Determination of cytokine protein levels in cervical mucus samples from young women by a multiplex immunoassay method and assessment of correlates.

Cytokines in cervical mucus are likely to play important roles in controlling pathogens. The cervical mucosal environment is complex, however, with many endogenous and exogenous factors that may affect cytokine levels. We used a multiplex, suspension-array-based immunoassay method to measure 10 proinflammatory (interleukin-1beta [IL-1beta], IL-6, and IL-8) and immunoregulatory (gamma interferon [IFN-gamma], IL-2, IL-4, IL-5, IL-10, IL-12, and IL-13) cytokines in cervical mucus specimens collected via ophthalmic sponge from 72 healthy, nonpregnant women and correlate their levels with biologic and behavioral covariates in a cross-sectional design. Proinflammatory and immunoregulatory cytokines were readily detected, although proinflammatory cytokines were present at markedly higher levels than were immunoregulatory cytokines. Among the covariates examined, the most striking finding was the significant (P < or = 0.05) association between depressed levels of the cytokines IFN-gamma, IL-1beta, IL-6, and IL-10 and cigarette smoking. Also, nonsignificant trends toward lower cytokine levels were found in the settings of incident and persistent human papillomavirus infection. The ready detection of proinflammatory cytokines may be reflective of the female genital tract as an anatomic site that is constantly exposed to immunogenic stimulation. Cigarette smoking appears to downregulate cytokine responses in the cervical mucosa, which may help explain the implicated role of tobacco use as a cofactor for cervical cancer development.

Asprich: A novel aspartic acid-rich protein family from the prismatic shell matrix of the bivalve Atrina rigida.

Almost all mineralized tissues contain proteins that are unusually acidic. As they are also often intimately associated with the mineral phase, they are thought to fulfill important functions in controlling mineral formation. Relatively little is known about these important proteins, because their acidic nature causes technical difficulties during purification and characterization procedures. Much effort has been made to overcome these problems, particularly in the study of mollusk-shell formation. To date about 16 proteins from mollusk-shell organic matrices have been sequenced, but only two are unusually rich in aspartic and glutamic acids. Here we screened a cDNA library made from the mRNA of the shell-forming cells of a bivalve, Atrina rigida, using probes for short Asp-containing repeat sequences, and identified ten different proteins. Using more specific probes designed from one subgroup of conserved sequences, we obtained the full sequences of a family of seven aspartic acid-rich proteins, which we named "Asprich"; a subfamily of the unusually acidic shell-matrix proteins. Polyclonal antibodies raised against a synthetic peptide of the conserved acidic1 domain of these proteins reacted specifically with the matrix components of the calcitic prismatic layer, but not with those of the aragonitic nacreous layer. Thus the Asprich proteins are constituents of the prismatic layer shell matrix. We can identify different domains within these sequences, including a signal peptide characteristic of proteins destined for extracellular secretion, a conserved domain rich in aspartic acid that contains a sequence very similar to the calcium-binding domain of Calsequestrin, and another domain rich in aspartic acid, that varies between the seven sequences. We also identified a domain with DEAD repeats that may have Mg-binding capabilities. Although we do not know, as yet, the function of these proteins, their generally conserved sequences do indicate that they might well fulfill basic functions in shell formation.

The sea urchin stem-loop-binding protein: a maternally expressed protein that probably functions in expression of multiple classes of histone mRNA.

Following the completion of oogenesis and oocyte maturation, histone mRNAs are synthesized and stored in the sea urchin egg pronucleus. Histone mRNAs are the only mRNAs that are not polyadenylated but instead end in a stem-loop which has been conserved in evolution. The 3' end binds the stem-loop-binding protein (SLBP), and SLBP is required for histone pre-mRNA processing as well as translation of the histone mRNAs. A cDNA encoding a 59 kDa sea urchin SLBP (suSLBP) has been cloned from an oocyte cDNA library. The suSLBP contains an RNA-binding domain that is similar to the RNA-binding domain found in SLBPs from other species, although there is no similarity between the rest of the suSLBP and other SLBPs. The suSLBP is present at constant levels in eggs and for the first 12 h of development. The levels of suSLBP then decline and remain at a low level for the rest of embryogenesis. The suSLBP is concentrated in the egg pronucleus and is released from the nucleus only when cells enter the first mitosis. SuSLBP expressed by in vitro translation does not bind the stem-loop RNA, suggesting that suSLBP is modified to activate RNA binding in sea urchin embryos.

Cyclin D and cdk4 are required for normal development beyond the blastula stage in sea urchin embryos.

cdk4 mRNA and protein are constitutively expressed in sea urchin eggs and throughout embryonic development. In contrast, cyclin D mRNA is barely detectable in eggs and early embryos, when the cell cycles consist of alternating S and M phases. Cyclin D mRNA increases dramatically in embryos at the early blastula stage and remains at a constant level throughout embryogenesis. An increase in cdk4 kinase activity occurs concomitantly with the increase in cyclin D mRNA. Ectopic expression of cyclin D mRNA in eggs arrests development before the 16-cell stage and causes eventual embryonic death, suggesting that activation of cyclin D/cdk4 in cleavage cell cycles is lethal to the embryo. In contrast, blocking cyclin D or cdk4 expression with morpholino antisense oligonucleotides results in normal development of early gastrula-stage embryos but abnormal, asymmetric larvae. These results suggest that in sea urchins, cyclin D and cdk4 are required for normal development and perhaps the patterning of the developing embryo, but may not be directly involved in regulating entry into the cell cycle.