Phospholamban is a good substrate for cyclic GMP-dependent protein kinase in vitro, but not in intact cardiac or smooth muscle.

Protein residualizing labels facilitate localization of tissue sites of protein catabolism and the quantification of protein accumulation because of their prolonged intracellular retention of protein accumulation because of their prolonged intracellular retention times. Radioiodinated residualizing labels have been used to define the metabolism of a wide variety of proteins, but this has necessitated destructive analysis. Here we describe the implementation and validation of a novel 19F-containing residualizing label for protein, NN-dilactitol-3,5-bis(trifluoromethyl)benzylamine (DLBA), that permits the non-invasive assessment of protein accumulation and catabolism by n.m.r. spectroscopy in vivo. DLBA comprises a reporter molecule containing six equivalent 19F atoms. 19F is strongly n.m.r.-active, has 100% natural abundance, and is present in minimal background concentrations in soft tissues. We validated the use of DLBA as a protein-labelling compound by coupling to asialofetuin (ASF), a protein that is recognized exclusively by hepatic tissue via a saturable receptor-mediated process. Coupling of DLBA to ASF by reductive amination had no effect on the physiological receptor-mediated uptake of the protein in rat liver in vivo. The 19F-n.m.r. spectrum of DLBA exhibited a single peak that was subject to a small chemical-shift change and broadening after coupling to ASF. Pronase digestion of DLBA-ASF was performed to simulate intracellular degradation products, and resulted in a narrower set of resonances, with chemical shifts intermediate between those of uncoupled DLBA and DLBA-ASF. Intravenous administration of DLBA-ASF to rats followed by quantification of 19F in homogenates of liver tissue indicated that the half-life of residence time of degradation products from DLBA-ASF in liver was approx. 2 days. This intracellular half-life was comparable with that described for similar residualizing labels that contain radioiodide as a reporter. Similar results for the half-life of retention were obtained non-destructively and non-invasively in situ with the use of a whole-body radio-frequency antenna to acquire sequential spectra over 80 h after intravenous administration of DLBA-ASF. Quantification of these spectra demonstrated an initial accumulation of DLBA-ASF in liver followed by an expected gradual loss of 19F-labelled degradation products. The approach developed offers promise for the sequential and longitudinal characterization of metabolism of specific proteins in individual experimental animals and ultimately in human subjects.     

Interrelationships among various measures of upper body strength assessed by different contraction modes Evidence for a general strength component

Two studies were conducted in 83 college men to determine the degree of generality of individual differences in upper body muscular strength assessed by different testing modes. In study 1 (N = 43), correlations were computed between four measures of upper body strength using the bench press movement, maximal isokinetic (0.09 rad.s-1), maximal fast (0.126 m.s-1) and slow (0.037 m.s-1) hydraulic, and one repetition maximum (1-RM) free weight bench press (BP). Compared to free weight BP, maximal strength during isokinetic and slow hydraulic BP was approximately 29% and approximately 8% larger, and fast hydraulic BP strength was approximately 63% lower (p less than 0.05). Simple linear regression of isokinetic BP on 1-RM BP yielded r = 0.79, error of prediction (SE) = 12%, and generality = 81%. The corresponding averaged values for the regression of slow and fast hydraulic BP on free weight 1-RM BP were r = 0.77, SE = 13.5%, and generality = 84%. In Study 2 (N = 40), testing included maximal isokinetic concentric and eccentric arm flexion and extension at 0.524, 1.570, and 2.094 rad.s-1. The ratio of concentric to eccentric torque at the 3 speeds averaged 0.68 (flexion) and 0.70 (extension), and eccentric torques were 32% and 30% greater than concentric torques (p less than 0.05). The linear regression between concentric vs. eccentric flexion and extension torques at the three velocities yielded an average r = 0.80, SE = 13.7%, and generality = 73%.(ABSTRACT TRUNCATED AT 250 WORDS)     

Isolation and characterization of cDNA clones encoding the 17.9 and 8.1 kDa subunits of Photosystem I from Chlamydomonas reinhardtii

cDNA clones encoding two Photosystem I subunits of Chlamydomonas reinhardtii with apparent molecular masses of 18 and 11 kDa (thylakoid polypeptides 21 and 30; P21 and P30 respectively) were isolated using oligonucleotides, the sequences of which were deduced from the N-terminal amino acid sequences of the proteins. The cDNAs were sequenced and used to probe Southern and Northern blots. The Southern blot analysis indicates that both proteins are encoded by single-copy genes. The mRNA sizes of the two components are 1400 and 740 nucleotides, respectively. Comparison between the open reading frames of the cDNAs and the N-terminal amino acid sequences of the proteins indicates that the molecular masses of the mature proteins are 17.9 (P21) and 8.1 kDa (P30). Analysis of the deduced protein sequences predicts that both subunits are extrinsic membrane proteins with net positive charges. The amino acid sequences of the transit peptides suggest that P21 and P30 are routed towards the lumenal and stromal sides of the thylakoid membranes, respectively.Abbreviations OEE1, 2 and 3 oxygen evolution enhancer proteins 1, 2 and 3 - Rubisco ribulose bisphosphate carboxylase/oxygenase - PS photosystem - P21 and P30 C. reinhardtii thylakoid polypeptides 21 and 30     

Transformation of Claviceps purpurea using a bleomycin resistance gene

Summary To develop a DNA-mediated transformation system for Claviceps purpurea a vector was constructed using a bleomycin-resistance gene (bleo ^R) fused in frame to the Aspergillus nidulans trp C promoter as a dominant selection marker. The construct was shown to be functional in Aspergillus nidulans and Aspergillus niger and used to transform a wild strain of Claviceps purpurea. Transformats were obtained at low frequencies; they were shown to contain transforming DNA integrated into the chromosomal DNA, probably in multimeric copies and at multiple sites. Combined Southern, Northern and resistance level analysis indicate that the A. nidulans promoter is functional in C. purpurea.Dedicated to Professor Dr. Dr. h. c. K. Esser on the occasion of his 65^th birthday     

Some pharmacological activities of novel adenine-related compounds isolated from a marine sponge Agelas mauritiana

Agelasimine A and agelasimine B, two novel compounds related to adenine, have been isolated from the orange sponge, Agelas mauritiana, and have been tested for a variety of biological activities. Both compounds inhibited proliferation of cultured L1210 leukemia cells at nanomolar concentrations with accumulation in the G1 stage of the cell cycle. However, no prolongation of life was observed in mice bearing P388 leukemia treated with these compounds. In the rat isolated aorta, micromolar concentrations of agelasimines were very effective in inhibiting contractions elicited by potassium chloride but had little or no effect on responses for prostaglandin F2 alpha and had modest effects on the responses to noradrenaline and significant effects on 5-hydroxytryptamine. Agelsamines A and B appeared to be equipotent in causing relaxation in rabbit jejunum and bovine coronary artery, and they also inhibited nucleoside transport into rabbit erythrocytes in micromolar concentrations.     

Characterization of bovine mononuclear cell populations with natural cytolytic activity against bovine herpesvirus 1-infected cells

Freshly isolated or overnight cultured peripheral blood mononuclear cells from immune or nonimmune animals had natural cytolytic activity against bovine herpesvirus 1 (BHV-1)-infected tumor target cells. No lysis was demonstrated against tumor target cells alone. This natural cytolytic activity was present in mononuclear cells from the spleen, lymph node, and peripheral blood but little or no cytolytic activity was detected in bone marrow or thymus cells. When monoclonal antibodies and complement to deplete bovine mononuclear cell subpopulations from the nonadherent cells were used, results indicated the effector cell was not a T cell, B cell, or activated monocyte. From nonadherent populations separated on density gradients, it was determined that the effector cells were large, low density mononuclear cells. These results indicate the nonadherent effector cells mediating lysis of BHV-1-infected xenogeneic adherent target cells were large null lymphocytes and/or immature monocytes.     

Decreased atrial natriuretic factor receptors and impaired cGMP generation in glomeruli from the cardiomyopathic hamster

To determine a possible basis for the decreased action of atrial natriuretic factors (ANF) in congestive heart failure, we compared the cardiomyopathic hamster (CMH) in frank congestive failure, and the age-matched, normal, F1B strain of Golden Syrian Hamsters. Scatchard analysis of competitive binding studies revealed two classes of glomerular receptors. The CMH exhibited decreased binding overall and a markedly decreased number of high affinity receptors but comparable receptor affinity compared to the F1B. In contrast, the low affinity receptor population in the CMH had a much greater affinity compared to the F1B while receptor number was similar. Plasma ANF levels were substantially elevated in the CMH compared to the F1B and in-vitro generation of cGMP was significantly lower in the CMH. Such abnormalities could contribute to the resistance to ANF in this disease.     

Tissue distribution of beta 1- and beta 2-subunits of regulatory guanine nucleotide-binding proteins

The beta-subunit of G-proteins occurs in two forms (beta 1 and beta 2), which differ in their primary structure as derived from cDNA clones and in their mobilities on SDS gels (36 and 35 kDa, respectively). To assess the tissue distribution of the two forms of beta-subunits, we synthesized peptides corresponding to defined regions of beta 1- and beta 2-subunits and injected them into rabbits; the antisera obtained reacted either with both beta-subunits or specifically with the beta 1- or the beta 2-subunit. They were used to identify the two beta-subunits in membranes prepared from various rat tissues and from human placenta. The concentration of total beta-subunits was high in rat brain and lung, human placenta, rat kidney, liver and spleen; it was much lower in rat erythrocytes, cardiac and skeletal muscle. In all tissues studied, both beta 1- and beta 2-subunits were detectable. In most tested tissues, the two forms were about equally distributed, whereas in the placenta, the beta 2-subunit was found to occur in approx. 2-fold excess over the beta 1-subunit. Our results demonstrate that both beta-subunits are widely distributed. In the majority of tissues, levels of beta 2-subunits are very similar to those of beta 1-subunits. Thus, the abundance of beta 2-subunits as compared to that of the beta 1-subunit is considerably higher than was previously estimated by measuring the respective mRNA levels.