Enkephalin analogues containing beta-naphthylalanine at the fourth position

To examine the importance of the aromatic side chains of enkephalin on opiate activity, we report the synthesis and conformational analysis of a series of analogues related to enkephalin with beta-naphthylalanine in place of phenylalanine at the fourth position. Three linear analogues (Tyr-D-Ala-Gly-(L and D)-beta Nal(1)-Leu-NH2 and Tyr-D-Ala-Gly-beta Nal(2)-Leu-NH2) were initially synthesized to examine the effect of the substitution on biological activity. The increased activity of these peptides at the mu-opiate receptor, compared to native Leu-enkephalin, prompted us to examine the more conformational constrained analogues, Tyr-c[D-A2bu-Gly-(L and D)-beta Nal(1)-Leu], incorporating a alpha, gamma-diaminobutyric acid at the second position and cyclization to the carboxylic end of the leucine. These two cyclic analogues provide insight into the necessity for the L chirality of the aromatic residue at position 4. The Tyr-c[D-A2bu-Gly-L-beta Nal(1)-Leu] analogue is highly potent and displays a slight preference for the mu receptor. The conformational analysis indicates that despite the high flexibility of the tyrosine side chain, the aromatic rings of the tyrosine and naphthylalanine are relatively distant from each other. The presence of two intramolecular hydrogen bonds help maintain the conformation of the 14-membered backbone ring that keeps the side chains directed away from each other. These findings are in agreement with our model of an extended structure required for mu selectivity and a folded form with close aromatic ring placement for delta selectivity.     

The human serglycin gene. Nucleotide sequence and methylation pattern in human promyelocytic leukemia HL-60 cells and T-lymphoblast Molt-4 cells.

The complete nucleotide sequence of the 16.7-kb human gene that encodes the peptide core (serglycin) of a secretory granule proteoglycan was determined, thus representing the first proteoglycan peptide core gene to be sequenced in its entirety. The exons, intron 1, and intron 2 comprised 7, 53, and 40% of the gene, respectively. Nineteen Alu-repetitive DNA sequences were interspersed in the gene, accounting for 28% of the total nucleotides in intron 1 and 40% of the nucleotides in intron 2. The nucleotide sequence was then used in an examination of the methylation pattern of the human serglycin gene in human promyelocytic leukemia HL-60 cells that contain serglycin mRNA and in T-lymphoblast Molt-4 cells that do not. With polymerase chain reaction methodology, 13 DNA probes of 250-880 base pairs in length were generated that corresponded to unique, non-Alu sequences spaced throughout the entire human serglycin gene. When blots containing genomic DNA digested with HpaII or MspI were examined with these genomic probes, it was discovered that the 5'-flanking region and intron 1 of the serglycin gene in HL-60 cells were both substantially less methylated than intron 2. In contrast, the entire serglycin gene in Molt-4 cells was highly methylated. Because hypomethylated genes generally are transcribed more efficiently than hypermethylated genes, the high level of serglycin mRNA in HL-60 cells probably is a consequence of the low level of methylation of intron 1 and the 5'-flanking region of the serglycin gene in these cells.     

Characterization and complementation of a cephalosporin-deficient mutant of Streptomyces clavuligerus NRRL 3585

Summary We have characterized a mutant of Streptomyces clavuligerus NRRL 3585 which is almost completely blocked in cephalosporin biosynthesis and exhibits depressed activities of both the delta(l-alpha-aminoadipyl)-l-cysteinyl-d-valine (ACV) synthetase and cyclase enzymes of the cephalosporin pathway. A wild-type DNA region was cloned which partially restores antibiotic production, ACV synthetase and cyclase activities to this mutant. The recombinant plasmid exhibits a variable copy number in different transformants. Hybridization experiments indicate that sequences homologous to the cloned region are present in various -lactam-producing Streptomyces spp. but absent in species which are not known to produce this class of antibiotics. Furthermore, the chromosomal copy of the cloned region lies in close proximity to a gene coding for the isopenicilin N synthase gene of the cephalosphorin pathway.Offprint requests to: J. Piret     

The region of the HPV E7 oncoprotein homologous to adenovirus E1a and Sv40 large T antigen contains separate domains for Rb binding and casein kinase II phosphorylation.

Some genital human papillomavirus (HPV) types, such as 16 and 18, are highly associated with malignant cervical tumors while others, such as HPV 6, are only rarely found in these malignancies. The E7 oncoproteins of HPV 6, 16 and 18 each have a 17 amino acid region with striking homology to adenovirus E1a and SV40 LT. E1a, LT and the E7 oncoprotein of HPV16 all bind the cellular Rb protein in vitro, and for E1a and LT this region of homology contains sequences essential for interaction with Rb. We have now found that in HPV 16 E7 this region (amino acids 21-37) contains two separate biochemical activities, each of which contributes to E7-mediated transformation. Rb binding was localized to the N terminus of this region, while the C terminus was shown to serve as a substrate for casein kinase (CK) II, which phosphorylated serine-31 and serine-32. Replacement of the two serines by non-phosphorylatable amino acids led to a reduction in transforming activity and abolished phosphorylation but did not affect Rb binding. Rb binding and CK II phosphorylation were also examined for the E7 proteins of HPV 6 and HPV 18. HPV 16 and 18 E7 bound similar amounts of Rb, but HPV 6 E7 consistently bound less. Phosphorylation rates also varied, with HPV 18 E7 being 2-fold faster than HPV 16 E7, which in turn was 2-fold faster than HPV 6 E7. We conclude that Rb binding and phosphorylation of E7 by CKII are independent activities which are required for efficient transformation by E7 and that these activities correlate directly with the relative oncogenic potential of these viruses.     

Structure and sequence of the rfb (O antigen) gene cluster of Salmonella serovar typhimurium (strain LT2)

The rfb gene cluster of Salmonella LT2 has been cloned and sequenced. The genes rfbA, rfbB, rfbD, rfbF, rfbG, rfbK, rfbM and rfbP were located individually and the gene rfbL was located outside the cluster. Approximately 16 open reading frames were found in the region which is essential for the expression of O antigen. The gene products of rfbB and rfbG were found to have homology with the group of dehydrogenase and related enzymes described previously. Analysis of the G + C ratio of the rfb cluster extended the area of low-G + C composition previously found in the sequence of rfbJ to the whole rfb gene cluster. Three to five segments with discrete G + C contents and codon adaptation indices are present in the rfb region, indicating a heterogeneous origin of these segments. Potential promoters were found near the start of the rfb region, supporting the possibility that the rfb gene cluster is an operon.     

Intraaortic Balloon Pumping Assist Following Open Heart Surgery for Coronary Artery Disease

Intraaortic balloon pump (IABP) assist was employed in 36 patients after surgical operation for coronary artery disease. In 31 patients, the aid of IABP was required because cardiopulmonary bypass could not be terminated without it. In three of these patients, IABP assist was started before the surgical procedure because these patients were in cardiogenic shock due to myocardial infarction. In the remaining five patients, IABP assist was applied for refractory cardiogenic shock in the early postoperative period.The overall survival rate was 58 percent. IABP assist was used in 13 patients with an ejection fraction of 0.1 to 0.2 (normal 0.7). Nine of these patients survived.From our experience, it would appear that this temporary mechanical circulatory support provides a significant advantage in saving patients who might otherwise die after surgical procedures involving the coronary artery.     

Survival of Entamoeba and related amoebae at low temperature-II. Viability of amoebae and cysts stored in liquid nitrogen

It was found that E. histolytica, E. histolytica-like, E. hartmanni, E. invadens, E. terrapinae, E. moshkovskii grown with a mixed bacterial flora, could be recovered after prolonged storage in liquid nitrogen. The longest period yet tested for E. histolytica is 382 weeks (7·3 years). Storage of amoebae of E. ranarum and E. coli was less successful. E. histolytica amoebae grown axenically or monoxenically were less easily stored than those amoebae grown with a mixed bacterial flora. Cysts were non-viable after freezing.E. histolytica amoebae showed the same virulence to rats and sensitivity to emetine after storage in liquid nitrogen, as was observed before freezing.A summary of a recommended procedure for freezing Entamoeba and related amoebae is given.     

Changes in rhizosphere populations of selected physiological groups of bacteria related to substitution of specific pairs of chromosomes in spring wheat

Summary Rhizosphere population characteristics of two cultivars of spring wheat (Triticum aestivum L. emend Thell.), Rescue (R) and Cadet (C), and the related chromosome substitution lines, C-R₅ B and C-R₅ D, were investigated. Replacement of the chromosome pair, ₅ B, in Cadet with ₅ B from Rescue made many of the rhizosphere microbial characteristics of C-R₅ B similar to or the same as those in the rhizosphere of the donor parent, Rescue. In contrast, substitution of the functionally related chromosome pair, ₅ D, did not cause marked changes in the rhizosphere microbial population, demonstrating the specificity of the plant's control over factors governing the rhizosphere microbial environment.