Solution structure of the superactive monomeric des-[Phe(B25)] human insulin mutant: elucidation of the structural basis for the monomerization of des-[Phe(B25)] insulin and the dimerization of native insulin

The three-dimensional solution structure of des-[Phe(B25)] human insulin has been determined by nuclear magnetic resonance spectroscopy and restrained molecular dynamics calculations. Thirty-five structures were calculated by distance geometry from 581 nuclear Overhauser enhancement-derived distance constraints, ten phi torsional angle restraints, the restraints from 16 helical hydrogen bonds, and three disulfide bridges. The distance geometry structures were optimized using simulated annealing and restrained energy minimization. The average root-mean-square (r.m.s.) deviation for the best 20 refined structures is 1.07 angstroms for the backbone and 1.92 angstroms for all atoms if the less well-defined N and C-terminal residues are excluded. The helical regions are more well defined, with r.m.s. deviations of 0.64 angstroms for the backbone and 1.51 angstroms for all atoms. It is found that the des-[Phe(B25)] insulin is a monomer under the applied conditions (4.6 to 4.7 mM, pH 3.0, 310 K), that the overall secondary and tertiary structures of the monomers in the 2Zn crystal hexamer of native insulin are preserved, and that the conformation-averaged NMR solution structure is close to the structure of molecule 1 in the hexamer. The structure reveals that the lost ability of des-[Phe(B25)] insulin to self-associate is caused by a conformational change of the C-terminal region of the B-chain, which results in an intra-molecular hydrophobic interaction between Pro(B28) and the hydrophobic region Leu(B11)-Leu(B15) of the B-chain alpha-helix. This interaction interferes with the inter-molecular hydrophobic interactions responsible for the dimerization of native insulin, depriving the mutant of the ability to dimerize. Further, the structure displays a series of features that may explain the high potency of the mutant on the basis of the current model for the insulin-receptor interaction. These features are: a change in conformation of the C-terminal region of the B-chain, the absence of strong hydrogen bonds between this region and the rest of the molecule, and a relatively easy accessibility to the Val(A3) residue.     

Effect of duodenum-preserving resection of the head of the pancreas on endocrine and exocrine pancreatic function in patients with chronic pancreatitis

Two mobilizable cloning vectors, designated pABW1 and pAWB2, were constructed basing on the E. coli vector pBGS18 and oriT originating from RK2. In pABW2 the kanamycin resistance gene was replaced by a novel tetracycline resistance cassette derived from Tn1721. Both vectors, specific for E. coli, allow to perform the cloning steps in E. coli and then to efficiently transfer the constructs by conjugation to the host of choice. A vector which cannot propagate in the given host can be applied for identification of the host specific plasmid replicator regions. With the use of pABW2 we defined the minimal replicator region of pTAV202-a mini-derivative of the large pTAV1 plasmid of P. versutus. We also proved that RepC' encoded on this fragment is the principal initiator replication protein and that oriV is located along its coding sequence.     

Indocyanine green: intracellular uptake and phototherapeutic effects in vitro

Indocyanine green (ICG; absorption peak in human plasma 805 nm) was investigated for ICG-mediated phototherapy in vitro. The cellular uptake of ICG (1 microM-50 microM) into HaCaT keratinocytes after an incubation period of 24 h increased up to an intracellular ICG concentration of 12.1 +/- 1.3 nmol per 10(6) cells. To examine dose dependent phototoxic effects in vitro, keratinocytes were incubated with 0 microM-50 microM ICG for 24 h and irradiated by a diode laser (805 nm) with different energy densities (0, 12, 24, 48 J cm-2). All applied ICG concentrations except for 5 microM yielded a cell killing effect in combination with irradiation depending significantly on ICG concentration and light dose. Cell viability for dark control and cells incubated with 50 microM ICG and irradiated with 48 J cm-2 was 0.82 +/- 0.15 and 0.07 +/- 0.02, respectively. Sodium azide (100 mM), a quencher of reactive oxygen species, inhibited significantly the cell killing using 50 microM ICG and 24 J cm-2. Taken together, photoactivation of ICG by irradiation with a diode laser was shown to induce effectively cell killing of HaCaT keratinocytes. Moreover, this effect was inhibited by sodium azide, thus irradiation of ICG might induce a photodynamic reaction.     

Incidence of Lightspeed separation and the potential for bypassing

It is important for endodontic instruments to have a low fracture rate. If a fracture does occur, it would be desirable to have the ability to bypass the broken segment and complete the root canal treatment. One hundred sixty-two root canals in 52 maxillary and mandibular first molars were cleaned and shaped with Lightspeed instruments by three endodontists in their private practices. The canals were instrumented using the technique recommended by the manufacturer. All canals were instrumented to at least a size 45 at the working length. Six instruments separated during treatment. All six had been used more times than recommended by the manufacturer. Five of the six were easily bypassed and treatment completed.     

IsK and KvLQT1: mutation in either of the two subunits of the slow component of the delayed rectifier potassium channel can cause Jervell and Lange-Nielsen syndrome

The Jervell and Lange-Nielsen syndrome (JLNS) comprises profound congenital sensorineural deafness associated with syncopal episodes. These are caused by ventricular arrhythmias secondary to abnormal repolarisation, manifested by a prolonged QT interval on the electrocardiogram. Recently, in families with JLNS, Neyroud et al. reported homozygosity for a single mutation in KVLQT1 , a gene which has previously been shown to be mutated in families with dominantly inherited isolated long QT syndrome [Neyroud et al . (1997) Nature Genet ., 15, 186-189]. We have analysed a group of families with JLNS and shown that the majority are consistent with mutation at this locus: five families of differing ethnic backgrounds were homozygous by descent for markers close to the KVLQT1 gene and a further three families from the same geographical region were shown to be homozygous for a common haplotype and to have the same homozygous mutation of the KVLQT1 gene. However, analysis of a single small consanguineous family excluded linkage to the KVLQT1 gene, establishing genetic heterogeneity in JLNS. The affected children in this family were homozygous by descent for markers on chromosome 21, in a region containing the gene IsK . This codes for a transmembrane protein known to associate with KVLQT1 to form the slow component of the delayed rectifier potassium channel. Sequencing of the affected boys showed a homozygous mutation, demonstrating that mutation in the IsK gene may be a rare cause of JLNS and that an indistinguishable phenotype can arise from mutations in either of the two interacting molecules.     

Heart transplantation in Finland 1985-1995

BACKGROUND: Clearance of large molecules from the interstitial space is an important function of lymphatics and is affected by local pathologic changes. OBJECTIVE: To determine if the clearance rate of interstitially injected albumin is correlated to tumour characteristics and outcome in women with invasive breast cancer. METHOD: In a consecutive series of women coming to biopsy for suspected breast cancer, technetium-tagged albumin was injected into the tissue adjacent to the palpable mass. The isotope disappearance rate was measured over two hours. Also assessed were the maximum vessel density (MVD-using Factor VIII polyclonal antisera), the proliferation rate (using Ki-67 antisera), node status, tumour size, histologic and nuclear grade, mitotic rate, and p53 and c-erbB-2 oncoproteins. All patients were followed until relapse and for a minimum of 10 years. RESULTS: In multivariate analysis, an association between relapse-free survival and isotope clearance rate was suggested (p = 0.024). The best outcome was seen in patients with the least isotope clearance. Node status, size, histologic and nuclear grade, and mitotic rate correlated with survival. MVD did not correlate with survival and was inversely related to the isotope clearance rate. Tumour proliferation rate, and the c-erbB-2 and p53 oncoproteins did not relate to outcome. CONCLUSION: The role of lymphatics in breast cancer is difficult to study. Measurement of interstitial clearance may be a useful technique and could be a prognostic factor.     

Health trajectories: long-term dynamics among black and white adults

Flow cytometry is a unique technology useful in the examination of effects of immunotoxic agents on target cells of the immune system. The purpose of this workshop was to provide an overview of the use of flow cytometry in new and established models of immunotoxicity, with emphasis on the potential applications, assay validation, and potential pitfalls. This overview begins with a discussion of methods useful in the assessment of Ca2+-dependent mechanisms of lymphoid cell activation in surface marker-defined human B cells, T cells, and monocytes. A discussion of the use of flow cytometry in analysis of apoptosis is also presented in this paper. The second paper presents data on the development and use of flow cytometry as an alternative to a Cr51 release assay for an assessment of cytotoxic T cell activation. The use of surface markers for characterizing and distinguishing the effects of chemical irritants from sensitizers is next presented, followed by an overview of the use of fluorescent probes to assess cell thiol status and overall oxidant-induced injury to lymphoid cells. Finally, an interlaboratory study designed to compare and evaluate the use of flow cytometry procedures in rat splenic cell subtyping is presented. Overall, these studies demonstrate the utility of flow cytometry assays in immunotoxicologic research, but further efforts are needed in the validation of many of these assays for routine use in immunotoxicologic testing.