Cell surface galactosyltransferase structure and function during mesenchymal cell migration

In order to more fully understand the function of surface GalTase on mesenchymal cells, anti-GalTase IgG was used to (a) examine the role of surface GalTase during mouse mesenchymal cell migration on laminin and fibronectin; (b) define the plasma membrane distribution of GalTase by indirect immunofluorescence on migrating cells; (c) quantitate the level of surface GalTase on migrating cells; and (d) determine whether GalTase is associated with the cytoskeleton.^ Results show that anti-GalTase IgG was able to inhibit migration (48-80% as compared to basal rate) when cells were migrating on laminin-containing matrices. Monovalent Fab fragments inhibited migration on laminin by 90% after 4 hours. On the other hand, anti-GalTase IgG had no effect on cells migrating on fibronectin. This illustrates the substrate specificity of GalTase mediated-migration. When anti-GalTase IgG was used to localize surface GalTase on cells migratory on laminin, the enzyme was restricted to the leading and trailing edges of the cell. Assays indicate that GalTase is elevated approximately 3-fold when cells are migrating on laminin-containing matrices as compared to migratory cells on plastic or fibronectin, or as compared to stationary cells on any substrate. Laminin appears to recruit GalTase from preexisting intracellular pools to the growing lamellipodia.^ Double-label indirect immunofluorescence studies indicate that there is an apparent co-localization between some of the surface GalTase and some actin filaments. This relationship was explored by extracting cells prelabeled with anti-GalTase IgG and quantitated by radiolabeled second antibodies. Results show that 79% of the surface GalTase is associated with the cytoskeleton (as judged by detergent insolubility) when monovalent antibodies (Fab) are used. However virtually all (80-100%) of the surface GalTase can be induced to associate with the cytoskeleton when cross-linked with bivalent antibodies. Furthermore, when cells in suspension are incubated with divalent antibodies, an additional 66% of the surface GalTase can be induced to associate with the cytoskeleton. The elevated levels of surface GalTase detectable on cells migrating on laminin also appear to be associated with the cytoskeleton.^ Several lines of evidence suggest that GalTase is associated with F-actin. Data suggest that laminin induces the expression of surface GalTase to the growing lamellipodia where it becomes associated with the cytoskeleton leading to cell spreading and migration. (Abstract shortened with permission of author.) ^

Phototaxis signaling by the membrane complex of archaeal sensory rhodopsins and their transducers

Sensory rhodopsins I and II (SRI and SRII) are visual pigment-like phototaxis receptors in the archaeon Halobacterium salinarum. The receptor proteins each consist of a single polypeptide that folds into 7 $\alpha$-helical membrane-spanning segments forming an internal pocket where the chromophore retinal is bound. They transmit signals to their tightly bound transducer proteins, HtrI and HtrII, respectively, which in turn control a phosphotransfer pathway modulating the flagellar motors. SRI-HtrI mediates attractant responses to orange-light and repellent responses to UV light, while SRII-HtrII mediates repellent response to blue light. Experiments were designed to analyze the molecular processes in the SR-Htr complexes responsible for receptor activation, which previously had been shown by our laboratory to involve proton transfer reactions of the retinylidene Schiff base in the photoactive site, transfer of signals from receptor to transducer, and signaling specificity by the receptor-transducer complex.^ Site-directed mutagenesis and laser-flash kinetic spectroscopy revealed that His-166 in SRI (i) plays a role in the proton transfers both to and from the Schiffbase, either as a structurally critical residue or possibly as a direct participant, (ii) is involved in the modulation of SIU photoreaction kinetics by HtrI, and (iii) modulates the pKa of Asp-76, an important residue in the photoactive site, through a long-distance electrostatic interaction. Computerized cell tracking and motion analysis demonstrated that (iv) His-166 is crucial in phototaxis signaling: a spectrum of substitutions either eliminate signaling or greatly perturb the activation process that produces attractant and repellent signaling states of the receptor.^ The signaling states of SRI are communicated to HtrI, whose oligomeric structure and conformational changes were investigated by engineered sulfhydryl probes. It was found that signaling by the SRI-HtrI complex involves reversible conformational changes within a preexisting HtrI dimer, which is likely accomplished through a slight winding or unwinding of the two HtrT monomers via their loose coiled coil association. To elucidate which domains of the Htr dimers confer specificity for interaction with SRI or SRII, chimeras of HtrI and HtrII were constructed. The only determinant needed for functional and specific interaction with SRI or SRII was found to be the four transmembrane segments of the HtrI or HtrII dimers, respectively. The entire cytoplasmic parts of HtrI and HtrII, which include the functionally important signaling and adaptation domains, were interchangeable.^ These observations support a model in which SRI and SRII undergo conformational changes coupled to light-induced proton transfers in their photoactive sites, and that lateral helix-helix interactions with their cognate transducers' 4-helix bundle in the membrane relay these conformational changes into different states of the Htr proteins which regulate the down-stream phosphotransfer pathway. ^

Protein-protein interaction between phototaxis receptor sensory Rhodopsin I and its transducer HtrI

The molecular complex containing the seven transmembrane helix photoreceptor S&barbelow;ensory R&barbelow;hodopsin I&barbelow; (SRI) and transducer protein HtrI (H&barbelow;alobacterial Transducer for SRI&barbelow;) mediates color-sensitive phototaxis responses in the archaeon Halobacterium salinarum. Orange light causes an attractant response by a one-photon reaction and white light (orange + UV light) a repellent response by a two-photon reaction. Three aspects of SRI-HtrI structure/function and the signal transduction pathway were explored. First, the coupling of HtrI to the photoactive site of SRI was analyzed by mutagenesis and kinetic spectroscopy. Second, SRI-HtrI mutations and suppressors were selected and characterized to elucidate the color-sensing mechanism. Third, the signal relay through the transducer-bound histidine kinase was analyzed using an in vitro reconstitution system with known and newly identified taxis components. ^ Twenty-one mutations on HtrI were introduced by site-directed mutagenesis. Several replacements of charged residues perturbed the photochemical kinetics of SRI which led to the finding of a cluster of residues at the membrane/cytoplasm interface in HtrI electrostatically coupled to the photoactive site of SRI. We found by laser-flash kinetic spectroscopy that the transducer and these residues have specific effects on the light-induced proton transfer between the retinal chromophore and the protein. ^ One of the mutations showed an unusual mutant phenotype we called “inverted” signaling, in which the cell produces a repellent response to normally attractant light. Therefore, this mutant (E56Q of HtrI) had lost the color-discrimination by the SRI-HtrI complex. We used suppressor analysis to better understand the phenotype. Certain suppressors resulted in return of attractant responses to orange light but with inversion of the normally repellent response to white light to an attractant response. To explain this and other results, we formulated the Conformational Shuttling model in which the HtrI-SRI complex is poised in a metastable equilibrium of two conformations shifted in opposite directions by orange and white light. We tested this model by behavioral analysis (computerized cell tracking and motion study) of double mutants of inverting and suppressing mutations and the results confirmed the equilibrium-shift explanation. ^ We developed an in vitro system for measuring the effect of purified transducer on the histidine-kinase CheAH that controls the flagellar motor switch. The rate of kinase autophosphorylation was stimulated >2 fold in the reconstitution of the complete signal transduction system from purified components from H. salinarum. The in vitro assay also showed that the kinase activity was reduced in the absence and in the presence of high levels of linker protein CheWH. (Abstract shortened by UMI.) ^

THE DISCHARGED PSYCHIATRIC PATIENT: POST-HOSPITAL ADJUSTMENT AND FACTORS AFFECTING REHOSPITALIZATION

Discharged psychiatric patients were studied six months post-discharge to determine those demographic, social and clinical characteristics affecting positive or negative adjustment and the degree to which the use of mental health services and medication compliance mediated the effects. With the exception of those with primary or secondary diagnoses of OBS, substance abuse or mental retardation, sixty-three psychiatric subjects between the ages of eighteen and sixty-four were chosen from all admissions into the hospital and interviewed six months after discharge using a specially designed questionnaire.^ The subjects' adjustment to community living was found to be marginal. Although not engaged in destructive activities, over half were living with their family members who supported them financially and emotionally. Most were unemployed and had been so for a long time. Others worked sporadically and frequently changed residences. Most did have substantial social ties with extended family and with friends with whom they interacted regularly, but one-fourth were socially isolated. Almost three-quarters continued to obtain regular mental health services after discharge and followed medication instructions under the supervision of their physician. The use of mental health services after discharge and the use of medication did not appear to affect the subjects' community adaption or their rate of rehospitalization.^ Forty percent of those discharged were rehospitalized by the end of the follow-up period. Four levels of risk of rehospitalization emerged. The highest risk was associated with a history of five or more prior hospitalizations, living alone, and social isolation. One third or more of the subjects expressed a need for more counseling, leisure time activities, case-manager assistance, vocational guidance, supervised housing, and placement into a transitional residential treatment program.^ Recommendations were made to enhance the ability to predict recidivism, to develop interorganizational casework management programs linking the patient and family to the community mental health system and to create computerized tracking and monitoring programs that systematically report patient treatment regimen and progress cross-sectionally and longitudinally. ^

A process evaluation of the childhood lead poisoning prevention program in Houston, Texas, 1992--1995

A process evaluation of the Houston Childhood Lead Poisoning Prevention Program, 1992-1995, was conducted. The Program's goal is to reduce lead poisoning prevalence. The study proposed to determine to what extent the Program was implemented as planned by measuring how well Program services were actually: (1) received by the intended target population; (2) delivered to children with elevated blood lead levels; (3) delivered in compliance with the Centers for Disease Control and Prevention and Program guidelines and timetables; and (4) able to reduce lead poisoning prevalence among those rescreened. Utilizing a program monitoring design, the Program's pre-collected computer records were reviewed. The study sample consisted of 820 children whose blood lead levels were above 15 micrograms per deciLiter, representing approximately 2.9% of the 28,406 screened over this period. Three blood lead levels from each participant were examined: the initial elevated result; the confirmatory result; and the next rescreen result, after the elevated confirmatory level. Results showed that the Program screened approximately 18% (28,406 of 161,569) of Houston's children under age 6 years for lead poisoning. Based on Chi-square tests of significance, results also showed that lead-poisoned participants were more likely to be younger than 3 years, male and Hispanic, compared to those not lead poisoned. The age, gender and ethnic differences observed were statistically significant (p =.01, p =.00, p =.00). Four of the six Program services: medical evaluations, rescreening, environmental inspections and confirmation, had satisfactory delivery completion rates of 71%-98%. Delivery timetable compliance rates for three of the six services examined: outreach contacts, home visits and environmental inspections were below 32%. However, dangerously elevated blood lead levels fell and lead poisoning prevalence dropped from 3.3% at initial screening to 1.2% among those rescreened, after intervention. From a public health perspective, reductions in lead poisoning prevalence are very meaningful. Based on these findings, the following are recommendations for future research: (1) integrate Program database files by utilizing a computer database management program; (2) target services at Hispanic male children under age 3 years living in the highest risk neighborhoods; (3) increase resources to: improve tracking and documentation of service delivery and provide more non-medical case management and environmental services; and (4) share the evaluation methodology/findings with the Centers for Disease Control and Prevention administrators; the implications may be relevant to other program managers conducting such assessments. ^

Effects of congressional modernization laws on public access to innovative medical device technologies

The Federal Food and Drug Administration (FDA) and the Centers for Medicare and Medicaid (CMS) play key roles in making Class III, medical devices available to the public, and they are required by law to meet statutory deadlines for applications under review. Historically, both agencies have failed to meet their respective statutory requirements. Since these failures affect patient access and may adversely impact public health, Congress has enacted several “modernization” laws. However, the effectiveness of these modernization laws has not been adequately studied or established for Class III medical devices. ^ The aim of this research study was, therefore, to analyze how these modernization laws may have affected public access to medical devices. Two questions were addressed: (1) How have the FDA modernization laws affected the time to approval for medical device premarket approval applications (PMAs)? (2) How has the CMS modernization law affected the time to approval for national coverage decisions (NCDs)? The data for this research study were collected from publicly available databases for the period January 1, 1995, through December 31, 2008. These dates were selected to ensure that a sufficient period of time was captured to measure pre- and post-modernization effects on time to approval. All records containing original PMAs were obtained from the FDA database, and all records containing NCDs were obtained from the CMS database. Source documents, including FDA premarket approval letters and CMS national coverage decision memoranda, were reviewed to obtain additional data not found in the search results. Analyses were conducted to determine the effects of the pre- and post-modernization laws on time to approval. Secondary analyses of FDA subcategories were conducted to uncover any causal factors that might explain differences in time to approval and to compare with the primary trends. The primary analysis showed that the FDA modernization laws of 1997 and 2002 initially reduced PMA time to approval; after the 2002 modernization law, the time to approval began increasing and continued to increase through December 2008. The non-combined, subcategory approval trends were similar to the primary analysis trends. The combined, subcategory analysis showed no clear trends with the exception of non-implantable devices, for which time to approval trended down after 1997. The CMS modernization law of 2003 reduced NCD time to approval, a trend that continued through December 2008. This study also showed that approximately 86% of PMA devices do not receive NCDs. ^ As a result of this research study, recommendations are offered to help resolve statutory non-compliance and access issues, as follows: (1) Authorities should examine underlying causal factors for the observed trends; (2) Process improvements should be made to better coordinate FDA and CMS activities to include sharing data, reducing duplication, and establishing clear criteria for “safe and effective” and “reasonable and necessary”; (3) A common identifier should be established to allow tracking and trending of applications between FDA and CMS databases; (4) Statutory requirements may need to be revised; and (5) An investigation should be undertaken to determine why NCDs are not issued for the majority of PMAs. Any process improvements should be made without creating additional safety risks and adversely impacting public health. Finally, additional studies are needed to fully characterize and better understand the trends identified in this research study.^

Cardiovascular diseases risk factors during the first year of life

The pattern of change in cardiovascular risk factors, blood pressure (SBP and DBP) and plasma total cholesterol (TC), over time, their tracking and their relation to anthropometric measurements during the first year of life were investigated. Also, the effect of breast feeding on TC and the relationship of blood pressure measurements and family history of CVD risk factors were examined. One hundred five newborn term, healthy infants who were seen at a pediatric clinic in The Woodlands, Texas were followed longitudinally from 2 weeks to 1 year of age. TC, blood pressure, weight and length of the infants were measured at age 2 weeks, and again at 2, 4, 6, 9 and 12 months. In addition, family history, maternal and paternal, of CVD risk factors was obtained. Data analyses included only 40 infants who completed one year of follow up.^ At 2 weeks of age, the median value for TC was 23 mg/dl higher for females than for males. This difference disappeared as infants got older. For males, most of the increase in TC median levels, from 114 to 137 mg/dl, occurred between the ages of 2 weeks and 2 months, whereas for the female group, TC levels increased moderately, about 10 mg/dl, between 9 and 12 months of age. Tracking of TC was examined by using Spearman's correlation analysis. There were strong correlations between measurements taken as early as 2 weeks of age with later measurements. These correlations were stronger and more significant for males than for females (for males, r varied between 0.51 to 0.70, whereas for females, r varied between 0.11 to 0.70). The association of body measurements with TC is no more than modest and is closer for female infants than for male infants. Analysis, also, showed that infants who received breast milk had a TC mean value 47 mg/dl higher than that for infants who received formula milk only during the period of breast feeding and this difference disappeared by age 12 months.^ In both genders, most of the increase in blood pressure (about 10-15 mmHg in both SBP and DBP) occurred during the first 4 months of life. Most of the increase for male infants occurred during the first 2 months of life, while for females, the increase in SBP and DBP was between the age of 2 and 4 months. Neither SBP nor DBP track well during the first year of life and most of the correlations between measurements at different ages were not significant for either gender. The cross-sectional relationship of blood pressure measurements and selected body measurements was assessed. For females, only at age of 12 months did DBP have positive and significant correlations with weight, length and Quetelet index (r = 0.57, 0.60 and 0.57, respectively). There were no significant correlations between blood pressure and body measurements for males. Finally, analysis showed that maternal history of CV risk factors was significantly related to SBP in the female infant group, but not for males. For DBP, neither maternal nor paternal history was related. ^

Tracking of plasma lipids and lipoproteins, within-person and year-to-year variability in plasma cholesterol levels among participants from age 8 to 18 in Project HeartBeat!

Coronary heart disease remains the leading cause of death in the United States and increased blood cholesterol level has been found to be a major risk factor with roots in childhood. Tracking of cholesterol, i.e., the tendency to maintain a particular cholesterol level relative to the rest of the population, and variability in blood lipid levels with increase in age have implications for cholesterol screening and assessment of lipid levels in children for possible prevention of further rise to prevent adulthood heart disease. In this study the pattern of change in plasma lipids, over time, and their tracking were investigated. Also, within-person variance and retest reliability defined as the square root of within-person variance for plasma total cholesterol, HDL-cholesterol, LDL-cholesterol, and triglycerides and their relation to age, sex and body mass index among participants from age 8 to 18 years were investigated. ^ In Project HeartBeat!, 678 healthy children aged 8, 11 and 14 years at baseline were enrolled and examined at 4-monthly intervals for up to 4 years. We examined the relationship between repeated observations by Pearson's correlations. Age- and sex-specific quintiles were calculated and the probability of participants to remain in the uppermost quintile of their respective distribution was evaluated with life table methods. Plasma total cholesterol, HDL-C and LDL-C at baseline were strongly and significantly correlated with measurements at subsequent visits across the sex and age groups. Plasma triglyceride at baseline was also significantly correlated with subsequent measurements but less strongly than was the case for other plasma lipids. The probability to remain in the upper quintile was also high (60 to 70%) for plasma total cholesterol, HDL-C and LDL-C. ^ We used a mixed longitudinal, or synthetic cohort design with continuous observations from age 8 to 18 years to estimate within person variance of plasma total cholesterol, HDL-C, LDL-C and triglycerides. A total of 5809 measurements were available for both cholesterol and triglycerides. A multilevel linear model was used. Within-person variance among repeated measures over up to four years of follow-up was estimated for total cholesterol, HDL-C, LDL-C and triglycerides separately. The relationship of within-person and inter-individual variance with age, sex, and body mass index was evaluated. Likelihood ratio tests were conducted by calculating the deviation of −2log (likelihood) within the basic model and alternative models. The square root of within-person variance provided the retest reliability (within person standard deviation) for plasma total cholesterol, HDL-C, LDL-C and triglycerides. We found 13.6 percent retest reliability for plasma cholesterol, 6.1 percent for HDL-cholesterol, 11.9 percent for LDL-cholesterol and 32.4 percent for triglycerides. Retest reliability of plasma lipids was significantly related with age and body mass index. It increased with increase in body mass index and age. These findings have implications for screening guidelines, as participants in the uppermost quintile tended to maintain their status in each of the age groups during a four-year follow-up. The magnitude of within-person variability of plasma lipids influences the ability to classify children into risk categories recommended by the National Cholesterol Education Program. ^