Multilocus linkage identifies two new loci for a mendelian form of stroke, cerebral cavernous malformation, at 7p15-13 and 3q25.2-27

Cerebral cavernous malformation (CCM) is a Mendelian model of stroke, characterized by focal abnormalities in small intracranial blood vessels leading to hemorrhage and consequent strokes and/or seizures. A significant fraction of cases is inherited as an autosomal dominant trait with incomplete penetrance. Among Hispanic Americans, virtually all CCM is attributable to a founder mutation localized to 7q ( CCM1 ). Recent analysis of non-Hispanic Caucasian kindreds, however, has excluded linkage to 7q in some, indicating at least one additional CCM locus. We now report analysis of linkage in 20 non-Hispanic Caucasian kindreds with familial CCM. In addition to linkage to CCM1, analysis of linkage demonstrates linkage to two new loci, CCM2 at 7p13-15 and CCM3 at 3q25.2-27. Multilocus analysis yields a maximum lod score of 14.11, with 40% of kindreds linked to CCM1, 20% linked to CCM2 and 40% linked to CCM3, with highly significant evidence for linkage to three loci (linkage to three loci supported with an odds ratio of 2.6 x 10(5):1 over linkage to two loci and 1.6 x 10(9):1 over linkage to one locus). Multipoint analysis among families with high posterior probabilities of linkage to each locus refines the locations of CCM2 and CCM3 to approximately 22 cM intervals. Linkage to these three loci can account for inheritance of CCM in all kindreds studied. Significant locus-specific differences in penetrance are identified. These findings have implications for genetic testing of this disorder and represent an important step toward identification of the molecular basis of this disease.     

Lithium protection against oxygen toxicity in rats: ammonia and amino acid metabolism

1. The use of Li pre-treatment in rats before high pressure oxygen exposure has been reported effective in controlling convulsions. This is an effect which is better demonstrated if exposure to oxygen follows shortly after Li injection than exposure following several hours later. 2. This study has investigated the hypothesis that the protective action of Li may be exerted, in the short term, by its removing ammonia from the blood and alleviating the latter's known toxic action. 3. A normal Li distribution time profile in unstressed rat brain and blood following intraperitoneal injection has been established. Brain and blood ammonia, amino acids and Li concentrations were also measured in Li-treated animals exposed and convulsed by oxygen. These measurements were made both shortly (15 min) and also several hours after (24 hr) Li treatment. Ammonia and amino acid values in Li-protected groups were compared to normal unstressed animal values and also to values in animals convulsed by oxygen unprotected by Li pre-treatment. 4. In rat brain abd blood significant (P less than 0-001) elevation of ammonia and glutamine and depression of gamma-amino butyric acid (brain only) and glutamate was noted following oxygen treatment in unprotected animals. Prior injection of Li 15 min before high pressure oxygen exposure delayed convulsions twice as long. Additionally if these animals were only exposed to oxygen for a period of time equal to that which would normally produce convulsions in unprotected animals, brain and blood ammonia and amino acids were maintained near to unstressed animal levels. Concomitantly, blood Li concentrations were considerably depressed below the values one would expect from the previously determined Li distribution time profile. 5. In rats exposed to high pressure oxygen 24 hr after Li treatment there was no protective action against high pressure oxygen convulsion, rather a potentiating effect for convulsion was seen. 6. These data present compelling evidence for the controlling effect of Li in rats, on rising blood ammonia concentration which occurs in high pressure oxygen exposure. The effect might well be due to the known chelating properties of Li with ammonia.     

Physician reluctance to discuss advance directives. An empiric investigation of potential barriers

BACKGROUND: To determine the relative impact of five proposed barriers to physician usage of advance directives with the aim of increasing the number of advance directives generated. METHODS: Questionnaires were sent to 460 internal medicine resident and attending physicians at a large New York, NY, hospital. Of these, 277 (60%) responded. We used multiple regression to measure the impact of five barriers to physician-initiated discussions of advance directives (time constraints, compensation concerns, discomfort with the subject, beliefs about appropriateness, and lack of understanding) on respondents' estimates of the number of advance directives held by respondents' patients, the number of recent advance directive discussions, and the number of discussions initiated by physicians. RESULTS: Physician lack of understanding and erroneous beliefs about appropriateness had particularly strong effects, serving as barriers to recent advance directive discussions (P < .0001 and P < .0001, respectively) and total number of advance directives held (P < .0001 and P < .02). Physicians' lack of knowledge also served as a barrier to the percentage of discussions that were physician initiated (P < .003 and P < .04). Time constraints and lack of comfort affected only discussions that were physician initiated (P < .001). Compensation concerns did not appear to serve as a barrier. Respondents were supportive of the concept of advance directives but reported minimal use of them in appropriate situations. CONCLUSIONS: Attention can now be focused on methods to overcome the five barriers studied and thereby enhance the execution of advance directives.     

SPARC/osteonectin induces matrix metalloproteinase 2 activation in human breast cancer cell lines

Activation of the matrix metalloproteinase 2 (MMP-2) has been shown to play a major role in the proteolysis of extracellular matrix (ECM) associated with tumor invasion. Although the precise mechanism of this activation remains elusive, levels of the membrane type 1-MMP (MT1-MMP) at the cell surface and of the tissue inhibitor of MMP-2 (TIMP-2) appear to be two important determinants. Induction of MMP-2 activation in cells cultivated on collagen type I gels indicated that the ECM is important in the regulation of this process. In this study, we show that SPARC/osteonectin, a small ECM-associated matricellular glycoprotein, can induce MMP-2 activation in two invasive breast cancer cell lines (MDA-MB-231 and BT549) but not in a noninvasive counterpart (MCF-7), which lacks MT1-MMP. Using a set of peptides from different regions of SPARC, we found that peptide 1.1 (corresponding to the NH2-terminal region of the protein) contained the activity that induced MMP-2 activation. Despite the requirement for MT1-MMP, seen in MCF-7 cells transfected with MT1-MMP, the activation of MMP-2 by SPARC peptide 1.1 was not associated with increased steady-state levels of MT1-MMP mRNA or protein in either MT1-MMP-transfected MCF-7 cells or constitutively expressing MDA-MB-231 and BT549 cells. We did, however, detect decreased levels of TIMP-2 protein in the media of cells incubated with peptide 1.1 or recombinant SPARC; thus, the induction of MMP-2 activation by SPARC might be due in part to a diminution of TIMP-2 protein. We conclude that SPARC, and specifically its NH2-terminal domain, regulates the activation of MMP-2 at the cell surface and is therefore likely to contribute to the proteolytic pathways associated with tumor invasion.     

Transcription factor E2F controls the reversible gamma delta T cell growth arrest mediated through WC1

IL-2-stimulated expansion of T cells requires continued and sequential passage of the dividing cells through a major cell cycle check point in the G1 phase. We have previously shown that a gamma delta T cell-specific surface receptor, WC1, induces G0/G1 growth arrest, reversible with Con A, in proliferating IL-2-dependent gamma delta T cells. We now show that this reversible WC1-induced cell cycle arrest is correlated with induction of the cyclin kinase inhibitor p27kip1 and an associated down-regulation in cyclins A, D2, and D3 expression, along with dephosphorylation of pocket proteins p107, p130, and pRb. Together with diminished pocket protein phosphorylation, p107 expression levels are significantly down-regulated in response to WC1 stimulation. This coordinated sequence of signaling events is focused on E2F regulation so that, downstream of the pocket proteins, WC1 stimulation results in a diminished DNA binding activity for free E2F as a consequence of reduced E2F1 expression, whereas E2F4 expression is unaffected. Consistent with this interpretation, overexpression of E2F1 overcomes the growth-arresting effects induced by WC1 stimulation. Finally, in accordance with our previous observations at both the cellular and molecular level, subsequent mitogen stimulation can reverse all the above changes induced by WC1. These results, focused on E2F regulation, therefore provide a first insight into the effects of both positive (mitogen) and negative (anti-WC1) stimuli on cell cycle control in IL-2-dependent gamma delta T cells.     

Insights into T cell development and signal transduction provided by TCR-zeta chain deficient mice

The T cell antigen receptor (TCR) transduces signals that mediate different responses depending on the stage of development of the T cell and the nature of the ligand it engages. The presence of multiple signal transducing subunits (CD3-gamma-delta,-epsilon and zeta chain) suggests the potential to control these responses by altering the subunit composition of the TCR. zeta chain represents an especially important signalling molecule as it contains multiple signalling motifs within its cytoplasmic tail. The generation and analysis of zeta deficient (zeta-/-) and zeta-transgenic mice has provided insight into the role of zeta as well as the CD3 subunits in TCR surface expression, T cell activation and thymocyte development. Herein, we discuss the results from such experiments which suggest distinct roles for zeta chain and the CD3 components at different stages of T cell development.     

Critical roles for the Bcl-3 oncoprotein in T cell-mediated immunity, splenic microarchitecture, and germinal center reactions

Chromosomal translocations of bcl-3 are associated with chronic B cell lymphocytic leukemias. Previously, we have shown that Bcl-3, a distinct member of the I kappa B family, may function as a positive regulator of NF-kappa B activity, although its physiologic roles remained unknown. To uncover these roles, we generated Bcl-3-deficient mice. Mutant mice, but not their littermate controls, succumb to T. gondii owing to failure to mount a protective T helper 1 immune response. Bcl-3-deficient mice are also impaired in germinal center reactions and T-dependent antibody responses to influenza virus. The results reveal critical roles for Bcl-3 in antigen-specific priming of T and B cells. Altered microarchitecture of secondary lymphoid organs in mutant mice, including partial loss of B cells, may underlie the immunologic defects. The implied role of Bcl-3 in maintaining B cells in wild-type mice may related to its oncogenic potential.