Sequencing Alpha-1 MZ Individuals Shows Frequent Biallelic Mutations.

RATIONALE: Individuals with a single Z mutation in the SERPINA1 gene that codes for alpha-1 antitrypsin (AAT) are at increased risk for COPD if they have ever-smoked. Whether additional variants alter the risk for COPD in this population remains unknown. OBJECTIVES: To determine whether additional SERPINA1 variants impact COPD development in a previously identified MZ (carrier) cohort. METHODS: Individuals with prior MZ results and AAT serum level <16uM were recruited from the Alpha-1 Coded Testing study and Alpha-1 Foundation Research Registry. Participants completed smoking history, demographics, and COPD Severity Score (Range 0-33) using REDCap data capture. At-home finger-stick tests were performed for next generation sequencing (NGS) at the Biocerna LLC laboratory. A genetic counselor reviewed records and interviewed participants with additional variants by NGS. A Wilcoxon Rank Sum test was used to assess correlation between variants and the COPD severity score. RESULTS: A second SERPINA1 variant of known or possible significance was identified in 6 (5.8%) participants. One each of ZZ, SZ, FZ, ZSmunich, ZM2obernburg, and Z/c.922G>T genotypes were identified. ZZ, SZ, and FZ are known pathogenic genotypes. Smunich is a likely pathogenic variant. M2obernburg and c.922G>T are variants of uncertain significance. The ZZ individual was on augmentation therapy when determined MZ by protease inhibitor (Pi) phenotyping; the others had limited targeted genotyping with MZ results. These six participants with biallelic variants had positive COPD severity scores >1. Presence of additional variants was not significantly associated with COPD symptoms in this small sample size. CONCLUSIONS: Some diagnosed MZ individuals instead have biallelic variants. Larger studies are needed to determine COPD-risk liability of variants. Accurate diagnosis impacts medical management and familial risk assessment. Pi phenotyping can be confounded by augmentation therapy and liver transplantation. Because a normal M allele may be reported in the absence of tested mutation(s) in AATD genotyping, clinicians should consider clinical circumstances and laboratory methods when selecting and interpreting AATD tests. Advanced testing, including NGS, may be beneficial for select individuals with prior MZ results. CLINICAL TRIAL REGISTRATION: This study was registered with clinicaltrials.gov (NCT NCT02810327).

Genetically engineered large animal model for studying cone photoreceptor survival and degeneration in retinitis pigmentosa.

Patients with retinitis pigmentosa (RP) typically develop night blindness early in life due to loss of rod photoreceptors. The remaining cone photoreceptors are the mainstay of their vision; however, over years or decades, these cones slowly degenerate, leading to blindness. We created transgenic pigs that express a mutated rhodopsin gene (Pro347Leu). Like RP patients with the same mutation, these pigs have early and severe rod loss; initially their cones are relatively spared, but these surviving cones slowly degenerate. By age 20 months, there is only a single layer of morphologically abnormal cones and the cone electroretinogram is markedly reduced. Given the strong similarities in phenotype to that of RP patients, these transgenic pigs will provide a large animal model for study of the protracted phase of cone degeneration found in RP and for preclinical treatment trials.

Localization of regions of the Torpedo californica nicotinic acetylcholine receptor labeled with an aryl azide derivative of phosphatidylserine.

A photoactivatable analog of phosphatidylserine, 125I-labeled 4-azidosalicylic acid-phosphatidylserine (125I-ASA-PS) (Blanton, M. and Wang, H.H. (1990) Biochemistry 29, 1186-1194) was used to label the nicotinic acetylcholine receptor. The photoactivatable group of 125I-ASA-PS is attached directly to the phospholipid head group making it an excellent probe of regions of the AchR structure in contact with the negatively-charged head group of phosphatidylserine. The 'binding domains' were localized by chemically cleaving the labeled receptor with cyanogen bromide (CNBr), separating the generated peptides by reverse-phase HPLC, and N-terminal sequence analysis of radiolabeled material. CNBr fragments containing flanking regions of the transmembrane spanning region M4 as well as within M3 were identified within HPLC separated radiolabeled material. The results suggest a topological arrangement of the transmembrane helices in which the hydrophobic faces of M3 and M4 form the boundary of the receptor complex in contact with the lipid bilayer.

Identifying the cholesterol binding domain in the nicotinic acetylcholine receptor with [125I]azido-cholesterol.

A novel photoreactive analog of cholesterol, 3alpha-(4-azido-3-[125I]iodosalicylic)-cholest-5-ene ([125I]azido-cholesterol), was used to label both native acetylcholine receptor (AChR)-rich membranes from Torpedo californica and affinity-purified Torpedo AChRs reconstituted into lipid vesicles. In both cases all four AChR subunits incorporated [125I]azido-cholesterol on an equal molar basis and neither the pattern nor the extent of labeling was affected by the presence of the agonist carbamylcholine. Labeled regions in each of the AChR subunits were initially mapped by Staphylococcus aureus V8 protease digestion to large fragments which contain the AChR transmembrane segments. Sites of [125I]azido-cholesterol incorporation were further mapped by exhaustive tryptic digestion of the V8 protease subunit fragments alphaV8-20 (alphaSer-173-Glu-338), alphaV8-10 (alphaAsn-339-Gly-439), and gammaV8-14 (gammaLeu-373-Pro-489). The digests were separated by reverse-phase high-performance liquid chromatography and labeled peptides identified by amino-terminal sequence analysis. [125I]Azido-cholesterol labeling was localized to peptides that contain almost exclusively the alpha-M4, alpha-M1 and gamma-M4 membrane spanning segments. These results establish that the binding domain for cholesterol is at the lipid-protein interface of the AChR.

Identification and characterization of membrane-associated polypeptides in Torpedo nicotinic acetylcholine receptor-rich membranes by hydrophobic photolabeling.

To identify membrane-associated polypeptides present in Torpedo nicotinic acetylcholine receptor (AChR)-rich membranes, we used hydrophobic photolabeling with [(3)H]diazofluorene ([(3)H]DAF) and 1-azidopyrene (1-AP) to tag the membrane proteins which were then identified by amino-terminal sequence analysis of labeled fragments isolated from proteolytic digests by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by reverse-phase high-performance liquid chromatography. In addition to AChR subunits, identified polypeptides include the 95 kDa alpha-subunit of the (Na(+)+K(+))-ATPase, the 89 kDa voltage-gated chloride channel (CLC-0), the 105 kDa SITS-binding protein, and 32 and 34 kDa polypeptides identified as Torpedo homologues of the mitochondrial membrane ATP/ADP carrier protein and the voltage-dependent anion channel (VDAC), respectively. Further, individual amino acids that reacted with [(3)H]DAF and therefore likely to be in contact with lipid were identified in the transmembrane segment M3 of the alpha-subunit of the (Na(+)+K(+))-ATPase and in a putative transmembrane beta-strand in VDAC. Collectively these results demonstrate that [(3)H]DAF/1-AP photolabeling provides an effective method for tagging the membrane-associated segments of polypeptides in a way that makes it easy to isolate the labeled polypeptide or polypeptide fragments by fluorescence and then to identify amino acids at the lipid-protein interface by (3)H release.

Alpha-conotoxins.

alpha-Conotoxins (alpha-CgTxs) are a family of Cys-enriched peptides found in several marine snails from the genus Conus. These small peptides behave pharmacologically as competitive antagonists of the nicotinic acetylcholine receptor (AChR). The data indicate that (1) alpha-CgTxs are able to discriminate between muscle- and neuronal-type AChRs and even among distinct AChR subtypes; (2) the binding sites for alpha-CgTxs are located, like other cholinergic ligands, at the interface of alpha and non-alpha subunits (gamma, delta, and epsilon for the muscle-type AChR, and beta for several neuronal-type AChRs); (3) some alpha-CgTxs differentiate the high- from the low-affinity binding site found on either alpha/non-alpha subunit interface; and that (4) specific residues in the cholinergic binding site are energetically coupled with their corresponding pairs in the toxin stabilizing the alpha-CgTx-AChR complex. The alpha-CgTxs have proven to be excellent probes for studying the structure and function of the AChR family.

Ethnic differences in blood pressure, pulse rate, and related characteristics in young adults. The CARDIA study.

This study examined ethnic differences in blood pressure and pulse rate in young adults to see whether the differences, if they exist, can be explained by differences in body mass index, lifestyle, psychological, and socioeconomic characteristics. Data used were from the baseline examination of the Coronary Artery Risk Development in (Young) Adults Study (CARDIA). CARDIA is a longitudinal study of lifestyle and evolution of cardiovascular disease risk factors in 5,116 young adults, black and white, men and women, aged 18-30 years, of varying socioeconomic status. Young black adults had higher mean systolic blood pressure and slightly higher mean diastolic blood pressure than young white adults. For both men and women, the blood pressure differences between blacks and whites tended to be greater for the age group 25-30 than for the age group 18-24 years. Among the variables studied, body mass index, duration of exercise on the treadmill, number of cigarettes smoked per day, and number of alcoholic drinks per week were consistently associated with blood pressure. The blood pressure differences were greatly reduced after adjusting for these variables. Black participants had lower mean pulse rate than white participants. The differences tended to be greater for the age group 18-24 than for the age group 25-30 years. Among the variables studied, only duration on treadmill and number of cigarettes smoked per day were consistently correlated with pulse rate. With adjustment for duration on treadmill, the differences in pulse rate increased. These results suggest that differences in ethnic pattern of blood pressures and pulse rate with age may be due in part to obesity, physical fitness, alcohol consumption, and cigarette smoking.

Morphological differentiation of distinct neuronal classes in embryonic turtle cerebral cortex.

As a starting point for understanding the development of the cerebral cortex in reptiles and for determining how reptilian cortical development compares to that in other vertebrate classes, we studied the appearance and morphological differentiation of cerebral cortical neurons in embryonic turtles. 3H-thymidine birthdate labeling and focal injections of horseradish peroxidase (HRP) in in vitro cortical slices revealed that replicating cells occupy the outer ventricular zone, and subsequently migrate to the ventricular surface where they divide. Postmitotic neurons begin differentiating and elaborating neurites while migrating back through the ventricular zone. On their arrival at the top of the ventricular zone, pyramidal and nonpyramidal neurons can be distinguished morphologically. Cells with multipolar apical dendritic tufts ascending in the marginal zone resemble immature pyramidal neurons. Neurons morphologically similar to these early pyramidal cells were retrogradely labeled by injections of the lipophilic tracer 1,1-dioctadecyl-3,3,3',3'-tetramethyl indocarbocyanine perchlorate (diI) in a known pyramidal cell target, the thalamus. Nonpyramidal neurons, resembling Cajal-Retzius cells, had horizontally oriented long axons and dendrites coursing in the plexiform primordium, the future marginal zone. With further development morphological differences between cell types became accentuated, and pyramidal cell somata were segregated into a single cellular layer flanked by zones containing predominantly nonpyramidal cells. Axon elaboration occurred early in embryonic development, as pyramidal cells sent axonal branches to the septum, thalamus, and cortical targets soon after their generation, and the intracortical axonal plexus became increasingly dense during embryonic life. Over a similar time course the distribution of projecting neurons labeled by thalamic diI injections changed from an initial homogeneous distribution to a preferential location in the superficial half of the cellular layer. Results from this study demonstrate several features of cortical differentiation that are conserved in reptiles and mammals, including similar early morphological differentiation events, the early distinction of principal cell types, and the parallel development of pyramidal and nonpyramidal neurons. The context in which these similar developmental events occur, however, differs profoundly in reptiles and mammals, with differences in the timing and location of neurite elaboration and differences in the appearance and architectonic organization of the cortex. Comparison of cortical developmental patterns between reptiles and mammals shows that similar functional cortical circuits with balanced excitation and inhibition can emerge in diverse cortical structures.

Appearance of putative amino acid neurotransmitters during differentiation of neurons in embryonic turtle cerebral cortex.

Pyramidal and nonpyramidal neurons can be recognized early in the development of the cerebral cortex in both reptiles and mammals, and the neurotransmitters likely utilized by these cells, glutamate and gamma-aminobutyric acid, or GABA, have been suggested to play critical developmental roles. Information concerning the timing and topography of neurotransmitter synthesis by specific classes of cortical neurons is important for understanding developmental roles of neurotransmitters and for identifying potential zones of neurotransmitter action in the developing brain. We therefore analyzed the appearance of GABA and glutamate in the cerebral cortex of embryonic turtles using polyclonal antisera raised against GABA and glutamate. Neuronal subtypes become immunoreactive for the putative amino acid neurotransmitters GABA and glutamate early in the embryonic development of turtle cerebral cortex, with nonpyramidal cells immunoreactive for GABA and pyramidal cells immunoreactive for glutamate. The results of controls strongly suggest that the immunocytochemical staining in tissue sections by the GABA and glutamate antisera corresponds to fixed endogenous GABA and glutamate. Horizontally oriented cells in the early marginal zone (stages 15-16) that are GABA-immunoreactive (GABA-IR) resemble nonpyramidal cells in morphology and distribution. GABA-IR neurons exhibit increasingly diverse morphologies and become distributed in all cortical layers as the cortex matures. Glutamate-immunoreactive (Glu-IR) cells dominate the cellular layer throughout development and are also common in the subcellular layer at early stages, a distribution like that of pyramidal neurons and distinct from that of GABA-IR nonpyramidal cells. The early organization of embryonic turtle cortex in reptiles resembles that of embryonic mammalian cortex, and the immunocytochemical results underline several shared as well as distinguishing features. Early GABA-IR nonpyramidal cells flank the developing cortical plate, composed primarily of pyramidal cells, shown here to be Glu-IR. The earliest GABA-IR cells in turtles likely correspond to Cajal-Retzius cells, a ubiquitous and precocious cell type in vertebrate cortex. Glutamate-IR projection neurons in vertebrates may also be related. The distinctly different topographies of GABA and glutamate containing cells in reptiles and mammals indicate that even if the basic amino acid transmitter-containing cell types are conserved in higher vertebrates, the local interactions mediated by these transmitters may differ. The potential role of GABA and glutamate in nonsynaptic interactions early in cortical development is reinforced by the precocious expression of these neurotransmitters in turtles, well before they are required for synaptic transmission.(ABSTRACT TRUNCATED AT 400 WORDS)

Evidence for the inhibitory neurotransmitter gamma-aminobutyric acid in aspiny and sparsely spiny nonpyramidal neurons of the turtle dorsal cortex.

In order to learn more about the anatomical substrate for gamma-aminobutyric acid (GABA)-mediated inhibition in cortical structures, the intrinsic neuronal organization of turtle dorsal cortex was studied by using Golgi impregnation, immunohistochemical localization of GABA and its synthetic enzyme glutamic acid decarboxylase (GAD), and histochemical localization of the presynaptic GABA-degrading enzyme GABA-transaminase (GABA-T). GABAergic markers are found in neurons identical in morphology and distribution to Golgi-impregnated aspiny and sparsely spiny nonpyramidal neurons with locally arborizing axons and appear to label most if not all of the nonpyramidal neurons. In addition, the GABAergic markers are found in punctate structures in a distribution characteristic of presumed inhibitory terminals. The spine-laden pyramidal neurons, the principal projecting cell type in the dorsal cortex, are devoid of labelling for GABAergic markers but are surrounded by presumed GABAergic terminals. The data complement previous physiological and ultrastructural studies that implicate aspiny and sparsely spiny nonpyramidal neurons as mediators of intrinsic inhibition of pyramidal neurons in turtle cortex. The results also suggest similarities in the functional organization of intrinsic inhibitory elements in turtle and mammalian cortex.

The effect of donor-specific transfusions on rat heart allograft survival.

A rat heart allograft model employing donor-specific transfusions (DSTs) was used to investigate several questions relevant to their clinical usage. The effects of varying blood storage duration (stored vs. fresh), number and timing of DSTs as well as use of concomitant azathioprine and cyclosporine (CsA) were assessed in terms of allograft survival and recipient sensitization. A comparison of stored and fresh-blood DSTs revealed that blood stored for up to 5 weeks was as effective as fresh blood and that a 2-week storage was optimal. Increased storage appeared to be associated with decreased sensitization. Multiple DSTs were more effective than a single DST and the peak effect appeared after six. Transfusions given at the time of transplantation were ineffective. The addition of concomitant (preoperative) azathioprine or CsA resulted in a further decrease in sensitization but also resulted in a dose-dependent diminution of the transfusion effect.

Whole cell recording from neurons in slices of reptilian and mammalian cerebral cortex.

We describe methods for obtaining stable, whole-cell recordings from neurons in brain hemispheres from turtles and in brain slices from rats and turtles. Synaptic currents and membrane properties of central neurons can be studied in voltage and current clamp in cells maintained within their endogenous synaptic circuits. The methods described here are compatible with unmodified dissecting microscopes and recording chambers, and with brain slices of standard thickness (400-500 microns).

Managed care and low-income populations: recent state experiences.

This DataWatch examines the relationship between managed care enrollment and access to care for low-income adults with Medicaid and compares their experience with that of low-income, privately insured managed care enrollees. Medicaid managed care enrollees are more likely than low-income, privately insured managed care enrollees to be poorer, have health problems, and experience access problems. Compared with low-income populations in fee-for-service care, managed care enrollees, whether in Medicaid or privately insured, are not appreciably different in having a usual source of care, having a regular provider, or emergency room use but report more problems in obtaining care and are more likely to be dissatisfied with their health plans.

5-hydroxytryptamine interaction with the nicotinic acetylcholine receptor.

The present study examines the interaction of the neurotransmitter 5-hydroxytryptamine (5-HT) with muscle-type nicotinic acetylcholine receptors. 5-HT inhibits the initial rate of [125I]alpha-bungarotoxin binding to Torpedo acetylcholine receptor membranes (IC(50)=8.5+/-0.32 mM) and [3H]5-HT can be photoincorporated into acetylcholine receptor subunits, with labeling of the alpha-subunit inhibitable by both agonists and competitive antagonists. Within the agonist-binding domain, [3H]5-HT photoincorporates into alphaTyr(190), alphaCys(192) and alphaCys(193). Functional studies using the human clonal cell line TE671/RD, show that 5-HT is a weak inhibitor (IC(50)=1.55+/-0.25 mM) of acetylcholine receptor activity. In this regard, agonist-response profiles in the absence and presence of 5-HT indicate a noncompetitive mode of inhibition. In addition, 5-HT displaces high affinity [3H]thienylcyclohexylpiperidine binding to the desensitized Torpedo acetylcholine receptor channel (IC(50)=1.61+/-0.07 mM). Collectively, these results indicate that 5-HT interacts weakly with the agonist recognition site and inhibits receptor function noncompetitively by binding to the acetylcholine receptor channel.

Neighborhood environment and opportunity to use cocaine and other drugs in late childhood and early adolescence.

We hypothesized that neighborhood disadvantage might function as a determinant of "exposure opportunity', an intermediate step on a path toward starting to use drugs illicitly. Testing this hypothesis, we analyzed self-report data gathered in 1992 by means of confidential interviews with 1416 urban-dwelling middle-school participants in a longitudinal field study. Within this epidemiologic sample, 50 youths said that someone actively had offered them a chance to take cocaine or smoke crack; tobacco had been offered to 395 youths; alcohol to 429 youths. Using multiple logistic regression to hold constant grade, sex, minority status, and peer drug use, we found a moderately potent association between neighborhood disadvantage and exposure to cocaine: youths living in the most disadvantaged neighborhoods (highest tertile) were an estimated 5.6 times more likely to have been offered cocaine, as compared to those in relatively advantaged neighborhoods (P = 0.001). By comparison, there were weaker but statistically significant associations involving tobacco exposure opportunity (odds ratio, OR = 1.7, P = 0.004) and alcohol exposure opportunity (OR = 1.9, P = 0.0005). Future research will clarify the etiologic significance of neighborhood disadvantage in pathways leading toward illicit drug use.

Race, ethnicity, and the health care system: public perceptions and experiences.

To assess the public's perceptions and attitudes about racial and ethnic differences in health care, the Kaiser Family Foundation surveyed a nationally representative sample of 3,884 whites, African Americans, and Latinos in 1999. The survey found that the majority of Americans are uninformed about health care disparities--many were unaware that blacks fare worse than whites on measures such as infant mortality and life expectancy, and that Latinos are less likely than whites to have health insurance. Views on whether the health system treats people equally were strikingly different by race. For example, most minority Americans perceive that they get lower quality care than whites, but most whites think otherwise. Nonetheless, more minority Americans were concerned about the cost of care than racial barriers. Efforts to eliminate disparities will need to improve public awareness of the problems as well as address racial and financial barriers to care.

Conducting an assessment of health needs and resources in a racial/ethnic minority community.

This article examines strategies and methodologic issues for researchers to consider when conducting community-based research within a racial/ethnic minority community. Members of minority communities have considerable skepticism about the health care system and researchers who work under its auspices. To facilitate quality research, it is necessary to build a mutually beneficial partnership between the community and researchers. Suggested strategies for accomplishing this goal, such as seeking out information on the social and political forces shaping the community and developing the community's capacity to undertake research of this type, are described. Methodologic issues include the importance of community input in defining the minority population group and its leadership, the benefits and limitations of conducting comparative analysis, and the need for measurement tools and techniques that are culturally and socially appropriate. Minority and nonminority researchers must make a concerted effort to gain knowledge of and respect for a community whose culture, values, and beliefs may differ from their own.

Race/ethnicity, the social environment, and health.

International and national research has documented the relations between socio-economic conditions and health. Nonetheless, racial/ethnic group comparisons of health indices frequently are presented in the United States without stratifying or adjusting for socio-economic conditions that could affect interpretation of the data. This paper examines how racial/ethnic group identifiers have been used in past research. While some studies assume biologic differences; others presume that race/ethnicity is a proxy for socio-economic race factors. One consequence of these presumptions has been an underdevelopment of knowledge about racial/ethnic minority populations that could help shape public policies and preventive interventions to reduce disparities in health. Findings from studies that examine the influence of both race and social class on health are reviewed in an effort to clarify the state-of-knowledge. Although the findings vary for particular health indices, the studies provide considerable evidence that socio-economic conditions are a powerful, although not necessarily exclusive, explanatory variable for racial disparities in health. The findings are used as the basis for encouraging more theoretically grounded and methodologically rigorous research rather than avoiding an assessment of the influence of race/ethnicity on health.

Black-white differences in alcohol use by women: Baltimore survey findings.

Although black women suffer disproportionately from alcohol-related illnesses and causes of death, little is known about the extent to which poorer outcomes are a function of differences in drinking, the use of health services, or some combination of these factors. This study, using interview data obtained in the Baltimore Epidemiologic Catchment Area household survey, compares racial differences in alcohol use and abuse among a sample of 2,100 women. After controlling for differences in sociodemographic characteristics, black women were found to be at no greater risk than whites for heavy drinking or for suffering from alcohol abuse or dependence. Racial differences, however, were observed in heavy drinking by years of education. A similar percentage of black women and white women who had not completed high school were heavy drinkers, but black women with 12 or more years of education were less likely to be heavy drinkers than whites with comparable education. These findings raise questions about the extent to which differences in drinking contribute to the poorer alcohol-related health outcomes of black women in Baltimore. Additionally, the finding that education was inversely related to heavy drinking among black women may be helpful in shaping early alcohol abuse intervention and treatment services that target black women.

Rural and urban hospital closures, 1985-1988: operating and environmental characteristics that affect risk.

Due to congressional concern that rural hospitals were particularly disadvantaged by Medicare's Prospective Payment System, the U.S. General Accounting Office investigated the role of Medicare and other factors in hospitals' risk of closure. This paper reports on the findings of that study, which compared the risk of closure among urban and rural hospitals during 1985 to 1988, the period after implementation of PPS. When hospital operating and environmental characteristics were held constant, the odds of closure in rural and urban areas differed significantly only for private nonprofit hospitals. Although a number of factors were associated with hospitals' higher risk of closure, we did not find evidence that Medicare was a major factor associated with financial distress or closure during the 1985 to 1988 period.