cDNA cloning and expression of a novel cell surface-expressed \b heparan sulfate/heparin \b interacting \b protein (HIP) from human cell lines and functional studies of a synthetic HIP peptide

Heparan sulfate proteoglycans and their corresponding binding sites have been suggested to play an important role during the initial attachment of blastocysts to uterine epithelium and human trophoblastic cell lines to uterine epithelial cell lines. Previous studies on RL95 cells, a human uterine epithelial cell line, characterized a single class of cell surface heparin/heparan sulfate (HP/HS)-binding sites. Three major HP/HS-binding peptide fragments were isolated from RL95 cell surfaces by tryptic digestion and partial amino-terminal amino acid sequence from each peptide fragment was obtained. In the current study, using the approaches of reverse transcription-polymerase chain reaction and cDNA library screening, a novel cell surface $\rm\underline{H}$P/HS $\rm\underline{i}$nteracting $\rm\underline{p}$rotein (HIP) has been isolated from RL95 cells. The full-length cDNA of HIP encodes a protein of 259 amino acids with a calculated molecular weight of 17,754 Da and pI of 11.75. Transfection of HIP cDNA into NIH-3T3 cells demonstrated cell surface expression and a size similar to that of HIP expressed by human cells. Predicted amino acid sequence indicates that HIP lacks a membrane spanning region and has no consensus sites for glycosylation. Northern blot analysis detected a single transcript of 1.3 kb in both total RNA and poly(A$\sp+$) RNA. Examination of human cell lines and normal tissues using both Northern blot and Western blot analysis revealed that HIP is differentially expressed in a variety of human cell lines and normal tissues, but absent in some cell lines examined. HIP has about 80% homology, at the level of both mRNA and protein, to a rodent protein, designated as ribosomal protein L29. Thus, members of the L29 family may be displayed on cell surfaces where they participate in HP/HS binding events. Studies on a synthetic peptide derived from HIP demonstrate that HIP peptide binds HS/HP with high selectivity and has high affinity (Kd = 10 nM) for a subset of polysaccharides found in commercial HIP preparations. Moreover, HIP peptide also binds certain forms of cell surface, but not secreted or intracellular. HS expressed by RL95 and JAR cells. This peptide supports the attachment of several human trophoblastic cell lines and a variety of mammalian adherent cell lines in a HS-dependent fashion. Furthermore, studies on the subset of HP specifically recognized by HIP peptide indicate that this high-affinity HP (HA-HP) has a larger median MW and a greater negative charge density than bulk HP. The minimum size of oligosaccharide required to bind to HIP peptide with high affinity is a septa- or octasaccharide. HA-HP also quantitatively binds to antithrombin-III (AT-III) with high affinity, indicating that HIP peptide and AT-III may recognize the same or similar oligosaccharide structure(s). Furthermore, HIP peptide antagonizes HP action and promotes blood coagulation in both factor Xa- and thrombin-dependent assays. Finally, HA-HP recognized by HP peptide is highly enriched with anticoagulant activity relative to bulk HP. Collectively, these results demonstrate that HIP may play a role in the HP/HS-involved cell-cell and cell-matrix interactions and recognizes a motif in HP similar or identical to that recognized by AT-III and therefore, may modulate blood coagulation. ^

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. ^