Characterization of ribosome binding on Rous sarcoma virus RNA in vitro.

We determined the sites at which ribosomes form initiation complexes on Rous sarcoma virus RNA in order to determine how initiation of Pr76gag synthesis at the fourth AUG codon from the 5' end of Rous sarcoma virus strain SR-A RNA occurs. Ribosomes bind almost exclusively at the 5'-proximal AUG codon when chloride is present as the major anion added to the translational system. However, when chloride is replaced with acetate, ribosomes bind at the two 5'-proximal AUG codons, as well as at the initiation site for Pr76gag. We confirmed that the 5'-proximal AUG codon is part of a functional initiation site by identifying the seven-amino acid peptide encoded there. Our results suggest that (i) translation in vitro of Rous sarcoma virus virion RNA results in the synthesis of at least two polypeptides; (ii) the pattern of ribosome binding observed for Rous sarcoma virus RNA can be accounted for by the modified scanning hypothesis; and (iii) the interaction between 40S ribosomal subunits or 80S ribosomal complexes is stronger at the 5'-proximal AUG codon than at sites farther downstream, including the initiation site for the major viral proteins.     

Aerobic spore-forming bacteria as detrimental infectants in plant tissue cultures

Summary Aerobic spore-forming bacteria were isolated from plant tissue cultures from a commercial plant cultivation station. Bacillus circulans was found to be a detrimental infectant as a serious consequence of the heat-resistance of the endospores of these bacteria. They were extremely motile, utilized several growth-promoting factors, and could be eliminated by early microscopical identification, killing by heat treatment, or by using antibiotics or disinfecants.Dedicated to Professor Dr. Gustav Kortüm on the occasion of his 80th birthday     

Control of K+ conductance by cholecystokinin and Ca2+ in single pancreatic acinar cells studied by the patch-clamp technique

Summary The effect of cholecystokinin (CCK) and internal Ca²⁺ on outward K⁺ current in isolated pig pancreatic acinar cells has been investigated using the patch-clamp method for whole-cell current recording under voltage-clamp conditions. CCK (2 × 10^–10 M) applied to the bath evoked a marked increase in the outward K⁺ current associated with depolarizing voltage steps, and this effect was fully reversible and acutely dependent on the presence of external Ca²⁺. When strongly buffered Ca²⁺-EGTA solutions were used inside the cells CCK failed to evoke an effect. Increasing the internal Ca²⁺ concentration ([Ca²⁺] _i ) from 5 × 10^–10 M to 10^–7 and 5 × 10^–7 M mimicked the effect of CCK. It would appear therefore that CCK controls K⁺ conductance in the acinar cells via changes in the internal free ionized Ca²⁺ concentration.     

Monocyte suppression of antigen-specific lymphocyte responses in diffuse cutaneous leishmaniasis patients from the Dominican Republic

Patients from the Dominican Republic with diffuse cutaneous leishmaniasis showed in vivo and in vitro anergy to leishmanial antigen. Relatives of these DCL patients living in the same endemic area frequently showed skin test and lymphocyte reactivity to leishmanial antigens. This further supports the concept of specific anergy in patients with diffuse cutaneous leishmaniasis. Adherent suppressor cells modulate the antigen-specific lymphocyte proliferative response. Suppressor cells could also be isolated by Percoll gradient centrifugation. Co-culturing of lymphocytes and monocytes from HLA-identical leishmanin responders and nonresponders also identified the suppressor cell as a monocyte. In one patient, this suppression disappeared when clinical cure had been accomplished.     

Replication of adenovirus mini-chromosomes

We have isolated adenovirus origins of DNA replication from both the right and left ends of the genome, which are functional on linear autonomously replicating mini-chromosomes. The mini-chromosomes contain two cloned inverted adenovirus termini and require non-defective adenovirus as a helper. Replicated molecules are covalently attached to protein, and DNA synthesis is initiated at the correct nucleotide even when the origins are not located at molecular ends. The activity of embedded origins leads to the generation of linear minichromosomes from circular or linear molecules. These observations therefore suggest that sequences within the adenovirus origin of replication position the protein priming event at the adenovirus terminus. Experiments investigating the regeneration of deleted viral inverted terminal repeat sequences show a sequence-independent requirement for inverted sequences in this process. This result strongly suggests that repair results from the formation of a panhandle structure by a displaced single strand. On the basis of these observations we propose a model for the generation of adenovirus mini-chromosomes from larger molecules.     

Thymidine kinase, thymidylate synthase, and endothelial cell growth under hyperoxia

To determine the respective role of thymidine kinase and thymidylate synthase activities in the hyperoxia-induced decrease in DNA synthesis and their relationship with cell replication, we measured these two enzyme activities in primary cultures of porcine aortic endothelial cells under different O2 concentrations for various durations. In confluent cells, exposure to 95% O2 for 5 days reduced thymidine kinase activity to 15% of control values; thymidylate synthase activity was unaffected. In preconfluent cells exposed to 95% O2 for 2 days, similar results were obtained, together with evidence for arrest in cell proliferation. Thymidylate synthase activity could therefore not be related to decreased cell proliferation under hyperoxia. [3H]thymidine incorporation into DNA, thymidine kinase activity, and cell proliferation were all similarly affected under exposure to graded O2 concentration for 2 days. Thymidine kinase appears to be a key enzyme in the modulation of DNA synthesis from thymidine and in its replication in endothelial cells.     

Accuracy and reliability of flow cytometric DNA analysis using a simple, one-step ethidium bromide staining protocol

Sources of variation and error were investigated for a simple flow cytometric analysis of DNA content of detergent-isolated nuclei stained with ethidium bromide. Using the ploidy classes of mouse liver nuclei, deviations from linearity were assessed for three different instruments. In more extreme settings, the maximum deviations for a FACS instrument were up to 6 to 9%, but in general deviations were around 1% or lower for all instruments. As biological DNA standards, human peripheral lymphocytes and trout erythrocytes appeared to be suitable and easy to store frozen. The erythrocytes had dye-binding characteristics similar to those of human lymphocytes and a 20% lower fluorescence, thus being well suited as an internal standard, as was demonstrated in tumor ploidy analyses performed with varied tissue concentration. Staining homogeneity was improved when staining time was extended to 24 h, at which time male and female lymphocytes were completely separated with an average difference in DNA content of 1.9%. A small difference in fluorescence between mitogen-stimulated and unstimulated lymphocytes was reduced to less than 1% after 24 h of staining. In general, the manipulations of the conditions for the analysis resulted in maximum variations of around 1%, indicating the robustness and reliability of the technique.     

Distribution and characterization of the opioid octapeptide met5-enkephalin-arg6-gly7-leu8 in the gastrointestinal tract of the rat

Summary The distribution and characterization of the opioid octapeptide met⁵-enkephalin-arg⁶-gly⁷-leu⁸ (met⁵-enk-arg⁶-gly⁷-leu⁸) within the gastrointestinal tract of the rat has been determined by immunohistochemistry and radioimmunoassay by use of a newly developed antibody to met⁵-enk-arg⁶-gly⁷-leu⁸. With both techniques, met⁵-enk-arg⁶-gly⁷-leu⁸-immunoreactivity (met⁵-enk-arg⁶-gly⁷-leu⁸IR) was detected in all regions of the gastrointestinal (GI) tract except the esophagus. The highest concentration of immunoreactive met⁵-enk-arg⁶-gly⁷-leu⁸ was observed in the colon, while intermediate concentrations were found in the stomach, duodenum, jejunum, and ileum. Immunostained somata were observed chiefly in the myenteric plexus; immunostained processes were present primarily in the myenteric plexus and the circular muscle layer. This distribution pattern is similar to that previously observed with antiserum to met⁵-enkephalin-arg⁶-phe⁷ (met⁵-enk-arg⁶phe⁷). Chromatographic analysis of met⁵-enk-arg⁶-gly⁷leu⁸-immunoreactive peptides extracted from the GI tract revealed the presence of an immunoreactive peptide of high molecular weight which accounted for approximately three-quarters of met⁵-enk-arg⁶-gly⁷-leu⁸-IR in both stomach and colon. These findings suggest a role for peptides related to the octapeptide met⁵-enk-arg⁶-gly⁷-leu⁸ in the regulation of GI function.