The catalytic domain of protein kinase C chimeras modulates the affinity and targeting of phorbol ester-induced translocation

Emerging evidence suggests important differences among protein kinase C (PKC) isozymes in terms of their regulation and biological functions. PKC is regulated by multiple interdependent mechanisms, including enzymatic activation, translocation of the enzyme in response to activation, phosphorylation, and proteolysis. As part of our ongoing studies to define the factors contributing to the specificity of PKC isozymes, we prepared chimeras between the catalytic and regulatory domains of PKCalpha, -delta, and -epsilon. These chimeras, which preserve the overall structure of the native PKC enzymes, were stably expressed in NIH 3T3 fibroblasts. Their intracellular distribution was similar to that of the endogenous enzymes, and they responded with translocation upon treatment with phorbol 12-myristate 13-acetate (PMA). We found that the potency of PMA for translocation of the PKCalpha/x chimeras from the soluble fraction was influenced by the catalytic domain. The ED50 for translocation of PKCalpha/alpha was 26 nM, in marked contrast to the ED50 of 0.9 nM in the case of the PKCalpha/epsilon chimera. In addition to this increase in potency, the site of translocation was also changed; the PKCalpha/epsilon chimera translocated mainly into the cytoskeletal fraction. PKCx/epsilon chimeras displayed twin isoforms with different mobilities on Western blots. PMA treatment increased the proportion of the higher mobility isoform. The two PKCx/epsilon isoforms differed in their localization; moreover, their localization pattern depended on the regulatory domain. Our results emphasize the complex contributions of the regulatory and catalytic domains to the overall behavior of PKC.     

Lack of protection against avian cholera by vaccination with recombinant P6-like protein from Pasteurella multocida

The gene encoding the P6-like protein of Pasteurella multocida was cloned in the baculovirus expression system. Baculovirus-expressed recombinant protein was used to parenterally immunize 6-wk-old Nicholas broad-breasted white turkeys. Turkeys developed significant antibody titers to the recombinant protein as measured by enzyme-linked immunosorbent assay. Two weeks after the last immunizing injection, vaccinated turkeys were placed in contact with turkeys infected with P. multocida strain P1059, as were nonvaccinated control birds. No differences occurred in percent mortality between the two groups. We conclude that parenterally administered recombinant P6-like protein does not protect turkeys from avian cholera.     

Mapped infinite elements in soil consolidation

The problems of consolidation often involve unbounded continua. The most common solution is then achieved by limiting the studied area through the introduction, at finite distance, of a boundary on which adequate conditions on the field variables are imposed. In this paper mapped infinite elements are used to model the far-field solution. Spatial discretization is hence performed on the basis of both finite and infinite elements. In two examples, solutions involving finite and infinite elements are compared with known analytical solutions and it is shown that an excellent agreement can be achieved by the use of mapped infinite elements.     

A randomized controlled trial of a peer support intervention targeting antiretroviral medication adherence and depressive symptomatology in HIV-positive men and women.

Objective: To determine the efficacy of a peer-led social support intervention involving support groups and telephone contacts compared with standard clinical care to enhance antiretroviral medication adherence. Design: Randomized controlled trial with follow-up. Participants were 136 HIV-positive indigent mainly African American and Puerto Rican men and women recruited from an outpatient clinic in the Bronx, New York. The 3-month intervention was delivered by other HIV-positive clinic patients trained in addressing barriers to adherence and sensitively providing appraisal, spiritual, emotional, and informational adherence-related social support. Main outcome measures: Medical chart-abstracted HIV-1 RNA viral load, antiretroviral adherence according to electronic drug monitoring and participant self-report, and social support and depressive symptomatology. All assessments conducted at baseline, 3 months, and 6 months. Results: Intent-to-treat and as-treated analyses indicated no between-conditions intervention effects on the primary outcome of HIV-1 RNA viral load or any of the secondary outcomes at immediate postintervention or follow-up. Post hoc analyses within the intervention condition indicated greater intervention exposure was associated with higher self-reported adherence, higher social support, and lower depressive symptomatology at follow-up, even after controlling for baseline adherence. Conclusion: Null findings, consistent with the limited literature on efficacious highly active antiretroviral therapy (HAART) adherence interventions, may be due to insufficient exposure to the intervention, its low intensity, or the nature of the sample-a heterogeneous HAART-experienced group of patients with high levels of substance use and multiple other competing stressors. Overall, findings highlight the need for more comprehensive and intensive efforts to battle nonadherence. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Isolation of the PufX protein from Rhodobacter capsulatus and Rhodobacter sphaeroides: evidence for its interaction with the alpha-polypeptide of the core light-harvesting complex

Using mutant strains of Rhodobacter capsulatus and Rhodobacter sphaeroides in which the pufX gene had been deleted, it was possible to identify by HPLC membrane protein components present in pufX+ cells but absent in pufX- cells. In parallel preparations, membrane proteins soluble in chloroform/methanol containing ammonium acetate were first extracted from lyophilized membrane fractions of the pufX+ cells and separated from pigments and larger protein material by gel-filtration chromatography. Protein-containing fractions were examined by HPLC, and several peaks were collected from pufX+ material that were not present in pufX- material. From N-terminal amino acid sequencing, the PufX protein of Rb. capsulatus was identified, and from positive interaction with a PufX protein antibody, the Rb. sphaeroides PufX protein was identified. Although overall yields were very small, sufficient quantities of these proteins were isolated to evaluate their effect on the reconstitution of the core light-havesting antenna (LH1) and its subunit complex. From the behavior of the PufX protein and the alpha-polypeptide of LH1 on HPLC, qualitative evidence was obtained that the two proteins have a high affinity for each other. In reconstitution assays with bacteriochlorophyll (Bchl) and the LH1 alpha- and beta-polypeptides of Rb. capsulatus, the PufX protein of Rb. capsulatus was inhibitory to LH1 formation at low concentration. A similar inhibition was exhibited by Rb. sphaeroides PufX protein for reconstitution of LH1 with Bchl and the LH1 alpha- and beta-polypeptides of Rb. sphaeroides. In both cases, the ratios of concentrations of the PufX protein to the alpha-polypeptide causing 50% inhibition were approximately 0.5. Formation of the heterologous (alpha beta) subunit-type complex formed with Bchl and the alpha- and beta-polypeptides of LH1 of Rb. capsulatus was also inhibited by low concentrations of the Rb. capsulatus PufX protein (approximately 50% inhibition at PufX:alpha-polypeptide ratios = 0.5). However, neither PufX protein inhibited formation of a homologous (beta beta) subunit-type complex, which indicates that the PufX proteins do not interact with the beta-polypeptides.     

Effects of establishment of community psychiatry in Aalborg]

The aim of the present study was to illustrate the effects of community-based psychiatry. The catchment area was divided into three homogeneous districts, East, North and West. Teams were established on 1.9. 1990, 1.10.91 and 1.5.1992, respectively. Social, diagnostic and treatment related data were gathered from two cross-sectional investigations (I: February 1992 and II: February 1993) and from in-patients and out-patients files. In cross-section I a majority of long-term ward patients and hospital-based employment offers was found in the district where the community district team had not yet been established. In the district where the first community district team was established most primary target patients were treated. In cross-section II the hospital-based psychiatric service were more homogeneously distributed between the districts. The establishment of community-based psychiatric teams resulted in new referrals, and increasing numbers of patients becoming attached to the psychiatric teams, but crowding and use of compulsory measures in hospital also increased.     

Crystal structure at 1.95 A resolution of the breast tumour-specific antibody SM3 complexed with its peptide epitope reveals novel hypervariable loop recognition

The anti-breast tumour antibody SM3 has a high selectivity in reacting specifically with carcinoma-associated mucin. SM3 recognises the core repeating motif (Pro-Asp-Thr-Arg-Pro) of aberrantly glycosylated epithelial mucin MUC1, and has potential as a therapeutic and diagnostic tool. Here we report the crystal structure of the Fab fragment of SM3 in complex with a 13-residue MUC1 peptide antigen (Thr1P-Ser2P-Ala3P-Pro4P-Asp5P-Thr6P -Arg7P-Pro8P-Ala9P-Pro10P-Gly11P- Ser12P-Thr13P). The SM3-MUC1 peptide structure was solved by molecular replacement, and the current model is refined at 1.95 A resolution with an R-factor of 21.3% and R-free 28.3%. The MUC1 peptide is bound both by non-polar interactions and hydrogen bonds in an elongated groove in the antibody-combining site through interactions with Complimentarity Determining Regions (CDRs), three of the light chain (L1, L2, L3) and two of the heavy chain (H1 and H3). The conformation of the peptide is mainly extended with no discernable standard secondary structure. There is a single non-proline cis-peptide bond in H3 (Val95H-Gly96H-Gln97H-Phe98H-Ala101H-Ty r102H) between Gly96H and Gln97H, which appears to play a role in SM3-peptide antigen interactions, and represents the first such example within an antibody hypervariable loop. The SM3-MUC1 peptide structure has implications for rational therapeutic and diagnostic antibody engineering.     

Isolation of a second recombinant human respiratory syncytial virus monoclonal antibody fragment (Fab RSVF2-5) that exhibits therapeutic efficacy in vivo

A second human respiratory syncytial virus (RSV)-neutralizing monoclonal antibody was isolated and its binding site was identified. Fab F2-5 is a broadly reactive fusion (F) protein-specific recombinant Fab generated by antigen selection from a random combinatorial library displayed on the surface of filamentous phage. In an in vitro plaque-reduction test, the Fab RSVF2-5 neutralized the infectivity of a variety of field isolates representing viruses of both RSV subgroups A and B. The Fab recognized an antigenic determinant that differed from the only other human anti-F monoclonal antibody (RSV Fab 19) described thus far. A single dose of 4.0 mg of Fab RSVF2-5/kg of body weight administered by inhalation was sufficient to achieve a 2000-fold reduction in pulmonary virus titer in RSV-infected mice. The antigen-binding domain of Fab RSVF2-5 offers promise as part of a prophylactic regimen for RSV infection in humans.     

Regulation of kynurenic acid levels in the developing rat brain

Several brain-specific mechanisms control the formation of the endogenous excitatory amino acid receptor antagonist kynurenic acid (KYNA) in the adult rat brain. Two of these, dopaminergic neurotransmission and cellular energy metabolism, were examined in the brain of immature (postnatal day 7) rats. The results indicate that during the early postnatal period cerebral KYNA synthesis is exceptionally amenable to modulation by dopaminergic mechanisms but rather insensitive to fluctuations in cellular energy status. These findings may be of relevance for the role of KYNA in the function and dysfunction of the developing brain.     

Role of P-selectin cytoplasmic domain in granular targeting in vivo and in early inflammatory responses

P-selectin is an adhesion receptor for leukocytes expressed on activated platelets and endothelial cells. The cytoplasmic domain of P-selectin was shown in vitro to contain signals required for both the sorting of this protein into storage granules and its internalization from the plasma membrane. To evaluate in vivo the role of the regulated secretion of P-selectin, we have generated a mouse that expresses P-selectin lacking the cytoplasmic domain (DeltaCT mice). The deletion did not affect the sorting of P-selectin into alpha-granules of platelets but severely compromised the storage of P-selectin in endothelial cells. Unstored P-selectin was proteolytically shed from the plasma membrane, resulting in increased levels of soluble P-selectin in the plasma. The DeltaCT-P-selectin appeared capable of mediating cell adhesion as it supported leukocyte rolling in the mutant mice. However, a secretagogue failed to upregulate leukocyte rolling in the DeltaCT mice, indicating an absence of a releasable storage pool of P-selectin in the endothelium. Furthermore, the neutrophil influx into the inflamed peritoneum was only 30% of the wild-type level 2 h after stimulation. Our results suggest that different sorting mechanisms for P-selectin are used in platelets and endothelial cells and that the storage pool of P-selectin in endothelial cells is functionally important during early stages of inflammation.