Functional analysis of the nicotinate mononucleotide:5,6-dimethylbenzimidazole phosphoribosyltransferase (CobT) enzyme, involved in the late steps of coenzyme B12 biosynthesis in Salmonella enterica.

In Salmonella enterica, the CobT enzyme activates the lower ligand base during the assembly of the nucleotide loop of adenosylcobalamin (AdoCbl) and other cobamides. Previously, mutational analysis identified a class of alleles (class M) that failed to restore AdoCbl biosynthesis during intragenic complementation studies. To learn why class M cobT mutations were deleterious, we determined the nature of three class M cobT alleles and performed in vivo and in vitro functional analyses guided by available structural data on the wild-type CobT (CobT(WT)) enzyme. We analyzed the effects of the variants CobT(G257D), CobT(G171D), CobT(G320D), and CobT(C160A). The latter was not a class M variant but was of interest because of the potential role of a disulfide bond between residues C160 and C256 in CobT activity. Substitutions G171D, G257D, and G320D had profound negative effects on the catalytic efficiency of the enzyme. The C160A substitution rendered the enzyme fivefold less efficient than CobT(WT). The CobT(G320D) protein was unstable, and results of structure-guided site-directed mutagenesis suggest that either variants CobT(G257D) and CobT(G171D) have less affinity for 5,6-dimethylbenzimidazole (DMB) or access of DMB to the active site is restricted in these variant proteins. The reported lack of intragenic complementation among class M cobT alleles is caused in some cases by unstable proteins, and in others it may be caused by the formation of dimers between two mutant CobT proteins with residual activity that is so low that the resulting CobT dimer cannot synthesize sufficient product to keep up with even the lowest demand for AdoCbl.

Preventing depression in high-risk college women: a report of an 18-month follow-up.

The authors tested the long-term effectiveness of a cognitive-behavioral group intervention in reducing depressive symptoms, decreasing negative thinking, and enhancing self-esteem in 92 college women aged 18 to 24 years who ere at risk for clinical depression. The women were randomly assigned to either an experimental or a no-treatment control group. The experimental group participated in a 6-week cognitive-behavioral intervention that targeted identification and reduction of negative thinking, using such techniques as thought stopping and affirmations. Data on depressive symptoms, self-esteem, and negative thinking were collected before the intervention and at intervals of 1, 6, and 18 months postintervention. The women in the intervention group experienced a greater decrease in depressive symptoms and negative thinking and a greater increase in self-esteem than those in the control group. The beneficial effects continued over an 18-month follow-up period. These findings support the importance of thought stopping and affirmations as prevention interventions with at-risk college women.

Regulation of gene expression and cell growth in vivo by tetracycline using the hollow fiber assay.

The hollow fiber assay presents a potentially unique tool to study the effects of regulated gene expression in cell lines that do not form tumors in vivo. The hollow fibers allow small molecules to pass freely through while keeping the cells within the fibers and segregated from host cells. OSp16.1 cells, derived from the U24 clone of the U2-OS osteogenic sarcoma tumor line, express the p16INK4a tumor suppressor under the regulation of tetracycline (tet) (Mitra J et al. Mol Cell Bio 19:3916, 1999). The in vitro induction of p16 in the OSp16.1 cell line is regulated by tet. The hollow fiber assay was used to determine whether the regulation of the p16 gene could be achieved in vivo, since these cells did not grow in the xenograft model. There were no differences in the in vivo growth pattern of U24 cells loaded into the hollow fibers with and without tet: 807% and 839% net growth, respectively. OSp16.1 cells in fibers in mice receiving 3.33 mg/kg/day tet had a 644% net growth after 21 days. There was a 194% net growth without tet. Immunoblotting of extracts prepared from the hollow fibers confirmed that p16 was induced in the absence of tet. These data demonstrate this assay is a useful tool for studying the effects of regulated gene expression in vivo.

The archives of the Pioneer Health Centre, Peckham, in the Wellcome Library.

This article gives a brief description of this important British experiment in health and community (1926-50) and the historiography relating to it. The archives, which are now in the Wellcome Library, are described. While mention is made of the gaps in the record (and the possible reasons for them), the strengths of the collection are indicated.

Somites in zebrafish doubly mutant for knypek and trilobite form without internal mesenchymal cells or compaction.

In vertebrates, paraxial mesoderm is partitioned into repeating units called somites. It is thought that the mechanical forces arising from compaction of the presumptive internal cells of prospective somites cause them to detach from the unsegmented presomitic mesoderm [1-3]. To determine how prospective somites physically segregate from each other, we used time-lapse microscopy to analyze the mechanics underlying early somitogenesis in wild-type zebrafish and in the mutants trilobite(m209) (tri), knypek(m119) (kny), and kny;tri, which are defective in convergent extension during gastrulation. Formation of somite boundaries in all of these embryos involved segregation, local alignment, and cell-shape changes of presumptive epitheloid border cells along nascent intersomitic boundaries. Although kny;tri somites formed without convergence of the presomitic mesoderm and were composed of only two cells in their anteroposterior (AP) dimension, they still exhibited AP intrasegmental polarity. Furthermore, morphogenesis of somite boundaries in these embryos proceeded in a manner similar to that in wild-type embryos. Thus, intersomitic boundary formation in zebrafish involves short-range movements of presumptive border cells that do not require mechanical forces generated by internal cells or compaction of the presomitic mesoderm.

Negative thinking mediates the effect of self-esteem on depressive symptoms in college women.

BACKGROUND: One of every three women between 18 and 24 years of age may be significantly depressed. Younger women have shown increasing rates of unipolar depression since the 1950s, and the average age of onset continues to decline. OBJECTIVES: To examine the prevalence and correlates of high depressive symptoms in single college women 18 to 24 years of age. Negative thinking was posited to mediate the relationship between self-esteem and depressive symptoms. METHODS: A sample of 246 women was recruited from a university student body. Each woman completed a survey that included the Center for Epidemiologic Studies-Depression Scale, the Beck Depression Inventory, the Rosenberg Self-esteem Scale, the Crandell Cognitions Inventory, and the Automatic Thoughts Questionnaire. RESULTS: Of the women, 35% had high depressive symptoms. Negative thinking mediated the relationship between self-esteem and depressive symptoms. However, self-esteem also showed a weak direct effect on depressive symptoms. CONCLUSION: The findings suggest that negative thinking may play an important role in the development of depressive symptoms in college women.

Reducing negative thinking and depressive symptoms in college women.

PURPOSE: Although cognitive-behavioral interventions have been successful in treating depression, no studies were found that focused solely on reducing negative thinking via group intervention as a means of preventing depression in at-risk groups. The purpose of this randomized controlled trial was to test the effectiveness of a cognitive-behavioral group intervention in reducing depressive symptoms, decreasing negative thinking, and enhancing self-esteem in young women at risk for depression. DESIGN: A randomized controlled trial with 92 college women ages 18 to 24 who were at risk for depression was conducted. METHOD: Participants were randomly assigned to either the control or experimental group. The experimental group participated in a 6-week cognitive-behavioral group intervention. Data on self-esteem, depressive symptoms, and negative thinking were collected via self-report questionnaires from control and experimental groups at baseline, 1 month after the intervention, and at 6-month follow-up. Data were analyzed using mixed-model methodology and the Cochran-Mantel-Haenszel chi-square test. FINDINGS: Compared to those in the control group, women who received the intervention had a greater decrease in depressive symptoms and negative thinking and a greater increase in self-esteem, and these beneficial effects were maintained over 6-months. CONCLUSIONS: The findings document the effectiveness of this cognitive-behavioral group intervention and indicate empirical support for the beneficial effects of reducing negative thinking by the use of affirmations and thought-stopping techniques on women's mental health.

The hollow fiber assay: continued characterization with novel approaches.

The hollow fiber assay, a unique in vivo model, permits the simultaneous evaluation of compound efficacy against multiple cell lines in two physiological compartments. This assay has been used to characterize in vivo activity of cytotoxic compounds. The purpose of the present study was to characterize and optimize this assay for compounds with a defined mechanism of action, specifically cell cycle inhibition. Two human tumor cell lines and one normal human cell line were loaded into polyvinylidene fluoride hollow fibers at two or more cell concentrations and grown in mice for 3-10 days. The data demonstrate the importance of characterizing the initial loading density of various cell lines in the evaluation of compounds. All studies were performed with cells in the linear part of the cell growth curves. Initial loading densities of 1-2 x 10(4) cells/fiber gave the greatest opportunity for growth in the three human cell lines tested (HCT116 colon carcinoma, NCI-H460 non-small cell carcinoma, and AG 1523 normal fibroblast). Utilizing the MTT assay, standard curves were constructed to correlate the final number of cells with optical density (OD) readings at 540 nm in order to calculate cell numbers in the fibers. Insights into the mechanism of action of cisplatin have been gained using Western blot analysis of the cell cycle markers PCNA (a protein present throughout the cell cycle) and Rb (a protein that acts as a tumor suppressor gene product) from the hollow fiber cells. In cisplatin-treated NCI-H460 cells both PCNA and Rb phosphorylation decreased, suggesting the arrest of the cells prior to the S phase. Standard therapeutic agents, cisplatin, racemic flavopiridol, cyclophosphamide and mitomycin C, were evaluated independently in the hollow fiber assay and the xenograft model. The data demonstrate that compounds active in the hollow fiber assay are also active in the xenograft.

Kindergarten children's knowledge and perceptions of alcohol, tobacco, and other drugs.

Kindergarten children's knowledge and perceptions of alcohol, tobacco, and other drugs (ATODs) were assessed and the congruence between parent ATOD use and children's knowledge of ATODs was examined. Data were collected during the pre-intervention phase of an ATOD prevention trial with 5- and 6-year-old children and their parents. Three elementary schools were randomly selected from a population of 15 high-risk elementary schools in Lexington, Ky., (n = 126 parent-child dyads). Children were interviewed about their knowledge, feelings, and attitudes toward ATODs using the Child Drug Awareness Inventory. Parents self-reported ATOD use. Almost all (95%) kindergarten children recognized cigarettes; 56% correctly identified alcoholic beverages; and 17% recognized at least one illicit drug. Minority children were almost four times more likely to recognize illicit drugs than were non-minority children. Children's knowledge of ATODs was not correlated with the parents' reported drug use. ATOD prevention programs for young children merit greater emphasis.

Synthesis and structures of polymeric Mn, Co, Cu, and Zn complexes of 3-diphenylamino-4-hydroxycyclobut-3-ene-1,2-dione (diphenylaminosquarate) and of the salt [Ni(H2O)6][(C6H5)2NC4O3]2.2H2O.

Reaction of M(NO3)2.xH2O (M = Mn, Co, Ni, Cu, Zn) with 3-diphenylamino-4-hydroxycyclobut-3-ene-1,2-dione (diphenylaminosquarate) produces the neutral polymeric species (M[mu-(C6H5)2NC4O3]2[H2O]2)n [M = Mn (1), Cu (2)]; (M[mu-(C6H5)2NC4O3][(C6H5)2NC4O3][H2O]3)n [M = Co (3), Zn (4)]; and in the case of Ni, the salt [Ni(H2O)6][(C6H5)2NC4O3]2.2H2O (5). Complexes 1 and 2 are isomorphous and crystallize in the monoclinic space group P2(1)/c with, for 1, a = 13.138(1) A, b = 10.900(2) A, c = 9.269(2) A, beta = 96.07(1) degrees, and Z = 2. Complexes 3 and 4 are also isomorphous and crystallize in the space group P2(1)/c with, for 3, a = 13.211(1) A, b = 11.038(1) A, c = 18.748(1) A, beta = 97.75(1) degrees, and Z = 4. The nickel salt, 5, crystallizes in the triclinic space group P1 with a = 6.181(1) A, b = 9.417(1) A, c = 15.486(1) A, alpha = 101.37(1) degrees, beta = 95.51(1) degrees, gamma = 107.57(1) degrees, and Z = 1. In 1 and 2, the metal coordination is octahedral, comprising four mu-1,3-bridging diphenylaminosquarate ligands and two trans aqua ligands. In 3 and 4, the metal coordination is again octahedral, comprising two mu-1,3-bridging and one pendant diphenylaminosquarate ligands, the octahedron being completed by three aqua ligands in a meridional configuration. In 5, the hexaaquanickel(II) ion is linked by O-H...O hydrogen bonds to a pair of diphenylaminosquarate anions. These anion-cation units are linked via included water molecules to form hydrogen-bonded chains. The diphenylaminosquarate ligands in the polymeric complexes 1-4 display multiple-bond localization, a feature which is absent in 5. Complex 1 exhibits weak antiferromagnetic coupling, whereas 2 shows no significant magnetic interactions.

Effects of the quality of dyadic relationships on the psychological well-being of elderly care-recipients.

The purpose of this study was to examine the association of the quality of caregiver relationships with the psychological well-being of elderly care-recipients. Sociodemographic variables and characteristics of the care-recipient situation (e.g., self-rated physical health, amount of instrumental support needed) were explored as potential predictors of the psychological well-being of elderly individuals. A secondary analysis of data collected during in-home interviews with 37 community-dwelling older adults revealed no significant correlations between the quality of the primary intimate relationship and any dimension of psychological well-being. However, better self-rated health was associated with fewer depressive symptoms, higher morale, greater life satisfaction, and better quality of life. The more instrumental support needed by an individual, the greater their depressive symptoms and the lower their morale. The findings also revealed that the older the individual was, the greater the depressive symptoms were and the lower life satisfaction became. Self-rated physical health predicted each dimension of psychological well-being. The findings suggest that age, the amount of instrumental support needed, and perceptions of physical health are important indicators of the psychological well-being of elderly care-recipients.

In vitro synthesis of the nucleotide loop of cobalamin by Salmonella typhimurium enzymes.

In Salmonella typhimurium, the CobU, CobS, CobT, and CobC proteins have been proposed to catalyze the late steps in adenosylcobalamin biosynthesis, which define the nucleotide loop assembly pathway. This paper reports the in vitro assembly of the nucleotide loop of adenosylcobalamin from its precursors adenosylcobinamide, 5, 6-dimethylbenzimidazole, nicotinate mononucleotide, and GTP. Incubation of these precursors with the CobU, CobS, and CobT proteins resulted in the synthesis of adenosylcobalamin-5'-phosphate. This cobamide was isolated by HPLC, identified by UV-visible spectroscopy and mass spectrometry, and shown to support growth of a cobalamin auxotroph. Adenosylcobalamin-5'-phosphate was also isolated from reaction mixtures containing adenosylcobinamide-GDP (the product of the CobU reaction) and alpha-ribazole-5'-phosphate (the product of the CobT reaction) as substrates and CobS. These results allowed us to conclude that CobS is the cobalamin(-5'-phosphate) synthase enzyme in S. typhimurium. The CobC enzyme, previously shown to dephosphorylate alpha-ribazole-5'-phosphate to alpha-ribazole, was shown to dephosphorylate adenosylcobalamin-5'-phosphate to adenosylcobalamin. Adenosylcobinamide was converted to adenosylcobalamin in reactions where all four enzymes were present in the reaction mixture. This in vitro system offers a unique opportunity for the rapid synthesis and isolation of cobamides with structurally different lower-ligand bases that can be used to investigate the contributions of the lower-ligand base to cobalamin-dependent reactions.

Potential for abusive parenting by rural mothers with low-birth-weight children.

PURPOSE: To describe factors influencing the potential for abusive parenting by rural mothers of low-birth-weight children (< 2,500 gm). Low-birth-weight (LBW) children are at risk for child abuse. However, little is known about the added risks created for these children by rural residency. DESIGN: Data for this descriptive and exploratory study were collected using a cross-sectional design. A convenience sample of 48 rural mothers delivering LBW children participated in 1994. METHODS: In-home interviews were conducted using structured questionnaires to assess mothers' everyday stressors, depressive symptoms, social resources, and child abuse potential. FINDINGS: The prevalence of high depressive symptoms among the mothers was 54%. Higher everyday stressors and less functional social support predicted greater depressive symptoms. Everyday stressors had a direct effect on the mothers' potential for child abuse and an indirect effect via maternal depressive symptoms. CONCLUSIONS: Rural mothers of LBW children are at risk for abusive parenting. Health care providers should pay particular attention to the mental health of mothers living in rural, isolated areas. Assistance with mobilization of community resources, including social support and child care, may provide needed help for these mothers to improve parenting and thus child health outcomes.

The physical health of rural mothers and their low birth weight children.

The purpose of this research was to examine the physical health of rural mothers and their low birth weight children (< 2,500 gm). The health of rural mothers is of concern because rural families have poorer health than urban ones, and as the primary caregiver for the low birth weight child, mother's health affects the child's care and potentially the child's health. In this cross-sectional, exploratory study, in-home interviews were conducted with 48 mothers between 2 weeks and 18 months after their children were discharged from their birth hospitalization. Mothers' physical health and their perceptions of their children's health were assessed. The mothers were more concerned about their children's health than their own (p = .0005). The concerns included uncertainty about the children's future health, growth, and development. Suggestions for community health care providers are addressed.

Psychometrics for two short forms of the Center for Epidemiologic Studies-Depression Scale.

The Center for Epidemiologic Studies-Depression Scale (CES-D; L. S. Radloff, 1977) assesses the presence and severity of depressive symptoms occurring over the past week. Although it contains only 20 items, its length may preclude its use in a variety of clinical populations. This study evaluated psychometric properties of 2 shorter forms of the CES-D developed by F. J. Kohout, L. F. Berkman, D. A. Evans, and J. Cornoni-Huntley (1993): the Iowa form and the Boston form. Data were pooled from 832 women representing 6 populations. Internal consistency estimates, correlations with the original version of the CES-D, and omitted-included item correlations supported use of the Iowa form over the Boston form when a shortened version of the scale is desired. Regression statistics are provided for use in estimating scores on the original CES-D when either shortened form is used. Factor analytic results from two populations support a single-factor structure for the original CES-D as well as the short forms.