The characterization of mouse carboxypeptidase N small subunit gene structure and presence in developing embryos

Carboxypeptidase N (CPN) is a plasma zinc metalloprotease, which consists of two enzymatically active small subunits and two large subunits that protect the protein from degradation. CPN cleaves carboxy-terminal arginines and lysines from peptides found in the bloodstream such as complement anaphylatoxins, kinins, and creatine kinase MM. In this study, the mouse CPN small subunit (CPN1) coding region, gene structure, and chromosomal location were characterized and the expression of CPN1 was investigated in mouse embryos at different stages of development. The CPN1 gene, which was approximately 29 kb in length, contained nine exons and localized to mouse chromosome 19D2. The fifth and sixth exons of CPN1 encoded the amino acids necessary for substrate binding and catalytic activity. CPN1 RNA was expressed predominately in adult liver and contained a 1371 bp open reading frame encoding 457 amino acids. In the mouse embryo, CPN1 RNA was observed at 8.5 days post coitus (dpc), while its protein was detected at 10.5 dpc. In situ hybridization of the fetal liver detected CPN1 RNA in erythroid progenitor cells at 10.5, 13.5, and 16.5 dpc and in hepatocytes at 16.5 dpc. This was compared to the expression of the complement component C3, the parent molecule of complement anaphylatoxin C3a. Consistently throughout the experiments, CPN1 message and protein preceded the expression of C3. To obtain a better understanding of the biological significance of CPN1 in vivo, studies were initiated to produce a genetically engineered mouse in which the CPN1 gene was ablated. To facilitate this project a targeting vector was constructed by removing the functionally important fifth and sixth exons of the CPN1 gene. Collectively, these studies have: (1) provided important detailed information regarding the structure and organization of the murine CPN1 gene, (2) yielded insights into the developmental expression of mouse CPN1 in relationship to C3 expression, and (3) set the stage for the generation of a CPN1 “knock-out” mouse, which can be used to determine the biological significance of CPN1 in both normal and diseased conditions. ^

Objective assessment of the built environment and its relationship to physical activity and obesity

The role of physical activity in the promotion of individual and population health has been well documented in research and policy publications. Significant research activities have produced compelling evidence for the support of the positive association between physical activity and improved health. Despite the knowledge about these public health benefits of physical activity, over half of US adults do not engage in physical activity at levels consistent with public health recommendations. Just as physical inactivity is of significant public health concern in the US, the prevalence of obesity (and its attendant co-morbidities) is also increasing among US adults.^ Research suggests racial and ethnic disparities relevant to physical inactivity and obesity in the US. Various studies have shown more favorable outcomes among non-Hispanic whites when compared to other minority groups as far as physical activity and obesity are concerned. The health disparity issue is especially important because Mexican-Americans who are the fastest growing segment of the US population are disproportionately affected by physical inactivity and obesity by a significant margin (when compared to non-Hispanic whites), so addressing the physical inactivity and obesity issues in this group is of significant public health concern. ^ Although the evidence for health benefits of physical activity is substantial, various research questions remain on the potential motivators for engaging in physical activity. One area of emerging interest is the potential role that the built environment may play in facilitating or inhibiting physical activity.^ In this study, based on an ongoing research project of the Department of Epidemiology at the University of Texas M. D. Anderson Cancer Center, we examined the built environment, measured objectively through the use of geographical information systems (GIS), and its association with physical activity and obesity among a cohort of Mexican- Americans living in Harris County, Texas. The overall study hypothesis was that residing in dense and highly connected neighborhoods with mixed land-use is associated with residents’ increased participation in physical activity and lowered prevalence of obesity. We completed the following specific aims: (1) to generate a land-use profile of the study area and create a “walkability index” measure for each block group within the study area; (2) to compare the level of engagement in physical activity between study participants that reside in high walkability index block groups and those from low walkability block groups; (3) to compare the prevalence of obesity between study participants that reside in high walkability index block groups and those from low walkability block groups. ^ We successfully created the walkability index as a form of objective measure of the built environment for portions of Harris County, Texas. We used a variety of spatial and non-spatial dataset to generate the so called walkability index. We are not aware of previous scholastic work of this kind (construction of walkability index) in the Houston area. Our findings from the assessment of relationships among walkability index, physical activity and obesity suggest the following, that: (1) that attempts to convert people to being walkers through health promotion activities may be much easier in high-walkability neighborhoods, and very hard in low-walkability neighborhoods. Therefore, health promotion activities to get people to be active may require supportive environment, walkable in this case, and may not succeed otherwise; and (2) Overall, among individuals with less education, those in the high walkability index areas may be less obese (extreme) than those in the low walkability area. To the extent that this association can be substantiated, we – public health practitioners, urban designers, and policy experts – we may need to start thinking about ways to “retrofit” existing urban forms to conform to more walkable neighborhoods. Also, in this population especially, there may be the need to focus special attention on those with lower educational attainment.^

Developmental Changes in the Structure and Composition of the Postsynaptic Density

The development of the brain and its underlying circuitry is dependent on the formation of trillions of chemical synapses, which are highly specialized contacts that regulate the flow of information from one neuron to the next. It is through these synaptic connections that neurons wire together into networks capable of performing specific tasks, and activity-dependent changes in their structural and physiological state is one way that the brain is thought to adapt and store information. At the ultrastructural level, developmental and activity-dependent changes in the size and shape of dendritic spines have been well documented, and it is widely believed that structural changes in spines are a hallmark sign of synapse maturation and alteration of synaptic physiology. While changes in spine structure have been studied extensively, changes in one of its most prominent components, the postsynaptic density (PSD), have largely evaded observation. The PSD is a protein-rich organelle on the cytoplasmic side of the postsynaptic membrane, where it sits in direct opposition to the presynaptic terminal. The PSD functions both to cluster neurotransmitter receptors at the cell surface as well as organize the intracellular signaling molecules responsible for transducing extracellular signals to the postsynaptic cell. Much is known about the chemical composition of the PSD, but the structural arrangement of its molecular components is not well documented. Adding to the difficulty of understanding such a complex mass of protein machinery is the fact that its protein composition is known to change in response to synaptic activity, meaning that its structure is plastic and no two PSDs are identical. Here, immuno-gold labeling and electron tomography of PSDs isolated throughout development was used to track changes in both the structure and molecular composition of the PSD. State-of-the-art cryo-electron tomography was used to study the fine structure of the PSD during development, and provides an unprecedented glimpse into its molecular architecture in an un-fixed, unstained and hydrated state. Through this analysis, large structural and compositional changes are apparent and suggest a model by which the PSD is first assembled as a mesh-like lattice of proteins that function as support for the later recruitment of various PSD components. Spatial analysis of the recruitment of proteins into the PSD demonstrated that its assembly has an underlying order.

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.) ^

Structure and function study of prostaglandin H synthase

Prostaglandin H synthase (PGHS) is a key enzyme in biosynthesis of prostaglandins, thromboxane, and prostacyclin. It has two activities, cyclooxygenase and peroxidase. "PGHS" means PGHS-1. A current hypothesis considers the cyclooxygenase reaction to be a free radical chain reaction, initiated by interaction of the synthase peroxidase with hydroperoxides leading to the production of a tyrosyl free radical. According to this hypothesis, tyrosyl residue(s) may play a key role in the cyclooxygenase reaction. Tetranitromethane (TNM) can relatively selectively nitrate tyrosines at pH 8.0. The effect of TNM on both cyclooxygenase activity and peroxidase activity has been examined: reaction of the synthase holoenzyme with TNM at pH 8.0 led to inactivation of both activities, with the cyclooxygenase activity being lost rapidly and completely, while the peroxidase activity was lost more slowly. Indomethacin, a non-steroidal anti-inflammatory agent, can protect the synthase from the inactivation of TNM. Amino acid analyses indicated that a loss of tyrosine and formation of nitrotyrosine residues occurred during reaction with TNM, and that TNM-reacted holoenzyme with $<$10% residual cyclooxygenase activity had about 2.0 nitrotyrosine/subunit.^ PGH synthase is known to be an endoplasmic reticulum membrane-associated protein. Antibodies directed at particular PGHS peptide segments and indirect immunofluorescence have been used to characterize the membrane topology of crucial portions of PGHS. PGHS was expressed in COS-1 cells transfected with the appropriate cDNA. Stably-transfected human endothelial cells were also used for the topology study. The cells were treated with streptolysin-O, which selectively permeabilizes the plasma membrane, or with saponin to achieve general membrane disruption, before incubation with the antipeptide antibodies. Bound antipeptide antibody was stained by FITC-labelled secondary antibody and visualized by fluorescence microscopy. With the antipeptide antibodies against residues 51-66, 156-170 or 377-390, there was a significant reticular and perinuclear pattern of staining in cells permeabilized with saponin but not in cells permeabilized with SLO alone. Antibodies directed against the endogenous C-terminal peptide or against residues 271-284 produced staining in cells permeabilized with saponin, and also in a lower, but significant fraction of cells permeabilized with SLO. Similar results were obtained when COS-1 cells expressing recombinant PGHS with a viral reporter peptide inserted at the C-terminus were stained with antibody against the reporter epitope.^ The PGHS C-terminal sequence is similar to that of the consensus KDEL ER retention signal. The potential function of the PGHS C-terminus segment in ER retention was examined by mutating this segment and analyzing the subcellular distribution of the mutants expressed in COS-1 cells. None of the mutants had an altered subcellular distribution, although some had greatly diminished the enzyme activities. (Abstract shortened by UMI.) ^

The characterization of residential fungal spores and the relationship with housing characteristics in the Houston area

A number of indoor environmental factors, including bioaerosol or aeroallergen concentrations have been identified as exacerbators for asthma and allergenic conditions of the respiratory system. People generally spend 90% to 95% of their time indoors. Therefore, understanding the environmental factors that affect the presence of aeroallergens indoors as well as outdoors is important in determining their health impact, and in identifying potential intervention methods. This study aimed to assess the relationship between indoor airborne fungal spore concentrations and indoor surface mold levels, indoor versus outdoor airborne fungal spore concentrations and the effect of previous as well as current water intrusion. Also, the association between airborne concentration of indoor fungal spores and surface mold levels and the age of the housing structure were examined. Further, the correlation between indoor concentrations of certain species was determined as well. ^ Air and surface fungal measurements and related information were obtained from a Houston-area data set compiled from visits to homes filing insurance claims. During the sampling visit these complaint homes exhibited either visible mold or a combination of visible mold and water intrusion problems. These data were examined to assess the relationships between the independent and dependent variables using simple linear regression analysis, and independent t-tests. To examine the correlation between indoor concentrations of certain species, Spearman correlation coefficients were used. ^ There were 126 houses sampled, with spring, n=43 (34.1%), and winter, n=42 (33.3%), representing the seasons with the most samples. The summer sample illustrated the highest geometric mean concentration of fungal spores, GM=5,816.5 relative to winter, fall and spring (GM=1,743.4, GM=3,683.5 and GM=2,507.4, respectively). In all seasons, greater concentrations of fungal spores were observed during the cloudy weather conditions. ^ The results indicated no statistically significant association between outdoor total airborne fungal spore concentration and total living room airborne fungal spore concentration (β = 0.095, p = 0.491). Second, living room surface mold levels were not associated with living room airborne fungal spore concentration, (β= 0.011, p = 0.669). Third, houses with and without previous water intrusion did not differ significantly with respect to either living room (t(111) = 0.710, p = 0.528) or bedroom (t(111) =1.673, p = 0.162) airborne fungal spore concentrations. Likewise houses with and without current water intrusion did not differ significantly with respect to living room (t(109)=0.716, p = 0.476) or bedroom (t(109) = 1.035, p = 0.304) airborne fungal spore concentration. Fourth, houses with and without current water intrusion did not differ significantly with respect to living room (χ 2 (5) = 5.61, p = 0.346), or bedroom (χ 2 (5) = 1.80, p = 0.875) surface mold levels. Fifth, the age of the house structure did not predict living room (β = 0.023, p = 0.102) and bedroom (β = 0.023, p = 0.065) surface mold levels nor living room (β = 0.002, p = 0.131) and bedroom (β = 0.001, p = 0.650) fungal spore airborne concentration. Sixth, in houses with visually observed mold growth there was statistically significant differences between the mean living room concentrations and mean outdoor concentrations for Cladosporium (t (107) = 11.73, p < 0.0001), Stachybotrys (t (106)=2.288, p = 0.024, and Nigrosporia (t (102) = 2.267, p = 0.025). Finally, there was a significant correlation between several living room fungal species pairs, namely, Cladosporium and Stachybotrys (r = 0.373, p <0.01, n=65), Curvularia and Aspergillus/Penicillium (r = 0.205, p < 0.05, n= 111)), Curvularia and Stachybotrys (r = 0.205, p < 0.05, n=111), Nigrospora and Chaetomium (r = 0.254, p < 0.01, n=105) and Stachybotrys and Nigrospora (r = 0.269, p < 0.01, n=105). ^ This study has demonstrated several positive findings, i.e., significant pairwise correlations of concentrations of several fungal species in living room air, and significant differences between indoor and outdoor concentrations of three fungal species in homes with visible mold. No association was observed between indoor and outdoor fungal spore concentrations. Neither living room nor bedroom airborne spore concentrations and surface mold levels were related to the age of the house or to water intrusion, either previous or current. Therefore, these findings suggest the need for evaluating additional parameters, as well as combinations of factors such as humidity, temperature, age of structure, ventilation, and room size to better understand the determinants of airborne fungal spore concentrations and surface mold levels in homes. ^

Structure and biosynthesis of a pyruvate -dependent enzyme---phosphatidylserine decarboxylase from {\it Escherichia coli\/}

Phosphatidylserine decarboxylase of E. coli, a cytoplasmic membrane protein, catalyzes the formation of phosphatidylethanolamine, the principal phospholipid of the organism. The activity of the enzyme is dependent on a covalently bound pyruvate (Satre and Kennedy (1978) J. Biol. Chem. 253, 479-483). This study shows that the enzyme consists of two nonidentical subunits, $\alpha$ (Mr = 7,332) and $\beta$ (Mr = 28,579), with the pyruvate prosthetic group in amide linkage to the amino-terminus of the $\alpha$ subunit. Partial protein sequence and DNA sequence analysis reveal that the two subunits are derived from a proenzyme ($\pi$ subunit, Mr = 35,893) through a post-translational event. During the conversion of the proenzyme to the $\alpha$ and $\beta$ subunits, the peptide bond between Gly253-Ser254 is cleaved, and Ser254 is converted to the pyruvate prosthetic group at the amino-terminus of the $\alpha$ subunit (Li and Dowhan (1988) J. Biol. Chem. 263, 11516-11522).^ The proenzyme cannot be detected in cells carrying either single or multiple copies of the gene (psd), but can be observed in a T7 RNA polymerase/promoter and transcription-translation system. The cleavage of the wild-type proenzyme occurs rapidly with a half-time on the order of 2 min. Changing of the Ser254 to cysteine (S254C) or threonine (S254T) slows the cleavage rate dramatically and results in mutants with a half-time for processing of around 2-4 h. Change of the Ser254 to alanine (S254A) blocks the cleavage of the proenzyme. The reduced processing rate with the mutations of the proenzyme is consistent with less of the functional enzyme being made. Mutants S254C and S254T produce $\sim$15% and $\sim$1%, respectively, of the activity of the wild-type allele, but can still complement a temperature-sensitive mutant of the psd locus. Neither detectable activity nor complementation is observed by mutant S254A. These results are consistent with the hydroxyl-group of the Ser254 playing a critical role in the cleavage of the peptide bond Gly253-Ser254 of the pro-phosphatidylserine decarboxylase, and support the mechanism proposed by Snell and co-workers (Recsei and Snell (1984) Annu. Rev. Biochem. 53, 357-387) for the formation of the prosthetic group of pyruvate-dependent decarboxylases. ^

The effects of DNA cytosine methylation on H -ras promoter structure and function

The effect of DNA cytosine methylation on H-ras promoter activity was assessed using a transient expression system employing the plasmid H-rasCAT (NaeI H-ras promoter linked to the chloramphenicol acetyltransferase (CAT) gene). This 551 bp promoter is 80% GC rich, enriched with 168 CpG dinucleotides, and contains six functional GC box elements which represent major DNA methylation target sites. Prokaryotic methyltransferases HhaI (CGm$\sp5$CG) and HpaII (Cm$\sp5$CGG) alone or in combination with a human placental methyltransferase (HP MTase) were used to introduce methyl groups at different CpG sites within the promoter. To test for functional promoter activity, the methylated plasmids were introduced into CV-1 cells and CAT activity assessed 48 h post-transfection. Methylation at specific HhaI and HpaII sites reduced CAT expression by 70%, whereas more extensive methylation at generalized CpG sites with HP MTase inactivated the promoter $>$95%. The inhibition of H-ras promoter activity was not attributable to methylation-induced differences in DNA uptake or stability in the cell, topological form of the plasmid, or methylation effects in nonpromoter regions. We also observed that DNA cytosine methylation of a 360 bp promoter fragment by HP MTase induced a local change in DNA conformation. Using three independent methodologies (nitrocellulose filter binding assays, gel mobility shifts, and Southwestern blots), we determined that this change in promoter conformation affected the interaction of nuclear proteins with cis-regulatory sequences residing in the promoter region. The results provide evidence to suggest that DNA methylation may regulate gene expression by inducing changes in local promoter conformation which in turn alters the interactions between DNA and protein factors required for transcription. The results provide supportive evidence for the hypothesis of Cedar and Riggs, who postulated that DNA methylation may regulate gene expression by altering the binding affinities of proteins for DNA. ^