Human TNF receptor p75/80 gene structure and promoter characterization

Tumor necrosis factor receptor p75/80 ((TNF-R p75/80) is a 75 kDa type 1 transmembrane protein expressed predominately on cells of hematopoietic lineage. TNF-R p75/80 belongs to the TNF receptor superfamily characterized by cysteine-rich extracellular regions composed of three to six disulfide-linked domains. In the present report, we have characterized, for the first time, the complete gene structure for human TNF-R p75/80 which spans approximately 43 kbp. The gene consists of 10 exons (ranging from 34 bp to 2.5 kbp) and 9 introns (343 bp to 19 kbp). Consensus elements for transcription factors involved in T cell development and activation were noted in the 5$\sp\prime$ flanking region including TCF-1, Ikaros, AP-1, CK-2, IL-6RE, ISRE, GAS, NF-$\kappa$B and SP1, as well as an unusually high GC content and CpG frequency that appears characteristic of some TNF-R family members. The unusual (GATA)$\sb{\rm n}$ and (GAA)(GGA) repeats found within intron 1 may prove useful for further genome analysis within the 1p36 chromosomal locus. The human TNF-R p75/80 gene structure will permit further assessment of its involvement in normal hematopoietic cell development and function, autoimmune disease, and non-random translocations in hematopoietic malignancies. The region 1.8 kb 5$\sp\prime$ of the ATG was able to drive luciferase expression when transfected into cell lines expressing TNF-R p75/80. Further characterization of the 5$\sp\prime$-regulatory region will aid in determining factors and signal transduction pathways involved in regulating TNF-R p75/80 expression. ^

Knowledge, beliefs, risk behaviors, and social skills for HIV/STD prevention in junior college students (grades 11--12) in Bombay, India

1230 year 11 and 12 college students, modal age 16 and 17, in three colleges in Bombay, India, were studied on sexual behaviors or risk of sexual behaviors, beliefs about sex, HIV/STD knowledge, perceived norms regarding sexual behaviors, and the relationships between social skills/anxieties in HIV/STD prevention and actual and anticipated sexual behaviors. A quantitative questionnaire examining HIV/STD risk behaviors, knowledge, attitudes and beliefs, and the AIDS Social Assertiveness Scale (ASAS) were administered to these 1230 college students. Data indicated that 8% of males and 1% of females had had sexual experience, but over one third were not sure at all of being able to abstain from sexual activity with either steady or casual partners. Perceived norms were slanted toward sexual abstinence for the majority of the sample. Knowledge of protective effects of condoms was high, although half of those who had had sex did not use condoms. Logistic regression showed knowledge was higher among males, those who believed it was OK to have sex with a steady partner and that they should not wait until they were older, those who believed that condoms should be used even if the partner is known, and those who believed it was acceptable to have multiple partners. Gender differences in sexual activity and beliefs about sexual activity showed males were less likely to believe in abstaining from sexual activity. The 5 scales of the ASAS were scored and compared on ANOVA on: those who had had sexual experience (HS), those who anticipated being unable to refuse sex (AS), and those who did not anticipate problems in refusing sex (DS). Those in the AS group had greater anxieties about refusing sexual or other risk behaviors than HS and DS groups. There were greater anxieties about dealing with condoms in the AS and DS groups compared with the HS group. Confiding sexual or HIV/STD-related problems to significant others was more anxiety-provoking for the AS group compared with the HS group, and the AS group were more anxious about interactions with people with HIV. Factor analysis produced the same 5 factors as those found in previous studies. Of these, condom interactions and confiding in significant others were most anxiety provoking, and condom interactions most variable based on demographic and attitudinal factors.^ This age group is appropriate for HIV/STD reduction education given the low rate of sexual activity but despite knowledge of the importance of condom use, social skills to apply this knowledge are lacking. Social skills training in sexual negotiations, condom negotiations, and confiding HIV/STD-related concerns to significant others should reduce the risks of Indian college students having unwanted or unprotected sex. ^

RUNOFF FROM COAL PILES: EFFECTS ON THE ENVIRONMENT AND THE PUBLIC'S HEALTH

The use of coal for fuel in place of oil and natural gas has been increasing in the United States. Typically, users store their reserves of coal outdoors in large piles and rainfall on the coal creates runoffs which may contain materials hazardous to the environment and the public's health. To study this hazard, rainfall on model coal piles was simulated, using deionized water and four coals of varying sulfur content. The simulated surface runoffs were collected during 9 rainfall simulations spaced 15 days apart. The runoffs were analyzed for 13 standard water quality parameters, extracted with organic solvents and then analyzed with capillary column GC/MS, and the extracts were tested for mutagenicity with the Ames Salmonella microsomal assay and for clastogenicity with Chinese hamster ovary cells.^ The runoffs from the high-sulfur coals and the lignite exhibited extremes of pH (acidity), specific conductance, chemical oxygen demand, and total suspended solids; the low-sulfur coal runoffs did not exhibit these extremes. Without treatment, effluents from these high-sulfur coals and lignite would not comply with federal water quality guidelines.^ Most extracts of the simulated surface runoffs contained at least 10 organic compounds including polycyclic aromatic hydrocarbons, their methyl and ethyl homologs, olefins, paraffins, and some terpenes. The concentrations of these compounds were generally less than 50 (mu)g/l in most extracts.^ Some of the extracts were weakly mutagenic and affected both a DNA-repair proficient and deficient Salmonella strain. The addition of S9 decreased the effect significantly. Extracts of runoffs from the low-sulfur coal were not mutagenic.^ All extracts were clastogenic. Extracts of runoffs from the high-sulfur coals were both clastogenic and cytotoxic; those from the low-sulfur coal and the lignite were less clastogenic and not cytotoxic. Clastogenicity occurred with and without S9 activation. Chromosomal lesions included gaps, breaks and exchanges. These data suggest a relationship between the sulfur content of a coal, its mutagenicity and also its clastogenicity.^ The runoffs from actual coal piles should be investigated for possible genotoxic effects in view of the data presented in this study.^

The cDNA cloning of murine HIP /L29 and the functional analysis of HIP/L29 domains

Human heparin/heparan sulfate interacting protein/L29 (HIP/L29) is a heparin/heparan sulfate (Hp/HS) binding protein found in many adult human tissues. Potential functions of this protein are promotion of embryo adhesion, modulation of blood coagulation, and control of cell growth. While these activities are diverse, the ability of human HIP/L29 to interact with Hp/HS at the cell surface may be a unifying mechanism of action since Hp/HS influences all of these processes. A murine ortholog has been identified that has 78.8% homology over the entire sequence and identity over the N-terminal 64 amino acids when compared to human HIP/L29. Northern, Western, and immunohistochemical analysis shows that murine HIP/L29 mRNA and protein are expressed in a tissue specific manner. Murine HIP/L29 is enriched in the membrane fraction of NmuMG cells where it is eluted with high salt, suggesting that it is a peripheral membrane protein. The ability of murine HIP/L29 to bind Hp is verified by studies using native and recombinant forms of murine HIP/L29. A synthetic peptide (HIP peptide-2) derived from the identical N-terminal region of HIP/L29 proteins was tested for the ability to bind Hp and support cell adhesion. This peptide was chosen because it conforms to a proposed consensus sequence for Hp/HS binding peptides. HIP peptide-2 binds Hp in a dose-dependent, saturable, and selective manner and supports Hp-dependent cell adhesion. However, a scrambled form of this peptide displayed similar activities indicating a lack of peptide sequence specificity required for activity. Lastly, an unbiased approach was used to identify sequences within human and mouse HIP/L29 proteins necessary for Hp/HS binding. A panel of recombinant proteins was made that collectively are deficient in every human HIP/L29 domain. The activities of these deletion mutants and recombinant murine HIP/L29 were compared to the activity of recombinant human HIP/L29 in a number of assays designed to look at differences in the ability to bind Hp/HS. These studies suggest that each domain within human HIP/L29 is important for binding to Hp/HS and divergences in the C-terminus of human and mouse HIP/L29 account for a decrease in murine HIP/L29 affinity for Hp/HS. It is apparent that multiple domains within human and mouse HIP/L29 contribute to the function of Hp/HS binding. The interaction of multiple HIP/L29 domains with Hp/HS will influence the biological activity of HIP/L29 proteins. ^

A study of the biology of perlecan

Extracellular matrix (ECM) is a component of a variety of organisms that provides both structural support and influence upon the cells it surrounds. The importance of the ECM is becoming more apparent as matrix defects are linked to human disease. In this study, the large, extracellular matrix heparan sulfate proteoglycan, perlecan (Pln) is examined in two systems. First, the role of Pln in the interaction between a blastocyst and uterine epithelial cells is investigated. In mice, blastocyst attachment and implantation occurs at approximately d 4.5 post coitus. In addition, a delayed implantation model has been used to distinguish between the response of the blastocyst to that of hatching and of becoming attachment competent. ^ The second series of experiments described in this study focuses on the process of chondrogenesis in mice. Pln, commonly expressed with other basement membrane (BM) proteins, was found to be expressed in cartilaginous tissue without other BM proteins. This unusual expression pattern led to further study and the development of an in vitro chondrogenesis assay using the mouse embryonic fibroblast cell line, C3H/10T1/2. When cultured on Pln in vitro, these cells form aggregates and express the cartilage proteins, collagen type II and aggrecan. In examining the participation of the heparan sulfate (HS) chains in this process, the proteoglycan was enzymatically digested to remove the HS chains before the initiation of 10T1/2 cell culture. After digestion, the ability of Pln to stimulate aggregate formation was greatly diminished. Thus, the HS chains participate in the cell induction process. To determine which domain of Pln might be responsible for this activity, recombinant fragments of Pin were used in the cell culture assay. Of all recombinant protein fragments tested, only the domain including the HS chains, domain 1, was able to initiate the morphological change exhibited by the 10T1/2 cells. Similar to native Pln, when HS chains were removed from domain I, chondrogenic activity was abolished. A variant of domain I carrying both HS and chondroitin sulfate (CS) chains retained activity when only HS chains were removed. When both HS and CS chains were removed, then activity was lost. ^ The ability to rapidly stimulate differentiation of 10T1/2 cells in vitro may lead to better control of chondrogenesis in vitro and in vivo, providing better understanding and manipulation of the chondrogenic process. This greater understanding may have benefits for study of cartilage and bone diseases and subsequent treatment options. (Abstract shortened by UMI.)^

Expression regulation of AP -2 gamma gene

AP-2γ is a member of the AP-2 transcription factor family, is highly enriched in the trophoblast cell lineage, and is essential for placenta development. In an effort to identify factors regulating AP-2γ gene expression we isolated and characterized the promoter and 5′ flanking region of the mouse and human AP-2γ genes. The transcription start site of the mouse AP-2γ gene was mapped by primer extension and 5′ RACE. Transient gene transfer studies showed that basal promoter activity resides within a highly conserved ∼200 by DNA sequence located immediately upstream of the transcription start site. The conserved region is highly GC-rich and lacks typical TATA or CCAAT boxes. Multiple potential Sp and AP-2 binding sites are clustered within this region. Electrophoretic mobility shift assays demonstrated that Sp1 and Sp3 bind to three sites in the promoter region of the mouse AP-2γ gene. Combined mutation of the three putative Sp sites reduced promoter activity by 80% in trophoblast and non-trophoblast cells, demonstrating the functional importance of these sites in AP-2γ gene expression. ^ Mutational analysis of the 5′-flanking region revealed a 117-bp positive regulatory region of the mouse AP-2γ gene located between −5700 and −5583 upstream of the transcription start site. This 117-bp positive regulatory element provided approximately 7-fold enhancement of reporter gene expression in cultured trophoblast cells. A C/EBP-Sp1 transcription factor-binding module is located in this DNA sequence. Electrophoretic mobility shift assays demonstrated that transcription factors Sp1, Sp3 and C/EBP bind to the enhancer element. Mutation of each protein-binding site reduced the enhanced expression significantly. Mutagenesis assays showed that two other protein-binding sites also contribute to the enhancer activity. In summary, we have shown that Sp1 and Sp3 bind to cis-regulatory elements located in the promoter region and contribute to basal promoter activity. We have identified a 117-bp positive regulatory element of AP-2γ gene, and we have shown that Sp and C/EBP proteins bind to the cis -regulatory elements and contribute to the enhanced gene expression. ^