 cloning vector. In comparison to ABP lesions in double-stranded DNA, lesions in single-stranded DNA were approximately 70-fold more mutagenic and 50-fold more genotoxic. Sequencing analysis of ABP-induced mutations in the lacZ gene revealed exclusively base-pair substitutions, with over 80% of the mutations occurring at G sites; the G at position 6310 accounted for 25% of the observed mutations. Among the sequence changes at G sites, G-->T transversions predominated, followed by G-->C transversions and G-->A transitions. In order to further elucidate the mutagenic mechanism of ABP, an oligonucleotide containing the major DNA adduct, N-(deoxyguanosin-8-yl)-4-aminobiphenyl (dG(8-ABP)), was situated within the PstI site of a single-stranded M13 genome. After in vivo replication of the adduct containing ABP-modified and control (unadducted) genomes, the mutational frequency and mutational specificity of the dG(8-ABP) lesion were determined. The targeted mutational efficiency was approximately 0.01%, and the primary mutation observed was the G-->C transversion. Thus dG(8-ABP), albeit weakly mutagenic at the PstI site, can contribute to the mutational spectrum of ABP lesions.

On-line monitoring of intracellular ATP concentration in Escherichia coli fermentations.

A method was developed to provide a real-time measurement of intracellular adenosine 5'-triphosophate (ATP) concentrations in growing Escherichia coli. The bacteria to be monitored must first be modified by inserting the cDNA for firefly luciferase expressed from a constitutive promoter. Such a construct leads to constant specific activity of firefly luciferase during both the lag phase and exponential growth. When the luciferase substrate, D-luciferin, is added to the medium, ATP within the cells is utilized in the luciferase-catalyzed reaction that produces light. The light is carried from the bioreactor to a computer-based detector by an optical fiber. The detected per cell light emission varies during exponential growth. Analysis of cytoplasm extracts shows that this variance is related to changes in the ATP concentration, which ranges from 1 to 6 times the literature value for K(M). Experimental analyses demonstrated that inner filter effects are not a significant factor affecting the use of this system. The method was tested in a benchtop fermentor at cell densities above 13 g/L dry cell weight. A correction factor based on the accumulated light data is calculated and used in real time to account for consumption of luciferin from the culture broth by the light producing reaction. Dissolved oxygen concentrations must be kept above 15% of air saturation to ensure constant light output, but no detectable increase in oxygen demand is seen. The method does not significantly affect growth or production rates. (c) 1996 John Wiley & Sons, Inc.

Adolescent depressed mood and parental unhappiness.

A set of self-report scales on depression, parental happiness, intimate relationships, social support, self-esteem, and risk-taking behavior were administered to 455 adolescents to determine the relationship between depression and these other variables. Adolescents with depressed mood were found to be less intimate with both parents, felt less social support, and had lower self-esteem than their peers. Adolescents who perceived their mother or father as unhappy also reported less intimacy with both parents and less social support.

Under-eating and over-eating concerns among adolescents.

462 adolescents were given a set of scales to determine their concerns about eating (under-eating or over-eating), and perceptions of family and peer intimacy, social support, self-esteem, depression and exercise. Although only 10% stated that they were "underweight" and 21% that they were "overweight", as many as 50% reported having eating concerns. As compared to those who did not have concerns about eating, those who were concerned about undereating felt that they had poorer relationships with their mothers and fathers, less social support, lower self-esteem and low levels of exercise. In contrast, those who were concerned about overeating perceived having an intimacy problem only with their fathers. Like those concerned about undereating, the group concerned about overeating also had lower self-esteem and low levels of exercise. But, unlike the under-eating concern group, the over-eating concern group scored higher on the depression scale.

5'phosphorylation of DNA in mammalian cells: identification of a polymin P-precipitable polynucleotide kinase.

Proteins that catalyze 5' phosphorylation of an oligodeoxyribonucleotide substrate can be fractionated by polymin P treatment of whole cell extracts of calf thymus glands. Anion exchange chromatography on Q-Sepharose revealed three separable peaks of activity in the polymin P supernatant fraction, and one peak of activity in the Polymin P pellet fraction. The latter activity, Polymin P-precipitable polynucleotide kinase (PP-PNK), was further purified with a 1,500-fold increase of specific activity compared to the crude Polymin P pellet fraction. Oligonucleotides, a dephosphorylated 2.9-kb EcoRI fragment, and poly(A) were phosphorylated by the enzyme preparation, but thymidine 3' monophosphate was not a substrate. PP-PNK preparations exhibited an apparent KM of 52 microM for ATP and 8 microM for oligo dT25. The enzyme preparation displayed no detectable 3' phosphatase or cyclic 2',3' phosphohydrolase activities. The sedimentation coefficient of the PP-PNK activity was 3.8S as determined by sucrose density gradient analysis; the Stokes radius was 45 A, leading to an estimated molecular mass of 72 kDa. The enzyme had a pH optimum in the neutral to alkaline range in several buffer systems and is distinct from the DNA kinase with an acidic pH optimum previously described in calf thymus.

Adolescents' perceptions of their risk-taking behavior.

A questionnaire comprised of several self-report scales was administered to 440 adolescents to assess differences between high and low sports and danger risk takers on relationship and personality variables. Sports risk takers reported more danger-related risk taking and more drug use but higher self-esteem than did nonrisk takers. Danger risk takers reported greater sports-related risk taking and more drug use as well as less intimacy with their mothers, less family responsibility taking, and less depression than did their nonrisk-taking counterparts.

Genetic mapping of the breast-ovarian cancer syndrome to a small interval on chromosome 17q12-21: exclusion of candidate genes EDH17B2 and RARA.

A susceptibility gene for hereditary breast-ovarian cancer, BRCA1, has been assigned by linkage analysis to chromosome 17q21. Candidate genes in this region include EDH17B2, which encodes estradiol 17 beta-hydroxysteroid dehydrogenase II (17 beta-HSD II), and RARA, the gene for retinoic acid receptor alpha. We have typed 22 breast and breast-ovarian cancer families with eight polymorphisms from the chromosome 17q12-21 region, including two in the EDH17B2 gene. Genetic recombination with the breast cancer trait excludes RARA from further consideration as a candidate gene for BRCA1. Both BRCA1 and EDH17B2 map to a 6 cM interval (between THRA1 and D17S579) and no recombination was observed between the two genes. However, direct sequencing of overlapping PCR products containing the entire EDH17B2 gene in four unrelated affected women did not uncover any sequence variation, other than previously described polymorphisms. Mutations in the EDH17B2 gene, therefore do not appear to be responsible for the hereditary breast-ovarian cancer syndrome. Single meiotic crossovers in affected women suggest that BRCA1 is flanked by the loci RARA and D17S78.