Characterization of cDNAs encoding a new family of tetraspanins from schistosomes--the Sj25 family

Asr is a family of genes that maps to chromosome 4 of tomato. Asr2, a recently reported member of this family, is believed to be regulated by abscisic acid (ABA), stress and ripening. A genomic Asr2 clone has been fully sequenced, and candidate upstream regulatory elements have been identified. To prove that the promoter region is functional in vivo, we fused it upstream of the beta-glucuronidase (GUS) reporter gene. The resulting chimeric gene fusion was used for transient expression assays in papaya embryogenic calli and leaves. In addition, the same construct was used to produce transgenic tomato, papaya, tobacco, and potato plants. Asr2 upstream sequences showed promoter function in all of these systems. Under the experimental conditions tested, ABA stimulated GUS expression in papaya and tobacco, but not in tomato and potato systems.     

A role for the cadherin family of cell adhesion molecules in hippocampal long-term potentiation

The cadherins are a family of cell-cell adhesion molecules that mediate Ca2+-dependent homophilic interactions between cells and transduce signals by interacting with cytoplasmic proteins. In the hippocampus, immunostaining combined with confocal microscopy revealed that both neural- (N-) and epithelial- (E-) cadherin are present at synaptic sites, implying a role in synaptic function. Pretreatment of hippocampal slices with antibodies (Abs) raised against the extracellular domain of either N-cad or E-cad had no effect on basal synaptic properties but significantly reduced long-term potentiation (LTP). Infusion of antagonistic peptides containing the His-Ala-Val (HAV) consensus sequence for cadherin dimerization also attenuated LTP induction without affecting previously established LTP. Because the intense synaptic stimulation associated with LTP induction might transiently deplete extracellular Ca2+ and hence potentially destabilize cadherin-cadherin interactions, we examined whether slices could be protected from inhibition by N-cad Abs or HAV peptides by raising the extracellular Ca2+ concentration. Indeed, we found that high extracellular Ca2+ prevented the block of LTP by these agents. Taken together, these results indicate that cadherins are involved in synaptic plasticity, and the stability of cadherin-cadherin bonds may be regulated by synaptic stimulation.     

Characterization of Brx, a novel Dbl family member that modulates estrogen receptor action

Regulation of gene activation by the estrogen receptor (ER) is complex and involves co-regulatory proteins, oncoproteins (such as Fos and Jun), and phosphorylation signaling pathways. Here we report the cloning and initial characterization of a novel protein, Brx, that contains a region of identity to the oncogenic Rho-guanine nucleotide exchange (Rho-GEF) protein Lbc, and a unique region capable of binding to nuclear hormone receptors, including the ER. Western and immunohistochemistry studies showed Brx to be expressed in estrogen-responsive reproductive tissues, including breast ductal epithelium. Brx bound specifically to the ER via an interaction that required distinct regions of ER and Brx. Furthermore, overexpression of Brx in transfection experiments using an estrogen-responsive reporter revealed that Brx augmented gene activation by the ER in an element-specific and ligand-dependent manner. Moreover, activation of ER by Brx could be specifically inhibited by a dominant-negative mutant of Cdc42Hs, but not by dominant negative mutants of RhoA or Rac1. Taken together, these data suggest that Brx represents a novel modular protein that may integrate cytoplasmic signaling pathways involving Rho family GTPases and nuclear hormone receptors.     

ICAAR, a novel member of a new family of transmembrane, tyrosine phosphatase-like proteins

We have isolated a cDNA from human foetal brain cDNA library which encodes a putative transmembrane protein bearing an intracellular protein tyrosine phosphatase (PTPase) like domain. The PTPase like domain contains an alanine to aspartate amino acid change relative to other PTPases in the catalytic core domain. This amino acid change is found in only three other known proteins, islet cell autoantigens; human, murine and rat IA-2, murine IA-2b and its rat orthologue phogrin, which have a similar overall structure to ICAAR, and the recently identified X-linked myotubular myopathy (MTM1) gene. ICAAR, IA-2 and IA-2b clearly represent a new family of PTP-like proteins for which catalytic activity has yet to be demonstrated. An abundant ICAAR mRNA is detectable in the brain and pancreas but not in the other normal human tissues surveyed. We have localised ICAAR to human chromosome 7q36.     

Role of a new Rho family member in cell migration and axon guidance in C. elegans

Rho family GTPases are thought to regulate actin-dependent processes, but their functions in vivo are still poorly understood. We have investigated the function of a new, widely expressed Rho family member in C. elegans by analyzing mutations in the endogenous gene. Activated and null alleles all inhibit cell migration, demonstrating that this protein is required for cell migration in vivo. Only a small subset of the migrations inhibited by activating mutations are inhibited by null mutations, suggesting that considerable functional redundancy exists within this system. Our findings support this conclusion and show that mig-2 functions redundantly with another pathway to regulate nuclear migration. Surprisingly, activated alleles also cause misguided axon growth, suggesting that Rho family GTPases may couple guidance cues to process outgrowth.     

Characterization of a novel dentin matrix acidic phosphoprotein. Implications for induction of biomineralization

Acidic phosphorylated proteins have been shown to be prominent constituents of the extracellular matrix of bone and dentin. The acidic phosphoproteins of bone contain more glutamic acid than aspartic acid and a lower serine content than either. On the other hand, the major dentin acidic phosphoproteins, phosphophoryns, have been defined as aspartic acid- and serine-rich proteins, with a lesser content of glutamic acid. Both sets of phosphoproteins have been implicated as key participants in regulating mineralization, but it has been difficult to unify their mechanisms of action. We have now identified, by cDNA cloning, a new serine-rich acidic protein of the dentin matrix, AG1, with a composition intermediate between the bone acidic proteins and dentin phosphophoryns. AG1 has numerous acidic consensus sites for phosphorylation by both casein kinases I and II. Immunochemical and organ culture biosynthetic studies show that AG1 is present in phosphorylated form at low levels in the dentin matrix. If fully phosphorylated, AG1 would bear a net charge of -175/molecule of 473 residues. AG1 contains single RGD integrin binding and N-glycosylation sequences. The overall picture that emerges is that of a matrix-associated acidic phosphoprotein, with a potentially high calcium ion binding capacity, present at levels compatible with a regulatory role in dentin mineralization.     

Self-expressiveness within the family context: Psychometric support for a new measure.

The family appears to be an important setting for learning about emotions and how to express them within a social context. Because of the need for reliable and valid measures of emotional expressiveness in the family, the Self-Expressiveness in the Family Questionnaire is introduced and evaluated in four studies with 499 mothers and 362 fathers. Factor analyses indicate highly consistent patterns of loadings for a two-factor solution across the four studies. The resulting positive and negative scales are highly internally consistent and stable over time. Evidence of good convergent, discriminant, and construct validity was obtained, and a preliminary short form with good internal consistency and construct validity was also identified. Ideas for future research on marital and parent–child issues are suggested. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Relation of family subsystems to adolescent depression: Implementing a new family assessment strategy.

A multitrait-multimethod (or more precisely, a multidyad-multiperspective) approach to family assessment is used to investigate the relation of conflict, cohesion, and expressiveness in family subsystems to depression in a sample of 107 high school students. Confirmatory factor analysis and structural equation modeling revealed that (1) mothers' reports tended to be more reliable and adolescents' reports tended to be less reliable than others; (2) substantial differences existed between family dyads, thus calling into question the utility of global family constructs; (3) effects of marital conflict and cohesion on adolescent depression were entirely mediated by the parent–adolescent relationships; (4) father–adolescent conflict and cohesion were more strongly related to adolescent depression than were mother–adolescent conflict and cohesion; and (5) expressiveness was unrelated to adolescent depression. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Structural requirements for outside-in and inside-out signaling by Drosophila neuroglian, a member of the L1 family of cell adhesion molecules

Expression of the Drosophila cell adhesion molecule neuroglian in S2 cells leads to cell aggregation and the intracellular recruitment of ankyrin to cell contact sites. We localized the region of neuroglian that interacts with ankyrin and investigated the mechanism that limits this interaction to cell contact sites. Yeast two-hybrid analysis and expression of neuroglian deletion constructs in S2 cells identified a conserved 36-amino acid sequence that is required for ankyrin binding. Mutation of a conserved tyrosine residue within this region reduced ankyrin binding and extracellular adhesion. However, residual recruitment of ankyrin by this mutant neuroglian molecule was still limited to cell contacts, indicating that the lack of ankyrin binding at noncontact sites is not caused by tyrosine phosphorylation. A chimeric molecule, in which the extracellular domain of neuroglian was replaced with the corresponding domain from the adhesion molecule fasciclin II, also selectively recruited ankyrin to cell contacts. Thus, outside-in signaling by neuroglian in S2 cells depends on extracellular adhesion, but does not depend on any unique property of its extracellular domain. We propose that the recruitment of ankyrin to cell contact sites depends on a physical rearrangement of neuroglian in response to cell adhesion, and that ankyrin binding plays a reciprocal role in stabilizing the adhesive interaction.     

Evidence for a novel bursting mechanism in rodent trigeminal neurons

We investigated bursting behavior in rodent trigeminal neurons. The essential mechanisms operating in the biological systems were determined based on testable predictions of mathematical models. Bursting activity in trigeminal motoneurons is consistent with a traditional mechanism employing a region of negative slope resistance in the steady-state current-voltage relationship (Smith, T. G. 1975. Nature. 253:450-452). However, the bursting dynamics of trigeminal interneurons is inconsistent with the traditional mechanisms, and is far more effectively explained by a new model of bursting that exploits the unique stability properties associated with spike threshold (Baer, S. M., T. Erneux, and J. Rinzel. 1989. SIAM J. Appl. Math. 49:55-71).     

Evidence for a third component in neutrophil aggregation: potential roles of O-linked glycoproteins as L-selectin counter-structures

The homotypic aggregation of neutrophils requires the participation of L-selectin and the beta 2-integrins, but it has not been clear whether the two receptors recognize one another as counter-structures or whether other adhesion molecules are involved. We have examined aggregation of live neutrophils with target populations, manipulated to alter expression of adhesive epitopes, using flow cytometry. A target population depleted of both L-selectin and activatable beta 2-integrin displayed an ability to aggregate with live neutrophils, suggesting that these two molecules are not counter-structures. We also found that an O-sialoglycoprotease (GCP) from Pasteurella haemolytica is capable of inhibiting homotypic aggregation. Neutrophils treated with GCP lose O-glycosylated proteins but retain L-selectin and activatable beta 2-integrin. One or more of the GCP substrates appears to function in L-selectin-dependent binding but not in beta 2-integrin-dependent binding. Together the data suggest a mechanism of aggregation that is analogous to leukocyte-endothelial cell adhesion in which a low-affinity carbohydrate-dependent interaction precedes a high-affinity integrin-dependent adhesion.     

Identification of rCop-1, a new member of the CCN protein family, as a negative regulator for cell transformation

By using a model system for cell transformation mediated by the cooperation of the activated H-ras oncogene and the inactivated p53 tumor suppressor gene, rCop-1 was identified by mRNA differential display as a gene whose expression became lost after cell transformation. Homology analysis indicates that rCop-1 belongs to an emerging cysteine-rich growth regulator family called CCN, which includes connective-tissue growth factor, CYR61, CEF10 (v-src inducible), and the product of the nov proto-oncogene. Unlike the other members of the CCN gene family, rCop-1 is not an immediate-early gene, it lacks the conserved C-terminal domain which was shown to confer both growth-stimulating and heparin-binding activities, and its expression is lost in cells transformed by a variety of mechanisms. Ectopic expression of rCop-1 by retroviral gene transfers led to cell death in a transformation-specific manner. These results suggest that rCop-1 represents a new class of CCN family proteins that have functions opposing those of the previously identified members.     

Characterization of a novel Streptococcus thermophilus rolling-circle plasmid used for vector construction

STUDY DESIGN: Bone mineral density and bone cross-sectional area of human cadaveric vertebral bodies were investigated radiologically and histologically, respectively. After ventral instrumentation with ventral derotation spondylodesis screws, axial pullout force was measured and compared with radiologic and histologic data. OBJECTIVES: To elucidate how well ventral derotation spondylodesis screw fixation strength can be estimated before surgery by specified applications of dual-energy x-ray absorptiometry, quantitative computed tomography, T2*-relaxation time in magnetic resonance imaging, and histomorphometry. SUMMARY OF BACKGROUND DATA: It is postulated that bone quality plays a crucial role in initial strength of the instrumented spine. Bone quality is even more important in anterior fixation because of the prevalence of spongy bone in the vertebral body. METHODS: Bone mineral density of human cadaveric lumbar-vertebral bodies was assessed by dual-energy x-ray absorptiometry and quantitative computed tomography (cancellous and cortical bone separately). Cancellous bone was also characterized by T2*-relaxation time, measured by magnetic resonance imaging and histomorphometric study. Vertebral bodies were instrumented ventrally with VDS screws, and screw axial pullout force was measured and correlated with each of the nonmechanical measures. Patients with manifest osteoporosis, osteomalacia and tumors were excluded. For statistical analysis, the Mann-Whitney rank sum test was used with a significance value of P < 0.05. RESULTS: The highest correlation with pullout force was for density of cancellous bone determined by quantitative computed tomography (r = 0.72; P < 0.001), immediately followed that determined by dual-energy x-ray absorptiometry (r = 0.70; P < 0.001). Results of measurement of T2*-relaxation time and those of histomorphometric study correlated moderately (r = 0.55; r = 0.50), whereas cortical bone density determined by quantitative computed tomography showed negligible correlation (r = 0.2). CONCLUSIONS: The absorptiometric techniques, quantitative computed tomographic scan of cancellous bone and dual-energy x-ray absorptiometric study, provide more accurate readings for preoperative estimation of initial VDS screw fixation strength than do the other methods studied.     

Palmitoylation of neurofascin at a site in the membrane-spanning domain highly conserved among the L1 family of cell adhesion molecules

This report presents the first evidence that a member of the L1 family of nervous system cell-adhesion molecules is covalently modified by thioesterification with palmitate, and identifies a highly conserved cysteine in the predicted membrane-spanning domain as the site of modification. Neurofascin is constitutively palmitoylated at cysteine-1213 at close to a 1:1 molar stoichiometry. Kinetics of palmitate incorporation into neurofascin expressed in resting neuroblastoma cells indicate that the palmitate modification has the same turnover rate as the polypeptide chain and does not affect the protein stability of neurofascin. Palmitoylation of neurofascin expressed in dorsal root ganglion neurons is not required for delivery of neurofascin to the plasma membrane or targeting to axons. Palmitoylation also has no effect on ankyrin-binding activity of neurofascin, on the oligomeric state of neurofascin in solution, or on cell-adhesion activity of neurofascin expressed in neuroblastoma cells. A significant difference between native and C1213L neurofascin is that these proteins were localized in distinct fractions within a low-density membrane population enriched in signaling molecules. These results indicate a palmitate-dependent targeting of neurofascin to a specialized membrane microdomain.