Difficult conversations: adults with diabetes and the discussion of microvascular complications.

AIMS: To investigate the experiences among adults with diabetes of discussions of microvascular complications and provide recommendations for providers. METHODS: We performed a qualitative study in 148 adults with Type 1 and Type 2 diabetes (56% women, 95% white, mean age 60±13 years, 65% with Type 1 diabetes, 71% with ≥1 microvascular complication). Data were analysed using content analysis. RESULTS: At their first discussion of microvascular complications, 93% of participants (138/148) recalled providers using a preventative approach including clinical suggestions, factual information and warnings. At complication diagnosis, 78% of participants (82/105) perceived provider support through comprehensive interactive education, specific self-care guidance, reassuring messages, and referrals and follow-ups. In response to complication diagnosis, 48% (50/105) felt scared, 46% (48/105) had 'a wake-up call', and 86% (90/105) reported increasing ≥1 specific area of self-care. Participants recommended providers offer factual and complete information, specific self-care guidance, and positive honesty, with an individualized and collaborative approach that includes psychosocial assessment and referrals and lacks 'scare tactics' and blame. CONCLUSIONS: Adults with diabetes want to learn about diabetes microvascular complications and apply preventative strategies as early as possible. Paradoxically, the diagnosis of a diabetes microvascular complication in itself may represent a unique learning opportunity because 86% of participants improved diabetes self-care after this event. Recommendations offer providers simple but important clinical approaches to improve these difficult conversations and thus support necessary behaviour changes and psychosocial well-being. Training is needed to help providers discuss the threat of diabetes complications with honest but positive messages so that people with diabetes can be fully informed but also maintain hope in the face of complications.

Cloning and pharmacological characterization of a rabbit bradykinin B1 receptor.

A rabbit B1 bradykinin receptor cDNA was isolated from a rabbit aorta smooth muscle cell library. The 1223 bp cDNA clone encodes a protein of 352 amino acids which is 78% identical to the human bradykinin B1(3) receptor protein. Heterologous expression of the rabbit B1 receptor cDNA in COS-7 cells imparts a high affinity specific binding for 3H-labeled [des-Arg10,Leu9]kallidin. Scatchard analysis indicates that the receptor binds the radiolabeled ligand with a Kd of 0.5 nM. The ability of kallidin (Lys-bradykinin) and bradykinin analogues to compete with binding of 3H-labeled [des-Arg10,Leu9]kallidin was determined and defined a rank order of potency: [des-Arg10,Leu9]kallidin = [des-Arg10]kallidin > [des- Arg9]bradykinin = kallidin >> bradykinin. This receptor exhibits the classical B1 pharmacological property of preferentially binding to kinin analogues which lack the C-terminal arginine. In addition, the affinities for [des-Arg10]kallidin and [des-Arg10,Leu9]kallidin are 100-fold higher than those for the corresponding bradykinin analogues [des-Arg9]bradykinin and [des-Arg9,Leu8]bradykinin which lack the N-terminal lysine. This pharmacological profile is characteristic of the B1 receptor subtype.

Analysis of Streptomyces avermitilis genes required for avermectin biosynthesis utilizing a novel integration vector.

An integration vector for gene analysis in Streptomyces has been constructed. This vector replicates in Escherichia coli, and integrates into Streptomyces by homologous recombination between a cloned fragment and the genome. To overcome methylation-specific restriction barriers, an E. coli mutant triply defective in DNA methylation was constructed as a source for the integration plasmids. The frequency of integration of pVE616 derivatives into the Streptomyces avermitilis genome was proportional to the size of the cloned DNA. Derivatives of pVE616, containing fragments from pVE650, a plasmid with a 24-kb insert of S. avermitilis DNA, were used in complementation analyses of seven S. avermitilis mutants defective in glycosylation of avermectin (Av). Three complementation groups, located in a 7-kb region, were identified. Derivatives of pVE616, containing fragments from the 18-kb of DNA adjacent to the glycosylation region, were integrated into an Av producer. Av produced from the integrants was substantially reduced, indicating that the 18 kb also encodes gene products which are involved in Av biosynthesis.

Complex organization of the Streptomyces avermitilis genes encoding the avermectin polyketide synthase.

The avermectin (Av) polyketide synthase (PKS) and erythromycin (Er) PKS are encoded by modular repeats of DNA, but the genetic organization of the modules encoding Av PKS is more complex than Er PKS. Sequencing of several related DNA fragments from Streptomyces avermitilis that are part of the Av biosynthetic gene cluster, revealed that they encode parts of large multifunctional PKS proteins. The Av PKS proteins show strong similarity to each other, as well as similarity to Er PKS proteins [Donadio et al., Science 252 (1991) 675-679] and fatty acid synthases. Partial DNA sequencing of the 65-kb region containing all the related sequence elements in the avr genes provides evidence for twelve modular repeats encoding FAS-like domains. The genes encoding the Av PKS are organized as two sets of six modular repeats which are convergently transcribed.

Molecular analysis of a new splice variant of the human melanocortin-1 receptor.

The primary hormonal regulator of pigmentation is melanocyte stimulating hormone derived from proopiomelanocortin by proteolytic processing. The melanocortin-1 receptor serves a key role in the regulation of pigmentation. We describe the identification of the first intron within a melanocortin receptor. A new melanocortin-1 receptor isoform, generated by alternative mRNA splicing, encodes an additional 65 amino acids at the predicted intracellular, C-terminal tail of the melanocortin-1 receptor. When expressed in heterologous cells, the new spliced form of the melanocortin-1 receptor (melanocortin-1 receptor B) appears pharmacologically similar to the non-spliced melanocortin-1 receptor. Melanocortin-1 receptor B is expressed in testis, fetal heart and melanomas.

D-Amino acid scan of gamma-melanocyte-stimulating hormone: importance of Trp(8) on human MC3 receptor selectivity.

In our search for potent and receptor-selective agonists and antagonists, we report here the results of D-amino acid substitution at each position of the short peptide gamma-melanocyte-stimulating hormone (gamma-MSH). The native gamma-MSH shows weak binding at all three receptors (i.e., the human MC3, MC4, and MC5) and a selectivity of 1-2 orders of magnitude at the MC3R over the MC4R and MC5R. Sequential replacement of each residue in the gamma-MSH sequence with the corresponding D-isomer results in analogues which mostly have weaker binding affinity than the native peptide, except for two analogues. For the DTrp(8) analogue, there is an increase in binding affinity by about 1 order of magnitude (IC(50) = 6 nM) at the MC3R compared with that of the natural molecule and an increase in selectivity for the MC3R by 2 orders of magnitude compared with the activity at the MC4R and MC5R. The DPhe(6) analogue is about 10-fold more potent (IC(50) = 8.8 nM) at the MC3R compared with the native peptide but lacks subtype selectivity. Measurement of the intracellular cAMP accumulation in human MC3R, MC4R, and MC5R revealed that the native peptide shows potent activity at the MC3R (EC(50) = 5.9 nM) and is about 50-100-fold selective at this receptor compared with the MC4R and MC5R. The DArg(10) (EC(50) = 35 nM) and DPhe(11) (EC(50) = 11 nM) analogues are selective for the MC3R by 1 and 2 orders of magnitude compared with the MC4R and MC5R, respectively. The DTrp(8) compound (EC(50) = 0.33 nM) shows about 300- and 250-fold increase in selectivity at the MC3R compared with the MC4R and MC5R, respectively. Finally, the DTyr(1) peptide is selective for the MC3R (EC(50) = 12 nM) by 40-200-fold compared with the MC4R and MC5R. In general, the trend is that D-amino acid substitutions of the aromatic residues 1, 6, 8, and 11 and the basic residue Arg(10), but not Arg(7), result in an increase in MC3R selectivity over the MC4R and MC5R and only agonist activity is observed. Thus, the key residues of gamma-MSH identified in this study include the aromatic residues 1, 6, 8, and 11 and the basic residue Arg(10) (but not Arg(7)), as important for MC3 selectivity over the MC4 and MC5 subtypes. Further, the study reveals the extreme importance of DTrp at position 8 in imparting potency and selectivity since this is the most selective analogue for the human MC3R reported thus far.

Selective, high affinity peptide antagonists of alpha-melanotropin action at human melanocortin receptor 4: their synthesis and biological evaluation in vitro.

Peptide Ac-Nle(4)-cyclo(5beta-->10epsilon)(Asp(5)-His(6)-D-(2')Nal(7)-Arg(8)-Trp(9)-Lys(10))-NH(2), compound 1, a cyclic derivative of alpha-melanotropin, is a nonselective high affinity antagonist at human melanocortin receptors 3 and 4, and an agonist at melanocortin receptors 1 and 5. To differentiate between the physiological functions of these receptors, antagonists with improved receptor selectivity are needed. In this study, analogues of compound 1 without Ac-Nle(4) or His(6) and/or the amino group of Asp(5) were prepared and tested in binding assays and in functional assays on CHO cells expressing hMC3-5R. Several of these peptides were to be selective, high affinity hMC-4R antagonists. The most interesting was compound 10, named MBP10, cyclo(6beta-->10epsilon)(succinyl(6)-D-(2')Nal(7)-Arg(8)-Trp(9)-Lys(10))-NH(2), an antagonist (IC(50) = 0.5 nM) with 125-fold selectivity over hMC-3R (and of >300-fold selectivity over MC-1RB). This compound had no agonist activity at hMC-3R or hMC-4R and only weak agonist activity at hMC-5R. Examination of the sequences of these new peptides revealed that the D-(2')Nal(7)-Arg(8)-Trp(9) segment of peptide 1 forms the "essential core" required for high affinity and high selectivity of analogues of peptide 1 at hMC-4R, but the "extended core", His(6)-D-(2')Nal(7)-Arg(8)-Trp(9), is necessary for the maximum affinity for hMC-3R and hMC-5R.

Correlation of the avermectin polyketide synthase genes to the avermectin structure. Implications for designing novel avermectins.

Streptomyces avermitilis produces a series of eight potent anthelmintic compounds called avermectins (AVM). AVM are pentacyclic, macrocyclic lactone compounds containing an oleandrose disaccharide. Labeling studies have shown that AVM is a polyketide derived from the condensation of 12 acyl units (five propionates and seven acetates) to an isobutyl or 2-methylbutyryl starter unit. The genes required for AVM biosynthesis have been cloned, and deletion mapping has located the AVM gene cluster to a 95-kb region. Partial DNA sequencing of this region indicates two 30-kb segments encode large, multifunctional peptides of the AVM polyketide synthase (PKS). The PKS proteins contain at least 49 domains with homology to the domains in fatty acid synthase and erythromycin PKS. These domains are arranged as 12 modular repeats that each encode a PKS unit with various subsets of the FAS-like functions. The predicted functions required to form the side groups on the AVM macrocyclic ring were compared to the functions found in the 12 PKS units. This comparison suggests that each PKS unit is specific for condensation and reduction of one acyl unit. If the various domains can be manipulated without disrupting the PKS, it may be possible to synthesize a variety of AVM derivatives.

Structure-function studies on the cyclic peptide MT-II, lactam derivative of alpha-melanotropin.

The alanine-substituted and the retro, enantio, and retro-enantio analogs of MT-II, a potent agonist at melanocortin (MC) receptors, were prepared by solid-phase synthesis and evaluated for their ability to bind and activate human MC3, MC4, and MC5 receptors. Replacement of His with Ala resulted in [Ala6]-MT-II with affinity and agonist potency at human MC3, MC4, and MC5 receptors similar to MT-II. Substitution of Arg with Ala gave compound 100-fold less potent than MT-II, but replacement of Phe or Trp with Ala led to inactive compounds (at the micromolar concentrations). The significant drop of potency of the retro, enantio, and retro-enantio analogs of MT-II, demonstrated a crucial role of side-chain topology, and to a lesser degree, of peptide backbone in interactions of MT-II with the melanocortin receptors. The nuclear magnetic resonance analysis of MT-II suggested involvement of Phe and Arg residues in H-bonds stabilizing the bent conformations of the peptide backbone.

Cloning and pharmacological characterization of bradykinin receptors.

A human B2 bradykinin receptor cDNA was cloned from the lung fibroblast cell line, CCD-Lu. This clone was utilized to isolate a genomic clone of a mouse B2 bradykinin receptor. Both clones encode a protein that has the predicted characteristics of a seven transmembrane domain G-protein-coupled receptor. The DNA sequence of these two clones is 84% identical in the putative coding region. The clones have been heterologously expressed in a mammalian cell line lacking endogenous bradykinin receptors, COS-7, and a comparative analysis of their pharmacology was done. Both clones exhibit properties characteristic of the B2 bradykinin receptor, binding bradykinin with high affinity (KD = 0.1-0.2 nM) and binding des-Arg9 bradykinin with a very low affinity (IC50 > 5 microM). Interestingly, the mouse B2 bradykinin receptor has a 60-80 fold higher affinity than the human B2 bradykinin receptor for the peptide antagonists D-Arg0[Hyp3,Thi5,8,D-Phe7]bradykinin and D-Arg0[Hyp3,D-Phe7]bradykinin.

Fine-structure deletion map and complementation analysis of the glnA-glnL-glnG region in Escherichia coli.

A total of 399 independent mutants of Escherichia coli were obtained which have point and insertion mutations in the glnA region. Mutants isolated included Gln- and Reg- strains (unable to utilize arginine as a nitrogen source). Mutations were mapped with 73 deletion-containing derivatives of a lambda gln phage. Complementation analysis was performed with lambda gln derivatives containing point mutations which conferred a Gln- or Reg- phenotype. Deletion mapping and complementation analysis assigned 104 mutations in 24 deletion intervals to glnA. Mutations in Reg- strains were assigned to two genes, glnL and glnG. glnL contained 131 mutations in 12 deletion intervals, and glnG contained 164 mutations in 10 deletion intervals. The gene order is glnA-glnL-glnG, transcribed from left to right. Polarity of insertion mutations indicates that glnL and glnG form from left to right. Polarity of insertion mutations indicates that glnL and glnG form an operon. Complementation analysis of glnA insertion mutations with glnL and glnG mutations showed polarity of glnA onto most glnL and glnG alleles, suggesting that transcription of glnA may proceed into the glnL-glnG operon. All mutations analyzed in glnA conferred a Gln- phenotype. However, we also found that over half of the Gln- strains isolated ater chemical mutagenesis contained point mutations in glnG. Mutants which synthesized a high level of glutamine synthetase in the presence of ammonia (GlnC phenotype) were selected as revertants of a strain with a Tn10 insertion in glnD and were mapped with chromosomal deletions. Results indicate that mutations in 12 and 15 examined strains clearly map outside of glnA, probably in glnL.

Characterization of the Streptomyces plasmid pVE1.

pVE1 (11.0 kb) was isolated from Streptomyces venezuelae ATCC 14585 and characterized. pVE1 has a broad host range and an apparent copy number of about 100 during exponential growth, rising to 1500 during stationary phase. It is a pock-forming, self-transmissible fertility factor which promotes chromosome recombination in S. lividans at frequencies of about 0.1% per total parents. A detailed restriction map for 15 enzymes was determined. Genes for ThioR (thiostrepton resistance), NeoR (neomycin resistance), and pBR322 derivatives were inserted into pVE1 and the resulting plasmids were analyzed for self-transmissibility and stability. The plasmid has an essential region of approximately 2.5 kb and a region of 1.0-3.6 kb required for conjugation. NeoR and ThioR vectors were constructed with unique HindIII, PvuI, BamHI, EcoRI, BglII, and EcoRV sites available for insertion of foreign DNA.

Deletion analysis of the avermectin biosynthetic genes of Streptomyces avermitilis by gene cluster displacement.

Streptomyces avermitilis produces a group of glycosylated, methylated macrocyclic lactones, the avermectins, which have potent anthelmintic activity. A homologous recombination strategy termed gene cluster displacement was used to construct Neor deletion strains with defined endpoints and to clone the corresponding complementary DNA encoding functions for avermectin biosynthesis (avr). Thirty-five unique deletions of 0.5 to > 100 kb over a continuous 150-kb region were introduced into S. avermitilis. Analysis of the avermectin phenotypes of the deletion-containing strains defined the extent and ends of the 95-kb avr gene cluster, identified a regulatory region, and mapped several avr functions. A 60-kb region in the central portion determines the synthesis of the macrolide ring. A 13-kb region at one end of the cluster is responsible for synthesis and attachment of oleandrose disaccharide. A 10-kb region at the other end has functions for positive regulation and C-5 O methylation. Physical analysis of the deletions and of in vivo-cloned fragments refined a 130-kb physical map of the avr gene cluster region.

Bacteriophage mu-induced deletions in a plasmid containing the nif (N2 fixation) genes of Klebsiella pneumoniae.

Five plasmids with insertions of a heat-inducible Mu prophage in a Mu-sensitive and P1-sensitive derivative of plasmid pRD1, a recombinant R factor containing the his-nif region of Klebsiella pneumoniae, were isolated and characterized. In one plasmid containing the Mu prophage integrated at the his-distal end of nif, selection for heat resistance resulted in the generation of deletions extending from the Mu prophage into the nif region. Thirty of these deltions were used to map 26 point mutations in nif.

Fine-structure mapping and complementation analysis of nif (nitrogen fixation) genes in Klebsiella pneumoniae.

Four hundred and eighty-nine independent Nif- strains containing 260 point, 130 millimicron-induced, and 99 deletion mutations in nif in the Klebsiella pneumoniae chromosome were isolated. Three hundred and ninety insertion and point mutations were mapped with millimicron-induced deletions carried on 44 plasmids derived from pTM4010, a recombinant R factor containing the his-nif region of K. pneumoniae. The 99 chromosomal deletions in the nif region were mapped with 69 derivatives of pTM4010 carrying insertion and point mutations in nif. Complementation analysis between 84 derivatives of pTM4010 carrying nif mutations and Rec- derivatives of the 390 Nif- mutants identified 14 genes. The nif mutations were ordered into 49 deletion groups with a gene order of his...nifQBALFMVSNEKDHJ. Complementation analysis of millimicron-induced, amber, frameshift, and deletion mutations indicates there are five polycistronic and two monocistronic operons: nifQ nifB, nifA nifL, nifF, nifM nifV nifS, nifN nifE, nifK nifD nifH, and nifJ. Transcription is from right to left in all polycistronic operons.

Regulation and characterization of protein products coded by the nif (nitrogen fixation) genes of Klebsiella pneumoniae.

Two hundred and thirty-five Nif- strains of Klebsiella pneumoniae were characterized by two-dimensional polyacrylamide gel electrophoresis. Forty-two of these strains were tested further by in vitro acetylene reduction assays. By these techniques, nine nif-coded polypeptides were identified, and eight of these were assigned to specific nif genes. Nitrogenase component I required nifK and nifD, which coded for the beta and alpha subunits, and nifB, -E, and -N were required for the iron-molybdenum cofactor, which is a part of the active site of nitrogenase. nifH coded for the structural protein of component II, and nifM and nifS products seemed to be necessary for the synthesis of an active component II. There were two genes, nifF and nifJ, that were required for N2 fixation in vivo but not for N2 fixation in vitro. There were at least two cases (nifE and nifN, nifK and nifD) of two proteins that seemed to require each other for stability in vivo. Regulation of N2 fixation is apparently complex, and this is reflected by the assignment of regulatory functions to the gene products of nifA, nifL, nifK, nifD, nifH, and NIFJ.

A poison education program for primary school.

A program with which to teach children in grades 1 and 2 to identify poisons and hazard symbols, to learn where poisons should be stored and to recognize a poisoning and contact a poison control center in the event of a poisoning was developed. Each objective was taught in one 30-minute lesson by the children's school teachers. Evaluation of the approximately 400 children who received the program by pre- and post-tests demonstrated that they learned to identify poisons, to recognize hazard symbols and the telephone number of the local poison control center. The children were also asked to identify their source of knowledge about poisons and cited parents, television and school as their most important sources.

The agonist selectivity of a mouse B1 bradykinin receptor differs from human and rabbit B1 receptors.

A genomic clone encoding the mouse B1 receptor was isolated by homology to the human B1 receptor cDNA. The deduced amino acid sequence of the mouse B1 receptor is 72% identical to the human B1 receptor and 73% identical to the rabbit B1 receptor. Ligand binding studies of the mouse B1 receptor expressed in COS cells indicate that it has the pharmacological properties associated with the B1 receptor subtype. However the pharmacology of the mouse receptor is unique in that it possesses a 2-3-fold selectivity for the 'classical' B1 agonist des-Arg9BK over the agonist des-Arg10 kallidin. In contrast, the human and rabbit B1 receptors exhibit an approx. 2000- and 150-fold selectivity, respectively, for des-Arg10kallidin over des-Arg9BK. Thus relative to the human and rabbit B1 receptors the mouse B1 receptor has the opposite selectivity for kinin agonists. The DNA sequence of the region encoding bradykinin was determined for two different mouse kininogen cDNA clones, both encode the sequence Arg-BK. Antipeptide antibodies directed against a C-terminal peptide of the human B1 receptor were produced. Initial characterization of this antibody indicates that it detects specific bands by Western blot analyses that are present in membranes prepared from COS cells transfected with the human B1 receptor cDNA but not from mock transfected COS cells.

Potent and selective peptide agonists of alpha-melanotropin action at human melanocortin receptor 4: their synthesis and biological evaluation in vitro.

alpha-Melanotropin (alphaMSH) and several of its derivatives are potent but not selective agonists at melanocortin receptors 3, 4, and 5 present in the brain (MC3-5R). To differentiate between the physiological role of hMC-4R (believed to be involved in regulation of energy balance) from those of melanocortin receptors 3 and 5, potent and receptor-specific agonists are needed. Therefore, the cyclic derivatives of alphaMSH of a general structure, cyclo(X-His-d-Phe-Arg-Trp-Y)-NH(2), where X is succinic acid or an omega-amino-carboxylic acid, and Y is an alpha,omega-di-amino-carboxylic acid or an omega-carboxy-alpha-amino acid, were prepared and tested in binding assays and in cAMP assays on CHO cells expressing hMC3-5R. Several of the 21-membered or larger lactams turned out to be potent and hMC-4R-selective agonists. For instance, cyclo(CO-CH(2)-CH(2)-CO-His-d-Phe-Arg-Trp-Dab)-NH(2) (Dab: 2,4-di-amino-butyric acid) was a potent agonist at hMC-4R (EC(50) = 4 nM) with 55-fold selectivity over hMC-3R and greater than 1000-fold selectivity over hMC-5R. Another potent and selective compound was cyclo(NH-CH(2)-CH(2)-CO-His-d-Phe-Arg-Trp-Glu)-NH(2): EC(50) about 1 nM at hMC-4R, with 90-fold selectivity over hMC-3R and greater than 2000-fold selectivity over hMC-5R.

Analogs of lactam derivatives of alpha-melanotropin with basic and acidic residues.

A role of the aromatic and of the basic residues of the potent agonist (MTII) and antagonist (SHU9119) at the human melanocortin receptors 4 in the formation and stabilization of ligand-receptor complexes was examined. Analogs of MTII and SHU9119 with glutamic acid replacing one amino acid at a time were synthesized and tested for their ability to bind to and activate human melanocortin receptors 3, 4, and 5. Replacement of Phe (Nal) or Trp with Glu resulted in analogs of MTII and SHU9119 which were practically inactive at the receptors studied. The rather large (and unexpected) tolerance toward the presence of Glu in the position of His or Arg of MTII and SHU9119 clearly suggested that in the ligand receptor complexes these basic residues are not in contact with the receptors but probably face the extracellular environment. This identified the aromatic residues of MTII and SHU9119 as the primary structural features determining interactions of the agonist/antagonist with hMCR3-5.