Levels of complement factor H-related 4 protein do not influence susceptibility to age-related macular degeneration or its course of progression.

Dysregulation of the alternative pathway (AP) of the complement system is a significant contributor to age-related macular degeneration (AMD), a primary cause of irreversible vision loss worldwide. Here, we assess the contribution of the liver-produced complement factor H-related 4 protein (FHR-4) to AMD initiation and course of progression. We show that FHR-4 variation in plasma and at the primary location of AMD-associated pathology, the retinal pigment epithelium/Bruch's membrane/choroid interface, is entirely explained by three independent quantitative trait loci (QTL). Using two distinct cohorts composed of a combined 14,965 controls and 20,741 cases, we ascertain that independent QTLs for FHR-4 are distinct from variants causally associated with AMD, and that FHR-4 variation is not independently associated with disease. Additionally, FHR-4 does not appear to influence AMD progression course among patients with disease driven predominantly by AP dysregulation. Modulation of FHR-4 is therefore unlikely to be an effective therapeutic strategy for AMD.

Microstructure and point defects in CdTe nanowires for photovoltaic applications.

Defects in Au-catalysed CdTe nanowires vapour-liquid-solid-grown on polycrystalline underlayers have been critically evaluated. Their low-temperature photoluminescence spectra were dominated by excitonic emission with rarely observed above-gap emission also being recorded. While acceptor bound exciton lines due to monovalent metallic impurities (Ag, Cu or Na) were seen, only deeper, donor-acceptor-pair emission could be attributed to the Au contamination that is expected from the catalyst. Annealing under nitrogen acted to enhance the single crystal-like PL emission, whilst oxidizing and reducing anneals of the type that is used in solar cell device processing caused it to degrade. The incidence of stacking faults, polytypes and twins was related only to the growth axes of the wires (<111> 50%, <112> 30% and <110> 20%), and was not influenced by annealing. The potential electrical activity of the point and extended defects, and the suitability of these nanowire materials (including processing steps) for solar cell applications, is discussed. Overall they have a quality that is superior to that of thin polycrystalline films, although questions remain about recombination due to Au.

Glasshouse vs field experiments: do they yield ecologically similar results for assessing N impacts on peat mosses?

• Peat bogs have accumulated more atmospheric carbon (C) than any other terrestrial ecosystem today. Most of this C is associated with peat moss (Sphagnum) litter. Atmospheric nitrogen (N) deposition can decrease Sphagnum production, compromising the C sequestration capacity of peat bogs. The mechanisms underlying the reduced production are uncertain, necessitating multifactorial experiments. • We investigated whether glasshouse experiments are reliable proxies for field experiments for assessing interactions between N deposition and environment as controls on Sphagnum N concentration and production. We performed a meta-analysis over 115 glasshouse experiments and 107 field experiments. • We found that glasshouse and field experiments gave similar qualitative and quantitative estimates of changes in Sphagnum N concentration in response to N application. However, glasshouse-based estimates of changes in production--even qualitative assessments-- diverged from field experiments owing to a stronger N effect on production response in absence of vascular plants in the glasshouse, and a weaker N effect on production response in presence of vascular plants compared to field experiments. • Thus, although we need glasshouse experiments to study how interacting environmental factors affect the response of Sphagnum to increased N deposition, we need field experiments to properly quantify these effects.

Climatic modifiers of the response to nitrogen deposition in peat-forming Sphagnum mosses: a meta-analysis.

Peatlands in the northern hemisphere have accumulated more atmospheric carbon (C) during the Holocene than any other terrestrial ecosystem, making peatlands long-term C sinks of global importance. Projected increases in nitrogen (N) deposition and temperature make future accumulation rates uncertain. Here, we assessed the impact of N deposition on peatland C sequestration potential by investigating the effects of experimental N addition on Sphagnum moss. We employed meta-regressions to the results of 107 field experiments, accounting for sampling dependence in the data. We found that high N loading (comprising N application rate, experiment duration, background N deposition) depressed Sphagnum production relative to untreated controls. The interactive effects of presence of competitive vascular plants and high tissue N concentrations indicated intensified biotic interactions and altered nutrient stochiometry as mechanisms underlying the detrimental N effects. Importantly, a higher summer temperature (mean for July) and increased annual precipitation intensified the negative effects of N. The temperature effect was comparable to an experimental application of almost 4 g N m(-2) yr(-1) for each 1°C increase. Our results indicate that current rates of N deposition in a warmer environment will strongly inhibit C sequestration by Sphagnum-dominated vegetation.

Regulation of ZAP-70 intracellular localization: visualization with the green fluorescent protein.

To investigate the cellular dynamics of ZAP-70, we have studied the distribution and regulation of its intracellular location using a ZAP-70 green fluorescent protein chimera. Initial experiments in epithelial cells indicated that ZAP-70 is diffusely located throughout the quiescent cell, and accumulates at the plasma membrane upon cellular activation, a phenotype enhanced by the coexpression of Lck and the initiation of ZAP-70 kinase activity. Subsequent studies in T cells confirmed this phenotype. Intriguingly, a large amount of ZAP-70, both chimeric and endogenous, resides in the nucleus of quiescent and activated cells. Nuclear ZAP-70 becomes tyrosine phosphorylated upon stimulation via the T cell receptor, indicating that it may have an important biologic function.

Tissue plasminogen activator induction in Purkinje neurons after cerebellar motor learning.

The cerebellar cortex is implicated in the learning of complex motor skills. This learning may require synaptic remodeling of Purkinje cell inputs. An extracellular serine protease, tissue plasminogen activator (tPA), is involved in remodeling various nonneural tissues and is associated with developing and regenerating neurons. In situ hybridization showed that expression of tPA messenger RNA was increased in the Purkinje neurons of rats within an hour of their being trained for a complex motor task. Antibody to tPA also showed the induction of tPA protein associated with cerebellar Purkinje cells. Thus, the induction of tPA during motor learning may play a role in activity-dependent synaptic plasticity.

Data on dopant characteristics and band alignment of CdTe cells with and without a ZnO highly-resistive-transparent buffer layer.

Photovoltaic enhancement of cadmium telluride (CdTe) thin film solar cells using a 50 nm thick, atomic-layer-deposited zinc oxide (ZnO) buffer film was reported in "Enhancement of the photocurrent and efficiency of CdTe solar cells suppressing the front contact reflection using a highly-resistive ZnO buffer layer" (Kartopu et al., 2019) [1]. Data presented here are the dopant profiles of two solar cells prepared side-by-side, one with and one without the ZnO highly resistive transparent (HRT) buffer, which displayed an open-circuit potential (Voc) difference of 25 mV (in favor of the no-buffer device), as well as their simulated device data. The concentration of absorber dopant atoms (arsenic) was measured using the secondary ion mass spectroscopy (SIMS) method, while the density of active dopants was calculated from the capacitance-voltage (CV) measurements. The solar cell simulation data was obtained using the SCAPS software, a one-dimensional solar cell simulation programme. The presented data indicates a small loss (around 20 mV) of Voc for the HRT buffered cells.

Interdomain B in ZAP-70 regulates but is not required for ZAP-70 signaling function in lymphocytes.

The protein tyrosine kinase ZAP-70 plays an important role in T-cell activation and development. After T-cell receptor stimulation, ZAP-70 associates with the receptor and is phosphorylated on many tyrosines, including Y292, Y315, and Y319 within interdomain B. Previously, we demonstrated that Y292 negatively regulates ZAP-70 function and that Y315 positively regulates ZAP-70 function by interacting with Vav. Recent studies have suggested that Y319 also positively regulate ZAP-70 function. Paradoxically, removal of interdomain B (to create the construct designated Delta), containing the Y292, Y315, and Y319 sites, did not eliminate the ability of ZAP-70 to induce multiple gene reporters in Syk-deficient DT-40 B cells and ZAP-70/Syk-deficient Jurkat cells. Here we show that Delta still utilizes the same pathways as wild-type ZAP-70 to mediate NF-AT induction. This is manifested by the ability of Delta to restore induction of calcium fluxes and mitogen-activated protein kinase activation and by the ability of dominant negative Ras and FK506 to block the induction of NF-AT activity mediated by Delta. Biochemically we show that the stimulated tyrosine phosphorylation of Vav, Shc, and ZAP-70 itself is diminished, whereas that of Slp-76 is increased in cells reconstituted with Delta. Deletion of interdomain B did not affect the ability of ZAP-70 to bind to the receptor. The in vitro kinase activity of ZAP-70 lacking interdomain B was markedly reduced, but the kinase activity was still required for the protein's in vivo activity. Based on these data, we concluded that interdomain B regulates but is not required for ZAP-70 signaling function leading to cellular responses.

Pleiotropic contributions of phospholipase C-gamma1 (PLC-gamma1) to T-cell antigen receptor-mediated signaling: reconstitution studies of a PLC-gamma1-deficient Jurkat T-cell line.

Phospholipase C-gamma1 (PLC-gamma1) plays a crucial role in the coupling of T-cell antigen receptor (TCR) ligation to interleukin-2 (IL-2) gene expression in activated T lymphocytes. In this study, we have isolated and characterized two novel, PLC-gamma1-deficient sublines derived from the Jurkat T-leukemic cell line. The P98 subline displays a >90% reduction in PLC-gamma1 expression, while the J.gamma1 subline contains no detectable PLC-gamma1 protein. The lack of PLC-gamma1 expression in J.gamma1 cells caused profound defects in TCR-dependent Ca(2+) mobilization and NFAT activation. In contrast, both of these responses occurred at normal levels in PLC-gamma1-deficient P98 cells. Unexpectedly, the P98 cells displayed significant and selective defects in the activation of both the composite CD28 response element (RE/AP) and the full-length IL-2 promoter following costimulation with anti-TCR antibodies and phorbol ester. These transcriptional defects were reversed by transfection of P98 cells with a wild-type PLC-gamma1 expression vector but not by expression of mutated PLC-gamma1 constructs that lacked a functional, carboxyl-terminal SH2 [SH2(C)] domain or the major Tyr(783) phosphorylation site. On the other hand, the amino-terminal SH2 [SH2(N)] domain was not essential for reconstitution of RE/AP- or IL-2 promoter-dependent transcription but was required for the association of PLC-gamma1 with LAT, as well as the tyrosine phosphorylation of PLC-gamma1 itself, in activated P98 cells. These studies demonstrate that the PLC-gamma1 SH2(N) and SH2(C) domains play functionally distinct roles during TCR-mediated signaling and identify a non-Ca(2+)-related signaling function linked to the SH2(C) domain, which couples TCR plus phorbol ester-CD28 costimulation to the activation of the IL-2 promoter in T lymphocytes.

Genetic evidence for differential coupling of Syk family kinases to the T-cell receptor: reconstitution studies in a ZAP-70-deficient Jurkat T-cell line.

T-cell antigen receptor (TCR) engagement activates multiple protein tyrosine kinases (PTKs), including the Src family member, Lck, and the Syk-related PTK, ZAP-70. Studies in ZAP-70-deficient humans have demonstrated that ZAP-70 plays crucial roles in T-cell activation and development. However, progress toward a detailed understanding of the regulation and function of ZAP-70 during TCR signaling has been hampered by the lack of a suitable T-cell model for biochemical and genetic analyses. In this report, we describe the isolation and phenotypic characterization of a Syk- and ZAP-70-negative somatic mutant derived from the Jurkat T-cell line. The P116 cell line displays severe defects in TCR-induced signaling functions, including protein tyrosine phosphorylation, intracellular Ca2+ mobilization, and interleukin-2 promoter-driven transcription. These signaling defects were fully reversed by reintroduction of catalytically active versions of either Syk or ZAP-70 into the P116 cells. However, in contrast to ZAP-70 expression, Syk expression triggered a significant degree of cellular activation in the absence of TCR ligation. Transfection experiments with ZAP-70-Syk chimeric proteins indicated that both the amino-terminal regulatory regions and the carboxy-terminal catalytic domains of Syk and ZAP-70 contribute to the distinctive functional properties of these PTKs. These studies underscore the crucial role of ZAP-70 in TCR signaling and offer a powerful genetic model for further analyses of ZAP-70 regulation and function in T cells.

Characterization of the human XPA promoter.

We cloned the human xeroderma pigmentosum group A gene (XPA) and characterized the XPA promoter (pXPA) by transient cat expression. The pXPA is extraordinarily weak in human fibroblasts (1% of RSV-LTR) and appears to function without any of the usual promoter elements. Regions containing positive and negative control elements were localized.

Expression of human CD59 in transgenic pig organs enhances organ survival in an ex vivo xenogeneic perfusion model.

The serious shortage of available donor organs for patients with end stage organ failure who are in need of solid organ transplantation has led to a heightened interest in xenotransplantation. The major barrier to successful discordant xenotransplantation is hyperacute rejection. Hyperacute rejection results from the deposition of preformed antibodies that activate complement on the luminal surface of the vascular endothelium, leading to vessel occlusion and graft failure within minutes to hours. Endogenous membrane-associated complement inhibitors normally protect endothelial cells from autologous complement -- however, these molecules are species-restricted and therefore are ineffective at inhibiting activated xenogeneic complement. To address the pathogenesis of hyperacute rejection in the pig-to-human combination, F1 offspring were generated from a transgenic founder animal that was engineered to express the human terminal complement inhibitor hCD59. High-level cell surface expression of hCD59 was detected in the hearts and kidneys of these transgenic F1 animals, similar to expression levels in human kidney tissue. The hCD59 was expressed on both large vessel and capillary endothelium. Ex vivo perfusion experiments, using human blood as the perfusate, were performed with transgenic porcine hearts and kidneys to evaluate the ability of hCD59 to inhibit hyperacute rejection. These experiments demonstrated that transgenic organs expressing hCD69 resisted hyperacute rejection, as measured by increased organ function for both the hearts and the kidneys, as compared with control pig organs. Hearts from hCD59-expressing animals demonstrated a five-fold prolongation in function compared with controls, 109.8 +/- 20.7 min versus 21.2 +/- 2.9 min (P = 0.164). The hCD59-expressing kidneys also demonstrated significantly prolonged function at 157.8 +/- 27.0 min compared with 60.0 +/- 6.1 min for controls (P = 0.0174). Deposition of C9 neoantigen In the vasculature of porcine organs perfused with human blood was markedly reduced in organs expressing hCD59. These studies demonstrate that C5b-9 plays an important role in hyperacute rejection of a porcine organ perfused with human blood and suggest that donor pigs transgenic for hCD59 may be an integral component of successful clinical xenotransplantation.

Global intellectual deficits in cystinosis.

Fourteen families of children with infantile nephropathic cystinosis were evaluated using the Stanford-Binet Intelligence Scale, Fourth Edition [Thorndike et al., 1986: Stanford-Binet Intelligence Scale, Fourth Ed.]. The IQs of 15 children with cystinosis, their 23 sibs and 24 parents were compared in order to evaluate a potential effect of cystinosis on intelligence. Children with cystinosis had a significantly lower mean IQ than their sibs and their parents (P = .001). Thus, even though the mean IQ of the children with cystinosis (94.4 +/- 10) was within the average range, there is evidence that these children have a mild global intellectual deficit relative to their expected IQ based upon the IQs of other relatives. In addition, to a subset of the subjects we administered a measure of scholastic ability, the Wide Range Achievement Test-Revised [Jastak and Wilkinson, 1984: The Wide Range Achievement Test-Revised], which consists of spelling, reading, and arithmetic subtests. The 11 cystinosis subjects scored significantly lower (P = .01) than their 16 sibs and their 14 parents in the area of spelling, whereas they did not significantly differ in their performance in the areas of reading and arithmetic.

Rate limitations in posttranslational processing by the mammary gland of transgenic animals.

Our studies in transgenic animal bioreactors sought to determine the rate limitations in posttranslational processing of recombinant human protein C (rhPC) made in mammary gland of mice and pigs. Human protein C (hPC) is a complex plasma protein containing nine gamma-carboxylated glutamic acid (gla) residues that bind calcium at about 1 to 3 mM. Gamma carboxylation is a vitamin K-dependent posttranslational modification. The effect of rhPC synthesis rate on the extent of gamma-carboxylation of glutamic acid was studied. We have perturbed the biosynthesis of rhPC by using two different transgenes to direct mammary gland-specific expression. Promoter elements of the murine whey acid protein (mWAP) gene were used to drive the expression of hPC-cDNA and hPC-genomic transgenes. Transgenic mice with hPC-cDNA and hPC-genomic sequences gave expression levels of 11 +/- 4 micrograms rhPC/ml of milk and 895 +/- 21 micrograms rhPC/ml of milk, respectively. Transgenic pigs with hPC-cDNA and hPC-genomic sequences gave expression levels of 100 to 500 micrograms rhPC/ml of milk and 800 to 2000 micrograms rhPC/ml of milk, respectively. A monoclonal antibody (7D7B10-mAb) that binds an epitope in the gla domain of hPC in the absence of calcium was used to study the conformational behavior of immunopurified rhPC. Immunopurified rhPC from lower expressing mice and pigs gave a calcium-dependent binding inhibition by 7D7B10-mAb similar to that of hPC. Immunopurified rhPC from higher expressing mice and pigs gave a less calcium-dependent response. This study suggests that a rate limitation in gamma-carboxylation by the mammary gland occurs at expression levels about > 20 micrograms/ml in mice and > 500 micrograms/ml in pigs.

The porcine mammary gland as a bioreactor for complex proteins.

The similar biological activity of rhPC and hPC indicates that porcine mammary gland can perform many of the processing reactions necessary for recombinant synthesis of complex human proteins and produce them at levels suitable for industrial bioreactor applications. The health of the transgenic pigs appeared unaffected by the expression of high levels of the heterologous protein. We suggest that one of the advantages of using the mammary gland as a bioreactor appears to be the high cell density relative to that of cell culture.

Ischemic preconditioning protects against paraplegia after transient aortic occlusion in the rat.

BACKGROUND: Paraplegia can result from operations requiring transient occlusion of the thoracic aorta. A rat model of paraplegia with the characteristics of delayed paraplegia and transient ischemic dysfunction was developed to determine whether ischemic preconditioning (IPC) improved neurologic outcome. METHODS: Rats underwent balloon occlusion of the upper descending thoracic aorta. One group (2 minute IPC, n = 19) underwent 2 minutes of IPC and a second group (5 minute IPC, n = 19) had 5 minutes of IPC 48 hours before 10 minutes of occlusion. The control group (n = 31) had no IPC prior to 10 minutes of occlusion. RESULTS: Paraplegia occurred in 68% of the control animals (21 of 31 paraplegic: 6 delayed and 15 immediate paraplegia). Both the 2-minute IPC and 5-minute IPC groups had a decreased incidence of paraplegia when compared to controls (32%, p = 0.011 and 26%, p = 0.009, respectively). CONCLUSIONS: A rat model of spinal cord ischemia demonstrating both delayed paraplegia and transient ischemic dysfunction was characterized. Both 2-minute and 5-minute periods of IPC were found to protect against paraplegia.

A comparison of antimicrobial activity of four disclosant dyes.

Four disclosant dyes were tested for antimicrobial activity against 27 oral reference strains and five non-oral human pathogens. Erythrosine and fluorescein inhibited most gram-positive and gram-negative organisms associated with dental plaque, and were bactericidal to selected strains. Fast green and brilliant blue demonstrated little antimicrobial activity.

Inter- and intra-ethnic variation in water intake, contact, and source estimates among Tucson residents: Implications for exposure analysis.

Water-related exposures among Hispanics, particularly among Mexican Americans, are relatively unknown. Exposure and risk assessment is further complicated by the absence of good time-activity data (e.g., water intake) among this population. This study attempts to provide some insight concerning water-related exposure parameters among Hispanics. Determining the extent to which non-Hispanic whites and Hispanics living in the Tucson metropolitan area differ with respect to direct water intake and source patterns is the primary purpose of this investigation. Using random digit dialing, researchers conducted a cross-sectional telephone population survey of 1183 Tucson residents. Significant ethnic variation was observed in water intake patterns among this sample, particularly in terms of source. Hispanics reported much higher rates of bottled water consumption than did non-Hispanic whites. Ethnic variation in exposure parameters such as that observed in this study increases the potential for measurement error in exposure analysis. Erroneous assumptions that exposure estimates (i.e., water intake source) are generalizable across various ethnic groups may lead to both overestimation and underestimation of contaminant exposure.

Clinical, microbiological and immunological studies of a family with a high prevalence of early-onset periodontitis.

Extensive clinical, laboratory and microbiological studies were performed on members of a family with an unusually high prevalence of early-onset severe periodontitis. Clinical observations included intraoral photographs and assessment of inflammation, plaque, probing depths and bone loss. Pocket bacteria were sampled, cultivated and identified. Immunological studies included assessment in vitro of neutrophil (PMN) and monocyte (MN) chemotaxis, assessment of PMN phagocytosis and other functions using the iodination assay, measurement of serum opsonic and chemoattractant activities and determination of levels of serum antibodies specific to various putative periodontal pathogens. The proband, a 19-year-old white woman, had rapidly progressive periodontitis (RP). Of her six siblings available for study, all had juvenile periodontitis (JP), and both parents had been edentulous since early adulthood. Early edentulism and recurrent infections, especially otitis media, were prevalent in the forebearers, especially on the maternal side. Two married sisters of the proband had young male children with recurrent infections. Abnormalities in leukocyte chemotaxis were found in the proband, in two of her siblings and in both parents. The pocket flora was predominantly Gram-negative, anaerobic rods with a high prevalence of Bacteroides species, and serum antibodies specific to Bacteroides species were detected in the sera of five of the seven patients studied. Actinobacillus actinomycetemcomitans was not found in any of the pockets studied, nor were antibodies specific to any of the three known serotypes of this bacterium detected in the serum of any of the patients. There was a relatively good correlation between the bacterial species isolated from the periodontal pockets and the antibodies found in the serum. PMN iodination and serum opsonic activity were normal in all of the patients. Thus not all JP patients have detectable Actinobacillus species in their periodontal pockets, nor do all have antibody detectable with the techniques we used specific to these bacteria in their serum. In contrast, JP patients may have Bacteroides species in their periodontal pockets and antibody specific to Bacteroides species in their serum. Although abnormal leukocyte chemotaxis is generally common in RP and JP patients, in this family the correlation between this defect and the presence of these diseases was poor.

Clinical and laboratory studies of a family with a high prevalence of juvenile periodontitis.

The form of periodontitis with onset at puberty and affecting predominantly the first molars and incisors is called juvenile periodontitis (JP). The disease has been the object of intense study because from its analysis may come insights into understanding other, more common, forms of periodontitis. We recently had the opportunity to study an unusual family in which both parents developed JP in their teens. We did clinical examinations, measured leukocyte chemotaxis, analyzed the pocket microflora, looked for serum antibodies against a large panel of putative periodontal pathogens and correlated the results. The couple had two affected and two unaffected children. One of the unaffected children was not available for study. Neutrophil chemotaxis was abnormal in both parents and in the two affected children, but not in the unaffected child. Actinobacillus actinomycetemcomitans accounted for 17.5% of the pocket flora isolated from one affected child and 2.5% of that from the unaffected child, but was not detected in the remaining family members. Antibodies specific for A. actinomycetemcomitans, Hemophilus aphrophilus and Eikenella corrodens were present in the serum of both affected children and for Capnocytophaga sputigena and C. ochracea in the father, but no antibodies directed against any of the species studied were found in the mother and the unaffected child. The distribution of disease in this family was more compatible with an X-linked dominant than with an autosomal recessive mode of inheritance. The correlations among presence or absence of disease, abnormal neutrophil chemotaxis and presence of serum antibodies reacting with A. actinomycetemcomitans were excellent.(ABSTRACT TRUNCATED AT 250 WORDS)