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(1/989) Energy-based de novo protein folding by conformational space annealing and an off-lattice united-residue force field: application to the 10-55 fragment of staphylococcal protein A and to apo calbindin D9K.

The conformational space annealing (CSA) method for global optimization has been applied to the 10-55 fragment of the B-domain of staphylococcal protein A (protein A) and to a 75-residue protein, apo calbindin D9K (PDB ID code), by using the UNRES off-lattice united-residue force field. Although the potential was not calibrated with these two proteins, the native-like structures were found among the low-energy conformations, without the use of threading or secondary-structure predictions. This is because the CSA method can find many distinct families of low-energy conformations. Starting from random conformations, the CSA method found that there are two families of low-energy conformations for each of the two proteins, the native-like fold and its mirror image. The CSA method converged to the same low-energy folds in all cases studied, as opposed to other optimization methods. It appears that the CSA method with the UNRES force field, which is based on the thermodynamic hypothesis, can be used in prediction of protein structures in real time.  (+info)

(2/989) Expression of calcium binding protein D-9k messenger RNA in the mouse uterine endometrium during implantation.

To investigate the molecular mechanisms of implantation, we constructed a cDNA library of mouse uteri enriched with pregnancy-induced genes by subtractive hybridization and polymerase chain reaction (PCR). One of the isolated clones was the cDNA for the calcium binding protein D-9k (Cabp9k), which is considered to regulate intracytoplasmic concentration and transport of free calcium ions. Northern blot and in-situ hybridization analyses demonstrated that the Cabp9k mRNA was expressed in the endometrial epithelia, both luminal and glandular, in the uterus at the time of implantation. On pregnancy day 5 it was detected in the luminal, but not in the glandular, epithelia. In the oophorectomized adult mice, progesterone enhanced Cabp9k mRNA expression in the uterus, whereas oestrogen did not. Consistent with this, a nucleotide change was identified in the first intron of mouse Cabp9k gene corresponding to the oestrogen responsive element in the rat Cabp9k gene. Transfer of embryos into the uterine cavity of pseudopregnant mice reduced the expression of Cabp9k mRNA in the glandular epithelium, suggesting that Cabp9k mRNA expression is also regulated by embryonal signal(s). These findings demonstrated that Cabp9k mRNA is expressed in the endometrial epithelia during the implantation period under the control of progesterone and the presence of embryo, and suggest that CaBP9k plays a role in implantation by regulating the local calcium concentrations.  (+info)

(3/989) Distribution of cholinergic contacts on Renshaw cells in the rat spinal cord: a light microscopic study.

1. Cholinergic terminals in the rat spinal cord were revealed by immunohistochemical detection of the vesicular acetycholine transporter (VAChT). In order to determine the relationships of these terminals to Renshaw cells, we used dual immunolabelling with antibodies against gephyrin or calbindin D28k to provide immunohistochemical identification of Renshaw cells in lamina VII of the ventral horn. 2. A total of 50 Renshaw cells were analysed quantitatively using a computer-aided reconstruction system to provide accurate localization of contact sites and determination of somatic and dendritic surface area. Dendrites could be traced for up to 413 microm from the soma in calbindin D28k-identified Renshaw cells and up to 184 microm in gephyrin-identified cells. 3. A total of 3330 cholinergic terminals were observed on 50 Renshaw cells, with a range of 21-138 terminal appositions per cell (mean 66.6 +/- 25.56 contacts per cell). The vast majority (83.5 %) of the terminals were apposed to dendrites rather than the soma. The overall density of cholinergic contacts increased from a little above 1 per 100 microm2 on the soma and initial 25 microm of proximal dendrites to 4-5 per 100 microm2 on the surface of dendritic segments located 50-250 microm from the soma. Single presynaptic fibres frequently formed multiple contacts with the soma and/or dendrites of individual Renshaw cells. 4. VAChT-immunoreactive terminals apposed to Renshaw cells varied in size from 0.6 to 6.9 microm in diameter (mean 2.26 +/- 0.94; n = 986) and were on average smaller than the cholinergic C-terminals apposed to motoneurones, but larger than VAChT-immunoreactive terminals contacting other ventral horn interneurones. 5. The high density and relatively large size of many cholinergic terminals on Renshaw cells presumably correlates with the strong synaptic connection between motoneurones and Renshaw cells. The fact that the majority of contacts are distributed over the dendrites makes the motoneurone axon collateral input susceptible to inhibition by the prominent glycinergic inhibitory synapses located on the soma and proximal dendrites. The relative positions and structural features of the excitatory cholinergic and inhibitory glycinergic synapses may explain why Renshaw cells, although capable of firing at very high frequency following motor axon stimulation, appear to fire at relatively low rates during locomotor activity.  (+info)

(4/989) Characterization of nodular neuronal heterotopia in children.

Neuronal heterotopia are seen in various pathologies and are associated with intractable epilepsy. We examined brain tissue from four children with subcortical or periventricular nodular heterotopia of different aetiologies: one with severe epilepsy following focal brain trauma at 17 weeks gestation, one with hemimegalencephaly and intractable epilepsy, one with focal cortical dysplasia and intractable epilepsy, and one dysmorphic term infant with associated hydrocephalus and polymicrogyria. The connectivity of nodules was investigated using histological and carbocyanine dye (DiI) tracing techniques. DiI crystal placement adjacent to heterotopic nodules revealed numerous DiI-labelled fibres within a 2-3 mm radius of the crystals. Although we observed labelled fibres closely surrounding nodules, the majority did not penetrate them. Placement of DiI crystals within nodules also identified a limited number of projections out of the nodules and in one case there was evidence for connectivity between adjacent nodules. The cellular and neurochemical composition of nodules was also examined using immunohistochemistry for calretinin and neuropeptide Y (NPY), which are normally expressed in GABAergic cortical interneurons. Within heterotopic nodules from all cases, numerous calretinin-positive neurons were identified, along with a few cell bodies and many processes positive for NPY. Calretinin-positive neurons within nodules were less morphologically complex than those in the cortex, which may reflect incomplete differentiation into an inhibitory neuronal phenotype. There were also abnormal clusters of calretinin-positive cells in the overlying cortical plate, indicating that the migratory defect which produces heterotopic nodules also affects development of the cortex itself. Thus, heterotopic nodules consisting of multiple neuronal cell types are associated with malformation in the overlying cortical plate, and have limited connectivity with other brain regions. This abnormal development of connectivity may affect neuronal maturation and consequently the balance of excitation and inhibition in neuronal circuits, leading to their epileptogenic potential.  (+info)

(5/989) Measurements of [Ca2+] using fura-2 in glioma C6 cells expressing calretinin with GFP as a marker of transfection: no Ca2+-buffering provided by calretinin.

Glioma C6 cells were transfected with a plasmid containing the calretinin (CR) and green fluorescent protein (GFP) coding regions to analyze the effect of CR's presence on [Ca2+]i. Positive transfectants were identified by the detection of GFP and [Ca2+]i was measured using fura-2 as a probe. We found that neither the basic [Ca2+]i nor activated [Ca2+]i achieved by exposure to ionomycin, ADP or thapsigargin were affected by CR's presence in transfected cells, despite the ability of CR to bind Ca2+ as part of fusion protein. The level of expressed CR was estimated as at least 1 microM. The presented results suggest that CR's function is unlikely to be an intracellular Ca2+-buffer and support the hypothesis that CR might be involved in a specific Ca2+-dependent process. The results of this work also show that the S65T mutant of GFP is compatible with fura-2 measurements of intracellular [Ca2+]. We have demonstrated that the presence of GFP, as a transfection marker of glioma C6 cells, does not disturb fura-2 fluorescence, the basal or activated [Ca2+]i in these cells.  (+info)

(6/989) Molecular identification of the apical Ca2+ channel in 1, 25-dihydroxyvitamin D3-responsive epithelia.

In mammals, the extracellular calcium concentration is maintained within a narrow range despite large variations in daily dietary input and body demand. The small intestine and kidney constitute the influx pathways into the extracellular Ca2+ pool and, therefore, play a primary role in Ca2+ homeostasis. We identified an apical Ca2+ influx channel, which is expressed in proximal small intestine, the distal part of the nephron and placenta. This novel epithelial Ca2+ channel (ECaC) of 730 amino acids contains six putative membrane-spanning domains with an additional hydrophobic stretch predicted to be the pore region. ECaC resembles the recently cloned capsaicin receptor and the transient receptor potential-related ion channels with respect to its predicted topology but shares less than 30% sequence homology with these channels. In kidney, ECaC is abundantly present in the apical membrane of Ca2+ transporting cells and colocalizes with 1,25-dihydroxyvitamin D3-dependent calbindin-D28K. ECaC expression in Xenopus oocytes confers Ca2+ influx with properties identical to those observed in distal renal cells. Thus, ECaC has the expected properties for being the gatekeeper of 1,25-dihydroxyvitamin D3-dependent active transepithelial Ca2+ transport.  (+info)

(7/989) Immunohistological studies of metabotropic glutamate receptor subtype 6-deficient mice show no abnormality of retinal cell organization and ganglion cell maturation.

Immature retinal ganglion cells (RGCs) initially show a multistratified dendritic pattern, and, during the postnatal period, these dendrites gradually monostratify into ON and OFF sublaminae. The selective agonist of group III metabotropic glutamate receptors (mGluR), L-2-amino-4-phosphonobutyrate (L-AP-4), hyperpolarizes ON bipolar cells and reduces glutamate release. On the basis of L-AP-4-evoked inhibitory effects on ON-OFF segregation of developing RGCs, it has been hypothesized that glutamate-mediated synaptic activity is crucial for formation of the ON-OFF network. Gene-targeted ablation of mGluR6 specifically expressed in ON bipolar cells blocks normal ON responses but has been predicted to enhance glutamate release from ON bipolar cells. The mGluR6 knock-out mouse therefore provides a unique opportunity to investigate whether glutamate release and ON responses are important factors in the development of ON-OFF segregation. The combination of several different morphological analyses indicates that ON bipolar cells, as well as several distinct amacrine cells, in mGluR6 knock-out mice are normally distributed and correctly extend their terminals to defined retinal laminae. Importantly, both alpha and delta RGCs in adult mGluR6 knock-out mice are found monostratified into cell type-specific layers. Furthermore, no difference between wild-type and mGluR6 knock-out mice is observed in the maturation and dendritic stratification of developing RGCs. Hence, despite a deficit in normal ON responses, mGluR6 deficiency causes no abnormality in the retinal cellular organization nor in the stratifications of both ON bipolar cells and developing and mature RGCs. Based on these findings, we discuss several possible mechanisms that may underlie ON-OFF segregation of RGCs.  (+info)

(8/989) Expression of type 2 iodothyronine deiodinase in hypothyroid rat brain indicates an important role of thyroid hormone in the development of specific primary sensory systems.

Thyroid hormone is an important epigenetic factor in brain development, acting by modulating rates of gene expression. The active form of thyroid hormone, 3,5,3'-triiodothyronine (T3) is produced in part by the thyroid gland but also after 5'-deiodination of thyroxine (T4) in target tissues. In brain, approximately 80% of T3 is formed locally from T4 through the activity of the 5'-deiodinase type 2 (D2), an enzyme that is expressed mostly by glial cells, tanycytes in the third ventricle, and astrocytes throughout the brain. D2 activity is an important point of control of thyroid hormone action because it increases in situations of low T4, thus preserving brain T3 concentrations. In this work, we have studied the expression of D2 by quantitative in situ hybridization in hypothyroid animals during postnatal development. Our hypothesis was that those regions that are most dependent on thyroid hormone should present selective increases of D2 as a protection against hypothyroidism. D2 mRNA concentration was increased severalfold over normal levels in relay nuclei and cortical targets of the primary somatosensory and auditory pathways. The results suggest that these pathways are specifically protected against thyroid failure and that T3 has a role in the development of these structures. At the cellular level, expression was observed mainly in glial cells, although some interneurons of the cerebral cortex were also labeled. Therefore, the T3 target cells, mostly neurons, are dependent on local astrocytes for T3 supply.  (+info)