Syntaxin 3b is a t-SNARE specific for ribbon synapses of the retina.

Previous studies have demonstrated that ribbon synapses in the retina do not contain the t-SNARE (target-soluble N-ethylmaleimide-sensitive factor attachment protein receptor) syntaxin 1A that is found in conventional synapses of the nervous system. In contrast, ribbon synapses of the retina contain the related isoform syntaxin 3. In addition to its localization in ribbon synapses, syntaxin 3 is also found in nonneuronal cells, where it has been implicated in the trafficking of transport vesicles to the apical plasma membrane of polarized cells. The syntaxin 3 gene codes for four different splice forms, syntaxins 3A, 3B, 3C, and 3D. We demonstrate here by using analysis of EST databases, RT-PCR, in situ hybridization, and Northern blot analysis that cells in the mouse retina express only syntaxin 3B. In contrast, nonneuronal tissues, such as kidney, express only syntaxin 3A. The two major syntaxin isoforms (3A and 3B) have an identical N-terminal domain but differ in the C-terminal half of the SNARE domain and the C-terminal transmembrane domain. These two domains are thought to be directly involved in synaptic vesicle fusion. The interaction of syntaxin 1A and syntaxin 3B with other synaptic proteins was examined. We found that both proteins bind Munc18/N-sec1 with similar affinity. In contrast, syntaxin 3B had a much lower binding affinity for the t-SNARE SNAP25 compared with syntaxin 1A. By using an in vitro fusion assay, we could demonstrate that vesicles containing syntaxin 3B and SNAP25 could fuse with vesicles containing synaptobrevin2/VAMP2, demonstrating that syntaxin 3B can function as a t-SNARE.

The last centuries of the 3rd millennium in the Syrian Ǧezīra

Overview on the settlement history of the Syrian Jezirah and the Middle Euphrates Valley in the period ca. 2300-1900 BC.

Cardiolipin membrane domains in prokaryotes and eukaryotes.

Cardiolipin (CL) plays a key role in dynamic organization of bacterial and mitochondrial membranes. CL forms membrane domains in bacterial cells, and these domains appear to participate in binding and functional regulation of multi-protein complexes involved in diverse cellular functions including cell division, energy metabolism, and membrane transport. Visualization of CL domains in bacterial cells by the fluorescent dye 10-N-nonyl acridine orange is critically reviewed. Possible mechanisms proposed for CL dynamic localization in bacterial cells are discussed. In the mitochondrial membrane CL is involved in organization of multi-subunit oxidative phosphorylation complexes and in their association into higher order supercomplexes. Evidence suggesting a possible role for CL in concert with ATP synthase oligomers in establishing mitochondrial cristae morphology is presented. Hypotheses on CL-dependent dynamic re-organization of the respiratory chain in response to changes in metabolic states and CL dynamic re-localization in mitochondria during the apoptotic response are briefly addressed.

Somatic and cognitive domains of depression in an underserved region of Ecuador: some cultural considerations.

Not enough research efforts on depression have been carried out up to now in Latin America. The knowledge that has resulted from research activities in the United States or Europe offers limited generalizability to other regions of the world, including Latin America. In the Andean highlands of Ecuador, we found very high rates of moderate and severe depressive symptoms, a finding that must be interpreted within its cultural context. Somatic manifestations of depression predominated over cognitive manifestations, and higher education level was protective against depression. These findings call for an appreciation of culturally-specific manifestations of depression and the social factors that influence them. These factors must be further studied in order to give them the deserved priority, allocate resources appropriately, and formulate innovative psychosocial interventions.

Enforced expression of Tbx1 in fetal thymic epithelial cells antagonizes thymus organogenesis

Enforced expression of Tbx1 in fetal thymic epithelial cells antagonizes thymus organogenesis Kim T. Cardenas The thymus and parathyroid glands originate from organ-specific domains of 3rd pharyngeal pouch (PP) endoderm. At embryonic day 11.5 (E11.5), the ventral thymus and dorsal parathyroid domains can be identified by Foxn1 and Gcm2 expression respectively. Neural crest cells, (NCCs) play a role in regulating patterning of 3rd PP endoderm. In addition, pharyngeal endoderm influences fate determination via secretion of Sonic hedgehog (Shh), a morphogen required for Gcm2 expression and generation of the parathyroid domain. Gcm2 is a downstream target of the transcription factor Tbx1, which in turn is positively regulated by Shh. Although initially expressed throughout pharyngeal pouch endoderm, Tbx1 expression is excluded from the thymus-specific domain of the 3rd PP by E10.5, but persists in the parathyroid domain. Based on these observations, we hypothesized that Tbx1 expression is non-permissive for thymus fate specification and that enforced expression of Tbx1 in the fetal thymus would impair thymus development. To test this hypothesis, we generated knock-in mice containing a Cre-inducible allele that allows for tissue-specific Tbx1 expression. Expression of the R26iTbx1 allele in fetal and adult thymus using Foxn1Cre resulted in severe thymus hypoplasia throughout ontogeny that persisted in the adult. Thymic epithelial cell (TEC) development was impaired as determined by immunohistochemical and FACS analysis of various differentiation markers. The relative level of Foxn1 expression in fetal TECs was significantly reduced. TECs in R26iTbx1/+ thymi assumed an almost universal expression of Plet-1, a marker associated with a TEC stem/progenitor cell fate. In addition, embryonic R26iTbx1/+ mice develop a perithymic mesechymal capsule that appears expanded compared to control littermates. Interestingly, thymi from neonatal and adult R26iTbx1/+ but not R26+/+ mice were encased in adipose tissue. This thymic phenotype also correlated with a decrease in thymocyte cellularity and aberrant thymocyte differentiation. The results to date support the conclusion that enforced expression of Tbx1 in TECs antagonizes their differentiation and prevents normal organogenesis via both direct and indirect effects.

Measuring Consumer Satisfaction in Family Preservation Services: Identifying Instrument Domains

Measuring consumer satisfaction in the social services has become an important source of feedback for the improvement of service delivery. Consumer satisfaction has recently been incorporated into family preservation evaluation. This article reviews instruments used to measure consumer satisfaction in family preservation services and other related areas. Trends in current practice are examined and instrument dimensions are identified. Finally, some recommendations are made about the application of consumer satisfaction measurement in family preservation services.

Experimental dissection and characterization of the functional domains in cardiac troponin C

Contraction of vertebrate cardiac muscle is regulated by the binding of Ca$\sp{2+}$ to the troponin C (cTnC) subunit of the troponin complex. In this study, we have used site-directed mutagenesis and a variety of assay techniques to explore the functional roles of regions in cTnC, including Ca$\sp{2+}$/Mg$\sp{2+}$-binding sites III and IV, the functionally inactive site I, the N-terminal helix, the N-terminal hydrophobic pocket and the two cysteine residues with regard to their ability to form disulfide bonds. Conversion of the first Ca$\sp{2+}$ ligand from Asp to Ala inactivated sites III and IV and decreased the apparent affinity of cTnC for the thin filament. Conversion of the second ligand from Asn to Ala also inactivated these sites in the free protein but Ca$\sp{2+}$-binding was recovered upon association with troponin I and troponin T. The Ca$\sp{2+}$-concentrations required for tight thin filament-binding by proteins containing second-ligand mutations were significantly greater than that required for the wild-type protein. Mutation of site I such that the primary sequence was that of an active site with the first Ca$\sp{2+}$ ligand changed from Asp to Ala resulted in a 70% decrease in maximal Ca$\sp{2\sp+}$ dependent ATPase activity in both cardiac and fast skeletal myofibrils. Thus, the primary sequence of the inactive site I in cTnC is functionally important. Major changes in the sequence of the N-terminus had little effect on the ability of cTnC to recover maximal activity but deletion of the first nine residues resulted in a 60 to 80% decrease in maximal activity with only a minor decrease in the pCa$\sb{50}$ of activation, suggesting that the N-terminal helix must be present but that a specific sequence is not required. The formation of an inter- or intramolecular disulfide bonds caused the exposure of hydrophobic surfaces on cTnC and rendered the protein Ca$\sp{2+}$ independent. Finally, elution patterns from a hydrophobic interactions column suggest that cTnC undergoes a significant change in hydrophobicity upon Ca$\sp{2+}$ binding, the majority of which is caused by site II. These latter data show an interesting correlation between exposure of hydrophobic surfaces on and activation of cTnC. Overall, these results represent significant progress toward the elucidation of the functional roles of a variety of structural regions in cTnC. ^

Human TNF receptor p75/80 gene structure and promoter characterization

Tumor necrosis factor receptor p75/80 ((TNF-R p75/80) is a 75 kDa type 1 transmembrane protein expressed predominately on cells of hematopoietic lineage. TNF-R p75/80 belongs to the TNF receptor superfamily characterized by cysteine-rich extracellular regions composed of three to six disulfide-linked domains. In the present report, we have characterized, for the first time, the complete gene structure for human TNF-R p75/80 which spans approximately 43 kbp. The gene consists of 10 exons (ranging from 34 bp to 2.5 kbp) and 9 introns (343 bp to 19 kbp). Consensus elements for transcription factors involved in T cell development and activation were noted in the 5$\sp\prime$ flanking region including TCF-1, Ikaros, AP-1, CK-2, IL-6RE, ISRE, GAS, NF-$\kappa$B and SP1, as well as an unusually high GC content and CpG frequency that appears characteristic of some TNF-R family members. The unusual (GATA)$\sb{\rm n}$ and (GAA)(GGA) repeats found within intron 1 may prove useful for further genome analysis within the 1p36 chromosomal locus. The human TNF-R p75/80 gene structure will permit further assessment of its involvement in normal hematopoietic cell development and function, autoimmune disease, and non-random translocations in hematopoietic malignancies. The region 1.8 kb 5$\sp\prime$ of the ATG was able to drive luciferase expression when transfected into cell lines expressing TNF-R p75/80. Further characterization of the 5$\sp\prime$-regulatory region will aid in determining factors and signal transduction pathways involved in regulating TNF-R p75/80 expression. ^

Phototaxis signaling by the membrane complex of archaeal sensory rhodopsins and their transducers

Sensory rhodopsins I and II (SRI and SRII) are visual pigment-like phototaxis receptors in the archaeon Halobacterium salinarum. The receptor proteins each consist of a single polypeptide that folds into 7 $\alpha$-helical membrane-spanning segments forming an internal pocket where the chromophore retinal is bound. They transmit signals to their tightly bound transducer proteins, HtrI and HtrII, respectively, which in turn control a phosphotransfer pathway modulating the flagellar motors. SRI-HtrI mediates attractant responses to orange-light and repellent responses to UV light, while SRII-HtrII mediates repellent response to blue light. Experiments were designed to analyze the molecular processes in the SR-Htr complexes responsible for receptor activation, which previously had been shown by our laboratory to involve proton transfer reactions of the retinylidene Schiff base in the photoactive site, transfer of signals from receptor to transducer, and signaling specificity by the receptor-transducer complex.^ Site-directed mutagenesis and laser-flash kinetic spectroscopy revealed that His-166 in SRI (i) plays a role in the proton transfers both to and from the Schiffbase, either as a structurally critical residue or possibly as a direct participant, (ii) is involved in the modulation of SIU photoreaction kinetics by HtrI, and (iii) modulates the pKa of Asp-76, an important residue in the photoactive site, through a long-distance electrostatic interaction. Computerized cell tracking and motion analysis demonstrated that (iv) His-166 is crucial in phototaxis signaling: a spectrum of substitutions either eliminate signaling or greatly perturb the activation process that produces attractant and repellent signaling states of the receptor.^ The signaling states of SRI are communicated to HtrI, whose oligomeric structure and conformational changes were investigated by engineered sulfhydryl probes. It was found that signaling by the SRI-HtrI complex involves reversible conformational changes within a preexisting HtrI dimer, which is likely accomplished through a slight winding or unwinding of the two HtrT monomers via their loose coiled coil association. To elucidate which domains of the Htr dimers confer specificity for interaction with SRI or SRII, chimeras of HtrI and HtrII were constructed. The only determinant needed for functional and specific interaction with SRI or SRII was found to be the four transmembrane segments of the HtrI or HtrII dimers, respectively. The entire cytoplasmic parts of HtrI and HtrII, which include the functionally important signaling and adaptation domains, were interchangeable.^ These observations support a model in which SRI and SRII undergo conformational changes coupled to light-induced proton transfers in their photoactive sites, and that lateral helix-helix interactions with their cognate transducers' 4-helix bundle in the membrane relay these conformational changes into different states of the Htr proteins which regulate the down-stream phosphotransfer pathway. ^

The evolution of paired domains

Pax genes are important developmental control genes. They are involved in nervous system development, organogenesis and oncogenesis. A DNA specific binding domain called the paired domain, which is well conserved during evolution, defines Pax genes. Furthermore, Pax genes are also conserved in terms of their functions. For example, the Pax-6 gene has been showed to be one of the master control genes for eye development both in Drosophila and vertebrates. All of these properties of Pax genes make them an excellent model for studying the evolution of gene function. ^ Molecular evolutionary studies of paired domain are carried out in this study. Five Pax genes from cnidarians, which are the most primitive organisms possessing a nervous system, were isolated and characterized for their DNA binding properties. By combining data obtained from Genbank and this study, the phylogenetic relationship between Pax genes was studied. It was found that Pax genes could be divided into five groups: Pax-1/9, Pax-3 /7, Pax-A, Pax-2/5/ 8/B, and Pax- 4/6. Furthermore, Pax-2/5/8/ B, Pax-A and Pax-4/6 could be clustered into a supergroup I, while Pax-1/9 and Pax-3/7 could be clustered into supergroup II. The phylogeny was also supported by studies on DNA binding properties of paired domains from different groups. A statistical method was applied to infer the critical amino acid residue substitutions between two supergroups and within the supergroup I. It was found that two amino acid residues were mainly responsible for the difference of DNA binding between two supergroups, while only one amino acid was critical for the evolution of novel DNA binding properties of Pax-4/6 group from ancestor. Evolutionary implications of these data are also discussed. ^

Role of transducer proteins in photoaxis signaling of Halobacterium salinarum

In Halobacterium salinarum phototaxis is mediated by the visual pigment-like photoreceptors sensory rhodopsin I (SRI) and II (SRII). SRI is a receptor for attractant orange and repellent UV-blue light, and SRII is a receptor for repellent blue-green light, and transmit signals through the membrane-bound transducer proteins HtrI and HtrII, respectively. ^ The primary sequences of HtrI and HtrII predict 2 transmembrane helices (TM1 and TM2) followed by a hydrophilic cytoplasmic domain. HtrII shows an additional large periplasmic domain for chemotactic ligand binding. The cytoplasmic regions are homologous to the adaptation and signaling domains of eubacterial chemotaxis receptors and, like their eubacterial homologs, modulate the transfer of phosphate groups from the histidine protein kinase CheA to the response regulator CheY that in turn controls flagellar motor rotation and the cell's swimming behavior. HtrII and Htrl are dimeric proteins which were predicted to contain carboxylmethylation sites in a 4-helix bundle in their cytoplasmic regions, like eubacterial chemotaxis receptors. ^ The phototaxis transducers of H. salinarum have provided a model for studying receptor/tranducer interaction, adaptation in sensory systems, and the role of membrane molecular complexes in signal transduction. ^ Interaction between the transducer HtrI and the photoreceptor SRI was explored by creating six deletion constructs of HtrI, with progressively shorter cytoplasmic domains. This study confirmed a putative chaperone-like function of HtrI, facilitating membrane insertion or stability of the SRI protein, a phenomenon previously observed in the laboratory, and identified the smallest HtrI fragment containing interaction sites for both the chaperone-like function and SRI photocycle control. The active fragment consisted of the N-terminal 147 residues of the 536-residue HtrI protein, a portion of the molecule predicted to contain the two transmembrane helices and the first ∼20% of the cytoplasmic portion of the protein. ^ Phototaxis and chemotaxis sensory systems adapt to stimuli, thereby signaling only in response to changes in environmental conditions. Observations made in our and in other laboratories and homologies between the halobacterial transducers with the chemoreceptors of enteric bacteria anticipated a role for methylation in adaptation to chemo- and photostimuli. By site directed mutagenesis we identified the methylation sites to be the glutamate pairs E265–E266 in HtrI and E513–E514 in HtrII. Cells containing the unmethylatable transducers are still able to perform phototaxis and adapt to light stimuli. By pulse-chase analysis we found that methanol production from carboxylmethyl group hydrolysis occurs upon specific photo stimulation of unmethylatable HtrI and HtrII and is due to turnover of methyl groups on other transducers. We demonstrated that the turnover in wild-type H. salinarum cells that follows a positive stimulus is CheY-dependent. The CheY-feedback pathway does not require the stimulated transducer to be methylatable and operates globally on other transducers present in the cell. ^ Assembly of signaling molecules into architecturally defined complexes is considered essential in transmission of the signals. The spectroscopic characteristics of SRI were exploited to study the stoichiometric composition in the phototaxis complex SRI-HtrI. A molar ratio of 2.1 HtrI: 1 SRI was obtained, suggesting that only 1 SRI binding site is occupied on the HtrI homodimer. We used gold-immunoelectron microscopy and light fluorescence microscopy to investigate the structural organization and the distribution of other halobacterial transducers. We detected clusters of transducers, usually near the cell's poles, providing a ultrastructural basis for the global effects and intertransducer communication we observe. ^

Terminal short arm domains of basement membrane laminin are critical for its self-assembly

Laminin self-assembles into large polymers by a cooperative two-step calcium-dependent mechanism (Yurchenco, P. D., E. C. Tsilibary, A. S. Charonis, and H. Furthmayr. 1985. J. Biol. Chem. 260:7636-7644). The domain specificity of this process was investigated using defined proteolytically generated fragments corresponding to the NH2-terminal globule and adjacent stem of the short arm of the B1 chain (E4), a complex of the two short arms of the A and B2 chains attached to the proximal stem of a third short arm (E1'), a similar complex lacking the globular domains (P1'), and the distal half of the long arm attached to the adjacent portion of the large globule (E8). Polymerization, followed by an increase of turbidity at 360 nm in neutral isotonic TBS containing CaCl2 at 35 degrees C, was quantitatively inhibited in a concentration-dependent manner with laminin fragments E4 and E1' but not with fragments E8 and P1'. Affinity retardation chromatography was used for further characterization of the binding of laminin domains. The migration of fragment E4, but not of fragments E8 and P1', was retarded in a temperature- and calcium-dependent fashion on a laminin affinity column but not on a similar BSA column. These data are evidence that laminin fragments E4 and E1' possess essential terminal binding domains for the self-aggregation of laminin, while fragments E8 and P1' do not. Furthermore, the individual domain-specific interactions that contribute to assembly are calcium dependent and of low affinity.

High resolution immunoelectron microscopic localization of functional domains of laminin, nidogen, and heparan sulfate proteoglycan in epithelial basement membrane of mouse cornea reveals different topological orientations

Thin and ultrathin cryosections of mouse cornea were labeled with affinity-purified antibodies directed against either laminin, its central segments (domain 1), the end of its long arm (domain 3), the end of one of its short arms (domain 4), nidogen, or low density heparan sulfate proteoglycan. All basement membrane proteins are detected by indirect immunofluorescence exclusively in the epithelial basement membrane, in Descemet's membrane, and in small amorphous plaques located in the stroma. Immunoelectron microscopy using the protein A-gold technique demonstrated laminin domain 1 and nidogen in a narrow segment of the lamina densa at the junction to the lamina lucida within the epithelial basement membrane. Domain 3 shows three preferred locations at both the cellular and stromal boundaries of the epithelial basement membrane and in its center. Domain 4 is located predominantly in the lamina lucida and the adjacent half of the lamina densa. The low density heparan sulfate proteoglycan is found all across the basement membrane showing a similar uniform distribution as with antibodies against the whole laminin molecule. In Descemet's membrane an even distribution was found with all these antibodies. It is concluded that within the epithelial basement membrane the center of the laminin molecule is located near the lamina densa/lamina lucida junction and that its long arm favors three major orientations. One is close to the cell surface indicating binding to a cell receptor, while the other two are directed to internal matrix structures. The apparent codistribution of laminin domain 1 and nidogen agrees with biochemical evidence that nidogen binds to this domain.