p75(NTR) gene and suicide attempts in young adults with a history of childhood-onset mood disorder.

Recently, evidence has accumulated for the role of neurotrophic processes in mood disorders. Neurotrophins operate on receptors, one of which is the p75 neurotrophin receptor (p75(NTR)). We examined three p75(NTR) markers at the p75(NTR) gene, including a missense polymorphism that changes serine to leucine (S205L), for association with suicide attempt (SA) in 203 childhood-onset mood disorder (COMD) cases. There was no difference between COMD suicide attempters and COMD non-attempters with logistic regression models for any of the three markers. We also compared the three polymorphisms between 192 COMD cases and 192 matched healthy controls and found no significant differences between COMD and healthy controls. Our results do not support an association of the p75(NTR) S205L polymorphism with risk for COMD or SA in COMD.     

Recombinant troponin I substitution and calcium responsiveness in skinned cardiac muscle

Using treatment with vanadate solutions, we extracted native cardiac troponin I and troponin C (cTnI and cTnC) from skinned fibers of porcine right ventricles. These proteins were replaced by exogenously supplied TnI and TnC isoforms, thereby restoring Ca²⁺-dependent regulation. Force then depended on the negative logarithm of Ca²⁺ concentration (pCa) in a sigmoidal manner, the pCa for 50% force development, pCa₅₀, being about 5.5. For reconstitution we used fast-twitch rabbit skeletal muscle TnI and TnC (sTnI and sTnC), bovine cTnI and cTnC or recombinant sTnIs that were altered by site-directed mutagenesis. Incubation with TnI inhibited isometric tension in TnI-extracted fibers in the absence of Ca²⁺, but restoration of Ca²⁺ dependence required incubation with both TnI and TnC. Relaxation at low Ca²⁺ levels and the steepness of the force/pCa relation depended on the concentration of exogenously supplied TnI in the reconstitution solution (range 20–150 μM), while Ca²⁺ sensitivity, i.e. the pCa₅₀, was dependent on the isoform, and also on the concentration of TnC in the reconstitution solution. At pH 6.7, skinned fibers reconstituted with optimal concentrations of sTnC and sTnI (120 μM and 150 μM, respectively) were more sensitive to Ca²⁺ than those reconstituted with cTnC and cTnI (difference in pCa₅₀ approx. 0.2 units). Rabbit sTnI was cloned and expressed in Escherichia coli using a high yield expression plasmid. We introduced point mutations into the TnI inhibitory region comprising the sequence of the minimal common TnC/actin binding site (-G¹⁰⁴-K-F-K-R-P-P-L-R-R-V-R¹¹⁵-). The four mutants produced by substitution of T for P¹¹⁰, G for P¹¹⁰, G for L¹¹¹, and G for K¹⁰⁵ were chosen, based on previous work with synthetic peptides showing that single amino acid substitution in this region diminished the capacity of these peptides to inhibit acto-S₁ ATPase or contraction of skinned fibers. Therefore, all amino acid residues of the inhibitory region are thought to contribute to biological activity of TnI. However, each of the recombinant TnIs could substitute for endogenous TnI. In combination with exogenous TnC, Ca²⁺ dependence could be restored when ^gly110sTnI, ^thr110sTnI or ^gly111sTnI was used for reconstitution. The mutant ^gly105sTnI, on the other hand, reduced the ability of skinned fibers to relax at low Ca²⁺ concentrations and it caused an increase in Ca²⁺ sensitivity. Received: 5 October 1995/Received after revision and accepted: 1 December 1995     

Optimizing vaccine design for cellular processing,MHC binding and TCR recognition

In this review we describe the methods and processes that our group have developed while aiming to test and design multiepitope vaccines for infectious diseases and cancer. Testing the performance of vaccines composed of epitopes restricted by human leukocyte antigen (HLA) molecules is accomplished by in vitro antigenicity assays, as well as in vivo immunogenicity assays in HLA transgenics. The efficiency by which multiepitope vaccines are processed is optimized by spacer residues, which are designed to facilitate generation by natural processing of the various class I- and class II-restricted epitopes. Methods and strategies to test and optimize HLA binding affinity, patient coverage from the vaccine construct, and TCR recognition of HLA/epitope complexes are also discussed.     

Differential long-term subcellular responses in heart and liver to adriamycin stress. Exogenous L-carnitine cardiac and hepatic protection

In order to evaluate the heart and liver responses after adriamycin (ADR) toxic aggression, with and without exogenous L-carnitine (CAR) protection, female Sprague-Dawley rats, body weight 40-60 g, were randomized into four groups: CON, ADR, CAR and CAR-ADR. ADR was injected i.v. at a dose of 15-18 mg/kg body wt (0.1 ml). CAR was administered i.v. at a dose of 20 mg (0.1 ml) before each subdose of ADR, and then orally at 180 mg/kg body wt daily for 12 weeks. Long-term cardiac and hepatic subcellular damage were determined by transmission electron microscopic analysis of ultrathin sections. The ADR-induced long-term cardiac subcellular pathology included loss, disruption and disassembly of myofibrils, and mitochondrial swelling and condensation. On the other hand, the ADR-induced subcellular hepatic alterations consisted of polymorphic mitochondria, cytoplasmic vacuolization and accumulation of lipid droplets. Apparently, cardiac tissue was more affected by ADR toxic aggression than hepatic tissue. However, these alterations were of less severity in protected groups, in both heart and liver, suggesting CAR as a possible hepatoprotector agent against ADR toxicity. Because of the liver-L-carnitine-heart relationship, studying ADR-hepatotoxicity could be helpful in the further understanding of severe ADR-cardiotoxicity.     

Analysis of the steroidogenic acute regulatory protein (StAR) gene in Japanese patients with congenital lipoid adrenal hyperplasia

Genomic DNA from 19 Japanese patients with congenital lipoid adrenal hyperplasia (lipoid CAH) representing 16 different families was examined to identify the genetic alterations of steroidogenic acute regulatory protein (StAR). Ten of 19 patients had a 46,XX karyotype and nine had a 46,XY karyotype. Six of the 46,XX patients have experienced spontaneous pubertal changes including breast development and irregular menstruation whereas none of the 46,XY subjects displayed pubertal changes. Eight different mutations were identified. Sixteen patients were either homozygotes or compound heterozygotes for the Q258X mutation. The seven other mutations identified were 189delG, 246insG, 564del13bp, 838delA, Q212X, A218V and M225T. The 189delG, 246insG, 546del13bp and Q212X mutants encode truncated proteins. COS-1 cells transfected with expression vectors encoding cDNAs for the mutant StAR proteins which affect the C-terminus, 838delA, A218V and Q258X, exhibited no steroidogenesis enhancing activity. However, the M225T mutant retained some steroidogenic activity. The patient with the M225T mutation had late onset of this disorder and some capacity to secrete testosterone in response to hCG. These findings suggest: (i) that the Q258X mutation can be used as a genetic marker for the screening of Japanese for lipoid CAH, (ii) that the C-terminus of StAR plays an important role in the protein's activity and (iii) that there are differences in the extent of functional impairment of the testis and ovaries in lipoid CAH.      

The nonstructural proteins of Sindbis virus as studied with an antibody specific for the C terminus of the nonstructural readthrough polyprotein

A dodecapeptide containing the sequence of the C terminus of the nonstructural polyprotein of Sindbis virus has been synthesized and used to immunize rabbits. The antisera obtained precipitated polypeptides from cells infected with the HR strains of Sindbis or with temperature-sensitive mutants ts11 or ts18. Four different polypeptides, having apparent molecular weights of approximately 250,000, 220,000, 155,000, and 72,000, were immunoprecipitated by the antipeptide antiserum. The largest of these polypeptides is sufficiently large to represent a polyprotein translated from the entire nonstructural region of the genome. These data suggest that nsP4 of molecular weight 72,000 is produced by translation of the entire nonstructural region of the genome, which requires readthrough of an opal termination codon immediately upstream of nsP4, followed by post-translational cleavage of this polyprotein. The amounts of nsP4 and its precursors found in infected cells are small relative to the amounts of other nonstructural proteins present, as would be expected if readthrough of a termination codon is required. In addition, the relative amounts of nsP4 and of its precursors differ in HR-infected or ts mutant-infected cells and differ with temperature of infection, suggesting that temperature of infection or ts lesions affect translation and processing of the precursor polyprotein.     

Complete nucleotide sequence of the genomic RNA of Sindbis virus

The entire nucleotide sequence of the genomic RNA of the type virus of the alphavirus genus, Sindbis virus, has been determined. The genome is 11,703 nucleotides in length, exclusive of the 5' cap and the 3'-terminal poly(A) tract. After the 5'-terminal cap there are 59 nucleotides of 5' nontranslated nucleic acid followed by a reading frame of 7539 nucleotides that encodes the nonstructural polypeptides and which is open except for a single opal termination codon. Following 48 untranslated bases located in the junction region which separates the nonstructural and structural protein coding sequences, there is an open reading frame 3735 nucleotides long that encodes the structural proteins. Finally, the 3' untranslated region is 322 nucleotides long. The nonstructural proteins are translated from the genomic RNA as two polyprotein precursors. The first is 1896 amino acids in length and terminates at an opal codon at position 1897. This polyprotein is processed to produce three polypeptides called nsP1, nsP2, and nsP3. Sites of post-translational cleavage to produce these three proteins have been tentatively located using available N-terminal amino acid sequence data. In both cases cleavage probably occurs between the two alanine residues in the sequence Gly-Ala-Ala. The fourth nonstructural protein, nsP4, is produced when readthrough of the opal codon produces a second polyprotein precursor of length 2513 amino acids, which is also cleaved posttranslationally. The structural proteins are translated from a subgenomic message which begins at nucleotide 7598, is 4106 nucleotides in length (exclusive of the poly(A) tract), and is coterminal with the 3' end of the genomic RNA. The structural proteins are also translated as a polyprotein precursor which is cleaved to produce a nucleocapsid protein and two integral membrane glycoproteins as well as two small peptides not present in the mature virion. A replication strategy for Sindbis virus based upon the complete nucleotide sequence, as well as prior data, is presented.     

A calmodulin-binding peptide relaxes skinned muscle from guinea-pig taenia coli

During smooth muscle activation the calcium calmodulin complex interacts with myosin light chain kinase (MLCK) whereby activating it. A synthetic peptide analogue (RS20) corresponding to the calmodulin recognition sequence of MLCK has been synthesized and previously found to inhibit the calmodulin stimulated light chain kinase activity. Here we studied the effect of this peptide on skinned fibers from guinea pig taenia coli. Maximal contractions induced by 30 M Ca²⁺ at 0.1 M calmodulin could be completely relaxed by the peptide at 1 M. The inhibitory effect was accompanied by partial dephosphorylation only of the regulatory myosin light chain. Relaxation could be reversed by addition of calmodulin which also increased the extent of light chain phosphorylation.The calmodulin concentration required for reversing the inhibition depended on the concentration of the inhibitory peptide suggesting that the peptide competed with MLCK for the calmodulin binding site. As the calcium-calmodulin-peptide mixture constitutes a calmodulin buffer, our results suggest, that the peptide is a calmodulin antagonist unique in terms of its potency and that less than nanomolar concentrations of free calmodulin may be required for inducing smooth muscle contractions.