Insertion/deletion polymorphism in the angiotensin-converting enzyme gene associated with macroangiopathy and blood pressure in patients with non-insulin-dependent diabetes mellitus

In the search for new risk factors for diabetic macroangiopathy the insertion/deletion (I/D) polymorphism in the angiotensin-converting enzyme gene was studied in 237 consecutive patients (125 men and 112 women) with non-insulin-dependent diabetes. The female population showed an excess of ischemic electrocardiographic changes or definite myocardial infarctions in the patients homozygous for the deletion [D/D; odds ratio (OR) 2.8; 95% confidence interval (CI) 1.4-5.3] and in the insertion/deletion heterozygotes (I/D; OR 1.8; CI 1.1-3.1) compared with the patients homozygous for the insertion (I/I). In the total series coronary heart disease, cerebrovascular disease, and claudication were more often observed in the patients with I/D (OR 1.5; CI 1.0-2.2) or the D/D genotype patients (OR 1.7; CI 1.1-2.6) than in those with the genotype I/I. The systolic blood pressure was lower in patients with genotype I/I (138 +/- 19 mmHg) than in those with the genotype I/D (149 +/- 22 mmHg) or D/D (150 +/- 21 mmHg; P < 0.02). The prevalence of hypertension and the median urinary albumin excretion rate also tended to be lowest in the I/I genotype patients. Multiple logistic analysis revealed that in women the angiotensin-converting enzyme D/D genotype is independently associated with coronary heart disease. Our findings suggest that variation at the angiotensin-converting enzyme gene locus is one of the factors involved in the predisposition of diabetic patients to the development of arterial disease and hypertension.     

The function of the reproductive system in x-ray-irradiated male rats after the transplantation of a culture of testicular cells from newborn piglets

The effect exerted by transplantation of the 5 day culture of testicles from newly born piglets on the hypogonadal state of mature X-ray irradiated (3 Gy) rats was studied. It has been found that a month after transplantation (two months after irradiation) the hypogonadal state disappeared for a while, which is confirmed by normalization of the weight of seminal vesicles, content of testosterone and biologically active lutropine, restoration of the reproductive function of rats. No significant changes in steroidogenesis activity have been found, which proves a substituent character of transplantation. The effect disappears three months later but certain restoration of the content of nucleic acids in the testicles is observed.     

Induction of Fos immunoreactivity in the brain by exposure to the elevated plus-maze

The log-rank test is commonly used to assess therapeutic effect in prospective, randomised clinical trials. This test is sensitive to differences in survival between treatment groups at a specific endpoint, but cannot determine whether such a difference is due to an enhanced cure rate or an enhanced survival time among uncured patients. To investigate the clinical impact of such limitations, an algorithm was constructed to simulate clinical, randomised, adjuvant therapy trials in patients with a cured fraction of 0.27 and a median survival time for uncured patients of 3.4 years. Hypothetical therapies were introduced to increase rate of cure, increase median survival time, or achieve a combination of these effects. For 500 simulated patients recruited over a 5 year period and then followed for three additional years, a 50% enhancement of median survival time (to 5.1 years) led to a survival increase detectable at the P = 0.05 level in 780 of 1000 trials, whereas a 50% enhancement of cured fraction (to 40.5%) led to a detectable increase at the same level in only 449 of 1000 trials. These findings suggest that, in clinical trials of adjuvant therapy for stage 2 breast cancer, the log rank test may be more sensitive to increases in tumour-related survival time than to increases in cured fraction.     

Regulation in the heart field of zebrafish

In many vertebrates, removal of early embryonic heart precursors can be repaired, leaving the heart and embryo without visible deficit. One possibility is that this 'regulation' involves a cell fate switch whereby cells, perhaps in regions surrounding normal progenitors, are redirected to the heart cell fate. However, the lineage and spatial relationships between cells that are normal heart progenitors and those that can assume that role after injury are not known, nor are their molecular distinctions. We have adapted a laser-activated technique to label single or small patches of cells in the lateral plate mesoderm of the zebrafish and to track their subsequent lineage. We find that the heart precursor cells are clustered in a region adjacent to the prechordal plate, just anterior to the notochord tip. Complete unilateral ablation of all heart precursors with a laser does not disrupt heart development, if performed before the 18-somite stage. By combining extirpation of the heart precursors with cell labeling, we find that cells anterior to the normal cardiogenic compartments constitute the source of regulatory cells that compensate for the loss of the progenitors. One of the earliest embryonic markers of the premyocardial cells is the divergent homeodomain gene, Nkx2.5. Interestingly, normal cardiogenic progenitors derive from only the anterior half of the Nkx2.5-expressing region in the lateral plate mesoderm. The posterior half, adjacent to the notochord, does not include cardiac progenitors and the posterior Nkx2.5-expressing cells do not contribute to the heart, even after ablation of the normal cardiogenic region. The cells that can acquire a cardiac cell fate after injury to the normal progenitors also reside near the prechordal plate, but anterior to the Nkx2.5-expressing domain. Normally they give rise to head mesenchyme. They share with cardiac progenitors early expression of GATA 4. The location of the different elements of the cardiac field, and their response to injury, suggests that the prechordal plate supports and/or the notochord suppresses the cardiac fate.     

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.