Inequalities in self rated health in the 1958 birth cohort: lifetime social circumstances or social mobility?

OBJECTIVE: To investigate explanations for social inequalities in health with respect to health related social mobility and cumulative socioeconomic circumstances over the first three decades of life. DESIGN: Longitudinal follow up. SETTING: Great Britain. SUBJECTS: Data from the 1958 birth cohort study (all children born in England, Wales, and Scotland during 3-9 March 1958) were used, from the original birth survey and from sweeps at 16, 23, and 33 years. MAIN OUTCOME MEASURES: Subjects'' own ratings of their health; social differences in self rated health at age 33. RESULTS: Social mobility varied by health status, with those reporting poor health at age 23 having higher odds of downward mobility than of staying in same social class. Men with poor health were also less likely to be upwardly mobile. Prevalence of poor health at age 33 increased with decreasing social class: from 8.5% in classes I and II to 17.7% in classes IV and V among men, and from 9.4% to 18.8% among women. These social differences remained significant after adjustment for effects of social mobility. Health inequalities attenuated when adjusted for social class at birth, at age 16, or at 23 or for self rated health at age 23. When adjusted for all these variables simultaneously, social differences in self rated health at age 33 were substantially reduced and no longer significant. CONCLUSIONS: Lifetime socioeconomic circumstances accounted for inequalities in self reported health at age 33, while social mobility did not have a major effect on health inequalities.     

Protein structural plasticity exemplified by insertion and deletion mutants in T4 lysozyme.

To further investigate the ways in which proteins respond to changes in the length of the polypeptide chain, a series of 32 insertions and five deletions were made within nine different alpha-helices of T4 lysozyme. In most cases, the inserted amino acid was a single alanine, although in some instances up to four residues, not necessarily alanine, were used. Different insertions destabilized the protein by different amounts, ranging from approximately 1 to 6 kcal/mol. In one case, no protein could be obtained. An "extension" mutant in which the carboxy terminus of the molecule was extended by four alanines increased stability by 0.3 kcal/mol. For the deletions, the loss in stability ranged from approximately 3 to 5 kcal/mol. The structures of six insertion mutants, as well as one deletion mutant and the extension mutant, were determined, three in crystal forms nonisomorphous with wild type. In all cases, including previously described insertion mutants within a single alpha-helix, there appears to be a strong tendency to preserve the helix by translocating residues so that the effects of the insertion are propagated into a bend or loop at one end or the other of the helix. In three mutants, even the hydrophobic core was disrupted so as to permit the preservation of the alpha-helix containing the insertion. Translocation (or "register shift") was also observed for the deletion mutant, in this case a loop at the end of the helix being shortened. In general, when translocation occurs, the reduction in stability is only moderate, averaging 2.5 kcal/mol. Only in the most extreme cases does "bulging" or "looping-out" occur within the body of an alpha-helix, in which case the destabilization is substantial, averaging 4.9 kcal/mol. Looping-out can occur for insertions close to the end of a helix, in which case the destabilization is less severe, averaging 2.6 kcal/mol. Mutant A73-[AAA] as well as mutants R119-[A] and V131-[A], include shifts in the backbone of 3-6 A, extending over 20 residues or more. As a result, residues 114-142, which form a "cap" on the carboxy-terminal domain, undergo substantial reorganizations such that the interface between this "cap" and the rest of the protein is altered substantially. In the case of mutant A73-[AAA], two nearby alpha-helices, which form a bend of approximately 105 degrees in the wild-type structure, reorganize in the mutant structure to form a single, essentially straight helix. These structural responses to mutation demonstrate the plasticity of protein structures and illustrate ways in which their three-dimensional structures might changes during evolution.     

Disinfection Alternatives for Control of Ditylenchus dipsaci in Garlic Seed Cloves

Hot-water dips with and without the additives abamectin and sodium hypochlorite were evaluated for control of Ditylenchus dipsaci infection of garlic seed cloves. All treatments were compared to hot water-formalin clove dip disinfection and to nontreated infected controls for garlic emergence, midseason infection, bulb damage, and yield at harvest in field plots in 12 experiments. Hot-water treatments without additives only partially controlled D. dipsaci when a warming presoak dip (38 C) of 30, 45, or 60 minutes'' duration was followed by a hot-water dip (49 C) of 15-30 minutes'' duration. Exposure to 49 C for 30 minutes caused slight retardation of garlic emergence, although normal stand was established. Abamectin at 10-20 ppm as the 20-minute hot dip (49 C) or as a 20-minute cool dip (18 C) following a 20-minute hot-water dip and sodium hypochlorite at 1.052-1.313% aqueous solution as the 20-minute hot dip were highly effective in controlling D. dipsaci and were noninjurious to garlic seed cloves. None of these treatments was as effective as a hot water-formalin dip and were noneradicative, but showed high efficacy on heavily infected seed cloves relative to nontreated controls. Abamectin was most effective as a cool dip. These abamectin cool-dip (following hot-water dip) and sodium hypochlorite hot-dip treatments can be considered as effective alternatives to replace formalin as a dip additive for control of clove-borne D. dipsaci. Sodium hypochlorite was less effective as the cool dip, and at concentrations of 1.75-2.63% was phytotoxic to garlic.     

New renal scarring in children who at age 3 and 4 years had had normal scans with dimercaptosuccinic acid: follow up study.

OBJECTIVE: To determine up to what age children remain at risk of developing a new renal scar from a urinary tract infection. DESIGN: Follow up study. Families of children who had normal ultrasound scans and scanning with dimercaptosuccinic acid (DMSA) after referral with a urinary tract infection when aged 3 (209) or 4 (220) were invited to bring the children for repeat scans 2-11 years later. A history of infections since the original scan was obtained for children not having a repeat scan. SETTING: Teaching hospital. SUBJECTS: Children from three health districts in whom a normal scan had been obtained at age 3-4 years in 1985-1992 because of a urinary tract infection. MAIN OUTCOME MEASURE: Frequency of new renal scars in each age group. RESULTS: In each group, about 97% of children either had repeat scanning (over 80%) or were confidently believed by their general practitioner or parent not to have had another urinary infection. The rate of further infections since the original scan was similar in the 3 and 4 year old groups (48/176 (27%)) and 55/179 (31%)). Few children in either group known to have had further urinary infections did not have repeat scanning (3/209 (1.4%) and 4/220 (1.8%)). In the 3 year old group, 2.4% (5/209) had one or more new kidney scars at repeat scanning (one sided 95% confidence interval up to 5.0%), whereas none of the 4 year olds did (one sided 95% confidence interval up to 1.4%). The children who developed scars were all aged under 3.4 years when scanned originally. CONCLUSIONS: Children with a urinary tract infection but unscarred kidneys after the third birthday have about a 1 in 40 risk of developing a scar subsequently, but after the fourth birthday the risk is either very low or zero. Thus the need for urinary surveillance is much reduced in a large number of children.     

Probing minimal independent folding units in dihydrofolate reductase by molecular dissection.

Molecular dissection was employed to identify minimal independent folding units in dihydrofolate reductase (DHFR) from Escherichia coli. Eight overlapping fragments of DHFR, spanning the entire sequence and ranging in size from 36 to 123 amino acids, were constructed by chemical cleavage. These fragments were designed to examine the effect of tethering multiple elements of secondary structure on folding and to test if the secondary structural domains represent autonomous folding units. CD and fluorescence spectroscopy demonstrated that six fragments containing up to a total of seven alpha-helices or beta-strands and, in three cases, the adenine binding domain (residues 37-86), are largely disordered. A stoichiometric mixture of the two fragments comprising the large discontinuous domain, 1-36 and 87-159, also showed no evidence for folding beyond that observed for the isolated fragments. A fragment containing residues 1-107 appears to have secondary and tertiary structure; however, spontaneous self-association made it impossible to determine if this structure solely reflects the behavior of the monomeric form. In contrast, a monomeric fragment spanning residues 37-159 possesses significant secondary and tertiary structure. The urea-induced unfolding of fragment 37-159 in the presence of 0.5 M ammonium sulfate was found to be a well-defined, two-state process. The observation that fragment 37-159 can adopt a stable native fold with unique, aromatic side-chain packing is quite striking because residues 1-36 form an integral part of the structural core of the full-length protein.     

Intracellular heteroplasmy for disease-associated point mutations in mtDNA: implications for disease expression and evidence for mitotic segregation of heteroplasmic units of mtDNA

Studies in vitro have shown that a respiratorydeficient phenotype is expressed by cells when the proportion of mtDNA with a disease-associated mutation exceeds a threshold level, but analysis of tissues from patients with mitochondrial encephalomyopathy, lactic acidosis, and strokelike episodes (MELAS) have failed to show a consistent relationship between the degree of heteroplasmy and biochemical expression of the defect. One possible explanation for this phenomenon is that there is variation of heteroplasmy between individual cells that is not adequately reflected by the mean heteroplasmy for a tissue. We have confirmed this by study of fibroblast clones from subjects heteroplasmic for the MELAS 3243 (A G) mtDNA mutation. Similar observations were made with fibroblast clones derived from two subjects heteroplasmic for the 11778 (GA) mtDNA mutation of Leber's hereditary optic neuropathy. For the MELAS 3243 mutation, the distribution of mutant mtDNA between different cells was not randomly distributed about the mean, suggesting that selection against cells with high proportions of mutant mtDNA had occurred. To explore the way in which heteroplasmic mtDNA segregates in mitosis we followed the distribution of heteroplasmy between clones over approximately 15 generations. There was either no change or a decrease in the variance of intercellular heteroplasmy for the MELAS 3243 mutation, which is most consistent with segregation of heteroplasmic units of multiple mtDNA molecules in mitosis. After mitochondria from one of the MELAS 3243 fibroblast cultures were transferred to a mitochondrial DNA-free (⁰) cell line derived from osteosarcoma cells by cytoplast fusion, the mean level and intercellular distribution of heteroplasmy was unchanged. We interpret this as evidence that somatic segregation (rather than nuclear background or cell differentiation state) is the primary determinant of the level of heteroplasmy.     

Structural analysis of zinc substitutions in the active site of thermolysin.

Native thermolysin binds a single catalytically essential zinc ion that is tetrahedrally coordinated by three protein ligands and a water molecule. During catalysis the zinc ligation is thought to change from fourfold to fivefold. Substitution of the active-site zinc with Cd2+, Mn2+, Fe2+, and Co2+ alters the catalytic activity (Holmquist B, Vallee BL, 1974, J Biol Chem 249:4601-4607). Excess zinc inhibits the enzyme. To investigate the structural basis of these changes in activity, we have determined the structures of a series of metal-substituted thermolysins at 1.7-1.9 A resolution. The structure of the Co(2+)-substituted enzyme is shown to be very similar to that of wild type except that two solvent molecules are liganded to the metal at positions that are thought to be occupied by the two oxygens of the hydrated scissile peptide in the transition state. Thus, the enhanced activity toward some substrates of the cobalt-relative to the zinc-substituted enzyme may be due to enhanced stabilization of the transition state. The ability of Zn2+ and Co2+ to accept tetrahedral coordination in the Michaelis complex, as well as fivefold coordination in the transition state, may also contribute to their effectiveness in catalysis. The Cd(2+)- and Mn(2+)-substituted thermolysins display conformational changes that disrupt the active site to varying degrees and could explain the associated reduction of activity. The conformational changes involve not only the essential catalytic residue, Glu 143, but also concerted side-chain rotations in the adjacent residues Met 120 and Leu 144. Some of these side-chain movements are similar to adjustments that have been observed previously in association with the "hinge-bending" motion that is presumed to occur during catalysis by the zinc endoproteases. In the presence of excess zinc, a second zinc ion is observed to bind at His 231 within 3.2 A of the zinc bound to native thermolysin, explaining the inhibitory effect.     

Heteropolypeptides from hydrogen cyanide and water? Solid state15N NMR investigations

Cross-polarization magic-angle spinning¹⁵NMR spectra have been used to determine the composition of hydrogen cyanide polymers both before and after treatment with water. The unambiguous presence of secondary amide groups (as in peptide links) has been established by double-cross-polarization studies on the polymers synthesized from equimolar amounts of H¹³CN and HC¹⁵N. The NMR results are consistent with the hypothesis that the original heteropolypeptides on Earth were synthesized directly from hydrogen cyanide and water without the intervening formation of -amino acids.