Stimulation of T cells via the CD3±T-cell receptor (TCR) complex results in rapid increases in b1 integrin-mediated adhesion via poorly de®ned intracellular signaling events. We demonstrate that TCR-mediated activation of b1 integrins requires activation of the Tec family tyrosine kinase Itk and phosphatidylinositol 3-kinase (PI 3-K)-dependent recruitment of Itk to detergent-insoluble glycosphingolipid-enriched microdomains (DIGs) via binding of the pleckstrin homology domain of Itk to the PI 3-K product PI(3,4,5)-P 3 . Activation of PI 3-K and the src family kinase Lck, via stimulation of the CD4 co-receptor, can initiate b1 integrin activation that is dependent on Itk function. Targeting of Itk speci®cally to DIGs, coupled with CD4 stimulation, can also activate b1 integrin function independently of TCR stimulation. Changes in b1 integrin function mediated by TCR activation of Itk are also accompanied by Itkdependent modulation of the actin cytoskeleton. Thus, TCR-mediated activation of b1 integrins involves membrane relocalization and activation of Itk via coordinate action of PI 3-K and a src family tyrosine kinase.

Yeast U1 snRNA (568 nucleotides) is 3.5-fold larger than its mammalian counterpart (164 nucleotides) and contains apparent sequence homology only at the 5' and 3' ends. We have used deletion analysis to determine whether the yeast-specific U1 sequences play essential roles in vivo. Yeast cells carrying a deletion of more than 60% (355 nucleotides) of the single-copy U1 gene are viable, though slow-growing, while a deletion of 316 nucleotides allows essentially wild-type growth. The boundaries of the viable deletions define a dispensable internal domain which comprises sequences unique to yeast. In contrast, the essential 5' and 3' terminal domains correspond to phylogenetically conserved sequences and/or structures previously implicated in RNA:RNA and RNA:protein interactions. The minimal essential sequences of yeast U1 can be drawn in a secondary structure which resembles metazoan U1 in four of seven structural domains.

Abstract:The primary function of the female ovary is the production of a mature and viable oocyte capable of fertilization and subsequent embryo development and implantation. At birth, the ovary contains a fi nite number of oocytes available for folliculogenesis. This fi nite number of available oocytes is termed "the ovarian reserve". The determination of ovarian reserve is important in the assessment and treatment of infertility. As the ovary ages, the ovarian reserve will decline. Infertility affects approximately 15%-20% of reproductive aged couples. The most commonly used biomarker assay to assess ovarian reserve is the measurement of follicle stimulating hormone (FSH) on day 3 of the menstrual cycle. However, antimüllerian hormone and inhibin-B are other biomarkers of ovarian reserve that are gaining in popularity since they provide direct determination of ovarian status, whereas day 3 FSH is an indirect measurement. This review examines the physical tools and the hormone biomarkers used to evaluate ovarian reserve.

The CD2 molecule is one of several lymphocyte receptors that rapidly initiates signaling events regulating integrin-mediated cell adhesion. CD2 stimulation of resting human T cells results within minutes in an increase in ␤1-integrin-mediated adhesion to fibronectin. We have utilized the HL60 cell line to map critical residues within the CD2 cytoplasmic domain involved in CD2 regulation of integrin function. A panel of CD2 cytoplasmic domain mutants was constructed and analyzed for their ability to upregulate integrin-mediated adhesion to fibronectin. Mutations in the CD2 cytoplasmic domain implicated in CD2-mediated interleukin-2 production or CD2 avidity do not affect CD2 regulation of integrin activity. A proline-rich sequence, K-G-P-P-L-P (amino acids 299 to 305), is essential for CD2-mediated regulation of ␤1 integrin activity. CD2-induced increases in ␤1 integrin activity could be blocked by two phosphoinositide 3-kinase (PI 3-K) inhibitors or by overexpression of a dominant negative form of the p85 subunit of PI 3-K. In addition, CD2 cytoplasmic domain mutations that abrogate CD2-induced increases in integrin-mediated adhesion also ablate CD2-induced increases in PI 3-K enzymatic activity. Surprisingly, CD2 cytoplasmic domain mutations that inhibit CD2 regulation of adhesion do not affect the constitutive association of the p85 subunit of PI 3-K association with CD2. Mutation of the proline residues in the K-G-P-P-L-P motif to alanines prevented CD2-mediated activation of integrin function and PI 3-K activity but not mitogen-activated protein (MAP) kinase activity. Furthermore, the MEK inhibitor PD 098059 blocked CD2-mediated activation of MAP kinase but had no effect on CD2-induced adhesion. These studies identify a proline-rich sequence in CD2 critical for PI 3-K-dependent regulation of ␤1 integrin adhesion by CD2. In addition, these studies suggest that CD2-mediated activation of MAP kinase is not involved in CD2 regulation of integrin adhesion.T lymphocytes continuously migrate throughout the body, mediating immune responses to foreign antigens. The capacity for normal T cells to function, develop, and migrate depends upon adhesive contacts with other cells as well as with extracellular matrix (ECM) components (76). These adhesive contacts are regulated by and initiate a complex series of elegantly controlled molecular signaling events. Members of the integrin superfamily of adhesion molecules are involved in the cell-cell and cell-ECM interactions that are essential for T-lymphocyte function and migration. The integrins are a family of ␣␤ heterodimeric cell surface receptors with a vast tissue distribution (67). There are at least 20 different integrin heterodimers, with each heterodimer containing 1 of 8 ␤ chains and 1 of 14 ␣ chains. Members of the ␤1 integrin subfamily bind to components of the ECM, such as fibronectin (FN), as well as cell surface counter-receptors, such as VCAM-1 (34). The functional activity of integrins on circulating leukocytes is dynamically regulated by signaling events (21...

Virtually all pre-mRNA introns begin with the sequence /GU and end with AG/ (where / indicates a border between an exon and an intron). We have previously shown that the G residues at the first and last positions of the yeast actin intron interact during the second step of splicing. In this work, we ask if other highly conserved intron nucleotides also take part in this /G-G/ interaction. Of special interest is the penultimate intron nucleotide (AG/), which is important for the second step of splicing and is in proximity to other conserved intron nucleotides. Therefore, we tested interactions of the penultimate intron nucleotide with the second intron nucleotide (/GU) and with the branch site nucleotide. We also tested two models that predict interactions between sets of three conserved intron nucleotides. In addition, we used random mutagenesis and genetic selection to search for interactions between nucleotides in the pre-mRNA. We find no evidence for other interactions between intron nucleotides besides the interaction between the first and last intron nucleotides.