Impairment of LTD and cerebellar learning by Purkinje cell-specific ablation of cGMP-dependent protein kinase I

The molecular basis for cerebellar plasticity and motor learning remains controversial. Cerebellar Purkinje cells (PCs) contain a high concentration of cGMP-dependent protein kinase type I (cGKI). To investigate the function of cGKI in long-term depression (LTD) and cerebellar learning, we have generated conditional knockout mice lacking cGKI selectively in PCs. These cGKI mutants had a normal cerebellar morphology and intact synaptic calcium signaling, but strongly reduced LTD. Interestingly, no defects in general behavior and motor performance could be detected in the LTD-deficient mice, but the mutants exhibited an impaired adaptation of the vestibulo-ocular reflex (VOR). These results indicate that cGKI in PCs is dispensable for general motor coordination, but that it is required for cerebellar LTD and specific forms of motor learning, namely the adaptation of the VOR.     

Partial molar volumes of mRNA 5' cap analogues

Partial molar volumes in aqueous solution of eleven selected 7-methylguanine cap-analogues and their guanine counterparts were determined by means of density measurements. Hydrophobicity of the investigated compounds regarding their structural features was analysed within the framework of the solute-solvent interaction model, based on the relative density of the molecular solvation shell.     

Noncatalytic assembly of ribonuclease III with double-stranded RNA

Ribonuclease III (RNase III) represents a family of double-stranded RNA (dsRNA) endonucleases. The simplest bacterial enzyme contains an endonuclease domain (endoND) and a dsRNA binding domain (dsRBD). RNase III can affect RNA structure and gene expression in either of two ways: as a dsRNA-processing enzyme that cleaves dsRNA, or as a dsRNA binding protein that binds but does not cleave dsRNA. We previously determined the endoND structure of Aquifex aeolicus RNase III (Aa-RNase III) and modeled a catalytic complex of full-length Aa-RNase III with dsRNA. Here, we present the crystal structure of Aa-RNase III in complex with dsRNA, revealing a noncatalytic assembly. The major differences between the two functional forms of RNase III.dsRNA are the conformation of the protein and the orientation and location of dsRNA. The flexibility of a 7 residue linker between the endoND and dsRBD enables the transition between these two forms.     

Molecular analysis of TCR clonotypes in LGL: a clonal model for polyclonal responses

Large granular lymphocytic (LGL) leukemia is a clonal lymphoproliferative disorder of CTL associated with cytopenias resulting from an immune and cytokine attack on hemopoietic progenitor cells. Extreme clonality of CTL expansions seen in LGL leukemia makes it an ideal model to study the role of the T cell repertoire in other less-polarized immune-mediated disorders. Complementarity-determining region 3 (CDR3) of the TCR is a unique Ag-specific region that can serve as a molecular marker, or clonotype, of the disease-specific T cells. We studied the variable portion of the beta-chain spectrum in a cohort of LGL leukemia patients. The CDR3 sequences were determined for the immunodominant clones and used to design clonotype-specific primers. By direct and semi-nested amplification, clonotype amplicons were found to be shared by multiple patients and controls. Analysis of the generated sequences demonstrated that the original clonotypes are rarely encountered in normal control samples; however, high levels of homology were found in both controls and patients. Clonotypes derived from individual LGL patients can be used as tumor markers for the malignant clone. More generally, clonotypic analysis and comparison of the variable portion of the beta-chain CDR3-specific sequences from a large number of patients may lead to better subclassification of not only LGL but also other immune-mediated disorders.     

Individual and population penalized regression splines for accelerated longitudinal designs

In an accelerated longitudinal design (ALD), individuals enter the study at different points of their growth trajectory and are observed over a short time span relative to the entire time span of interest. ALD data are combined across independent units to provide an estimate of an overall population curve and predictions of individual patterns of change. As a modest extension of the work of Ruppert et al. (2003, Semiparametric Regression, Cambridge University Press), we develop a computationally efficient procedure for the application of longitudinal semiparametric methods under ALD sampling schemes. We compare balanced and complete longitudinal designs to ALDs using the Berkeley Growth Study data and apply our method to longitudinal magnetic resonance imaging (MRI) brain structure size (volume) measurements from an ongoing developmental study. Potential applications extend beyond growth studies to many other fields in which cost and feasibility constraints impose restrictions on sample size and on the numbers and timings of repeated measurements across subjects.     

Interaction between lipid monolayers and poloxamer 188: an X-ray reflectivity and diffraction study

The mechanism by which poloxamer 188 (P188) seals a damaged cell membrane is examined using the lipid monolayer as a model system. X-ray reflectivity and grazing-incidence x-ray diffraction results show that at low nominal lipid density, P188, by physically occupying the available area and phase separating from the lipids, forces the lipid molecules to pack tightly and restore the barrier function of the membrane. Upon compression to bilayer equivalent pressure, P188 is squeezed out from the lipid monolayer, allowing a graceful exit of P188 when the membrane integrity is restored.     

Differential effects of morphine and naltrexone on the in vitro LH secretion from male and female carp pituitary gland

The in vitro effects of morphine (10(-10), 10(-8), 10(-6) or 10(-5) M) or/and naltrexone (10(-6) or 10(-8) M) on LH release from male and female carp (Cyprinus carpio L.) dispersed pituitary cells (obtained from fish at the time of late gonad recrudescence) were investigated. Morphine alone at the lowest tested concentration (10(-10) M) increased LH secretion from the cells of males. On the contrary, in female cell incubations the highest concentrations of morphine (10(-6) or 10(-5) M) significantly lowered LH levels. Naltrexone alone (at both tested concentrations) had no influence on LH secretion, neither in males nor in females. However in the incubations of female cells it antagonised the influence of morphine at 10(-10) or 10(-8) M. In male cell incubations naltrexone abolished the stimulatory action of morphine at 10(-10) M. The results suggest that in the in vitro culture of carp pituitary cells LH secretion is modulated by the opioids which affect the release of this gonadotropin through the typical opioid receptors and that the mu type of these receptors is involved in this process. The effects of opioid agonist and antagonist depend on the stage of gonadal maturity and the sex of fish i.e. the actual level of sex steroids.     

Effect of the V3 loop deletion of envelope glycoprotein on cellular responses and protection against challenge with recombinant vaccinia virus expressing gp160 of primary human immunodeficiency virus type 1 isolates

The magnitude and breadth of cytotoxic-T-lymphocyte (CTL) responses induced by human immunodeficiency virus type 1 (HIV-1) envelope protein from which the hypervariable V3 loop had been deleted (DeltaV3) were evaluated in the HLA-A2/K(b) transgenic mice. It was demonstrated that vaccines expressing the DeltaV3 mutant of either HIV-1(IIIB) or HIV-1(89.6) envelope glycoprotein induced broader CD8(+) T-cell activities than those elicited by the wild-type (WT) counterparts. Specifically, the differences were associated with higher responses to conserved HLA-A2-restricted CTL epitopes of the envelope glycoprotein and could be correlated with an increased cell surface occupancy by the epitope-HLA-A2 complexes in target cells expressing the DeltaV3 mutant. Using recombinant vaccinia virus expressing heterologous gp160 of primary HIV-1 isolates in a murine challenge system, we observed that the extent of resistance to viral transmission was higher in animals immunized with the DeltaV3 than the WT envelope vaccine. The protection was linked to the presence of envelope-specific CD8(+) T cells, since depletion of these cells by anti-CD8 antibody treatment at the time of challenge abolished the vaccine-induced protection. The results from our studies provide insights into approaches for boosting the breadth of envelope-specific CTL responses.     

Network of protein interactions within the Drosophila inner kinetochore

The kinetochore provides a physical connection between microtubules and the centromeric regions of chromosomes that is critical for their equitable segregation. The trimeric Mis12 sub-complex of the Drosophila kinetochore binds to the mitotic centromere using CENP-C as a platform. However, knowledge of the precise connections between Mis12 complex components and CENP-C has remained elusive despite the fundamental importance of this part of the cell division machinery. Here, we employ hydrogen–deuterium exchange coupled with mass spectrometry to reveal that Mis12 and Nnf1 form a dimer maintained by interacting coiled-coil (CC) domains within the carboxy-terminal parts of both proteins. Adjacent to these interacting CCs is a carboxy-terminal domain that also interacts with Nsl1. The amino-terminal parts of Mis12 and Nnf1 form a CENP-C-binding surface, which docks the complex and thus the entire kinetochore to mitotic centromeres. Mutational analysis confirms these precise interactions are critical for both structure and function of the complex. Thus, we conclude the organization of the Mis12–Nnf1 dimer confers upon the Mis12 complex a bipolar, elongated structure that is critical for kinetochore function.