Soluble, prolonged-acting insulin derivatives. II. Degree of protraction and crystallizability of insulins substituted in positions A17, B8, B13, B27 and B30

It has previously been found that insulins, to which positive charge has been added by substitutions in position B30, thus raising the isoelectric point towards pH 7, had a prolonged action when injected as slightly acidic solutions because such derivatives crystallize very readily upon neutralization. Positive charge has now been added by substituting the B13 and A17 glutamic acid residues with glutamines and B27 threonine with lysine or arginine. These substitutions were introduced by site-specific mutagenesis in a gene coding for a single-chain insulin precursor. By tryptic transpeptidation the single-chain precursors were transformed to the double-chain insulin structure, concomitantly with incorporation of residue B30. Thus insulins combining B13 glutamine, A17 glutamine and B27 lysine or arginine with B30 threonine, threonine amide or lysine amide were synthesized. The time course of blood glucose lowering effect and the absorption were studied after subcutaneous injection in rabbits and pigs. The prolonged action of B30-substituted insulins was markedly enhanced by B27 lysine or arginine substitutions and by B13 glutamine. The B27 residue is located on the surface of the hexamer, so a basic residue in this position presumably promotes the packing of hexamers at neutral pH. The B13 residues cluster in the centre of the hexamer. When the electrostatic repulsive forces from six glutamic acid residues are abolished by substitution with glutamine, a stabilization of the hexamer can be envisaged.(ABSTRACT TRUNCATED AT 250 WORDS)     

Detection of gastrin and its messenger RNA in Zollinger-Ellison tumors by non-radioactive in situ hybridization and immunocytochemistry

Summary Gastrin immunocytochemistry and non-radioactive in situ hybridization, using biotinylated oligonucleotide probes, for gastrin mRNA have been used for studying a retrospective material of six gastrin-producing (Zollinger-Ellison) tumors. Hybridization results for gastrin mRNA were positive in all six, while gastrin immunoreactivity could be detected in five tumors. In one of the patients, different areas of the same tumor displayed differences in immunoreactivity to gastrin, but were uniformly hybridization positive. Weak hybridization signals were detected in liver metastases from a necropsy case, while the gastrin immunostaining was more pronounced. The results show that non-radioactive hybridization methods are applicable to routine clinical specimens stored for as long as 16 years and that in situ hybridization may be a useful complement to immunocytochemical diagnosis, particularly in cases where high synthesis and little storage of hormonal products occur.     

Trimethyloxonium modification of single batrachotoxin-activated sodium channels in planar bilayers. Changes in unit conductance and in block by saxitoxin and calcium

Single batrachotoxin-activated sodium channels from rat brain were modified by trimethyloxonium (TMO) after incorporation in planar lipid bilayers. TMO modification eliminated saxitoxin (STX) sensitivity, reduced the single channel conductance by 37%, and reduced calcium block of inward sodium currents. These effects always occurred concomitantly, in an all-or-none fashion. Calcium and STX protected sodium channels from TMO modification with potencies similar to their affinities for block. Calcium inhibited STX binding to rat brain membrane vesicles and relieved toxin block of channels in bilayers, apparently by competing with STX for the toxin binding site. These results suggest that toxins, permeant cations, and blocking cations can interact with a common site on the sodium channel near the extracellular surface. It is likely that permeant cations transiently bind to this superficial site, as the first of several steps in passing inward through the channel.     

Predictors of mortality in insulin dependent diabetes: 10 year observational follow up study.

OBJECTIVE: To evaluate the prognostic significance of microalbuminuria and overt diabetic nephropathy and other putative risk factors for cardiovascular and all cause mortality in insulin dependent diabetes. DESIGN: Ten year observational follow up study. SETTING: Outpatient diabetic clinic in a tertiary referral centre. SUBJECTS: All 939 adults with insulin dependent diabetes (duration of diabetes five years or more) attending the clinic in 1984; 593 had normal urinary albumin excretion (< or = 30 mg/24 h), 181 persistent microalbuminuria (31-299 mg/24 h), and 165 overt nephropathy (> or = 300 mg/24 h). MAIN OUTCOME MEASURE: All cause and cardiovascular mortality. RESULTS: Fifteen per cent of patients (90/593) with normoalbuminuria, 25% (45/181) with microalbuminuria, and 44% (72/165) with overt nephropathy at baseline died during follow up. Cox multiple regression analysis identified the following significant predictors of all cause mortality: male sex (relative risk 2.03; 95% confidence interval 1.37 to 3.02), age (1.07; 1.06 to 1.08), height (0.96; 0.94 to 0.98), smoking (1.51; 1.09 to 2.08), social class V versus social class IV (1.70; 1.25 to 2.31), log10 urinary albumin excretion (1.45; 1.18 to 1.77), hypertension (1.63; 1.18 to 2.25), log10 serum creatinine concentration (8.96; 3.34 to 24.08), and haemoglobin A1c concentration (1.11; 1.03 to 1.20). Age, smoking, microalbuminuria, overt nephropathy, and hypertension were significant predictors of cardiovascular mortality. Mortality in patients with microalbuminuria was only slightly increased compared with that in patients with normoalbuminuria. Median survival time after the onset of overt diabetic nephropathy was 13.9 years (95% confidence interval 11.8 to 17.2 years). CONCLUSIONS: Abnormally increased urinary albumin excretion and other potentially modifiable risk factors such as hypertension, smoking, poor glycaemic control, and social class predict increased mortality in insulin dependent diabetes. Microalbuminuria by itself confers only a small increase in mortality. The prognosis of patients with overt diabetic nephropathy has improved, probably owing to effective antihypertensive treatment.     

Endogenous polyamines are intimately associated with highly condensed chromatin in vivo. A fluorescence cytochemical and immunocytochemical study of spermine and spermidine during the cell cycle and in reactivated nuclei

Polyamines are low molecular weight aliphatic polycations essential for cell proliferation and differentiation. By immunocytochemistry, as well as by two independent fluorescence cytochemical methods, we show that polyamines are associated with highly condensed chromatin in nucleated erythrocytes and in metaphase and anaphase chromosomes. In other cells, polyamines mainly occur in cytoplasm. The association between polyamines and DNA in condensed chromatin is so close that DNase treatment is necessary for making polyamines available for reaction with antibodies. Studies of chick/HeLa cell heterokaryons reveal that polyamines disappear from the chick erythrocyte nuclei concomitantly with DNA decondensation and initiation of RNA synthesis. Our data strongly suggest that polyamines are important for chromatin condensation in vivo.     

Combined non-radioactive detection of peptide hormones and their mRNA's in endocrine cells

Summary Non-radioactive in situ hybridization employing detection of biotin-labeled probes by an alkaline phosphatase-based procedure has proven useful for demonstrating a wide variety of mRNA species. With certain developers, the alkaline phosphatase reaction product is both light microscopically visible and fluorescent. We have exploited this to perform simple double-stainings for mRNA's and their corresponding peptide products in human insulin and gastrin cells and in rat ACTH, MSH and gastrin cells. Such stainings show that nearly all of these cells simultaneously contain both the peptide hormone and its corresponding mRNA. Human gastrin cells show a differentiated localization of gastrin mRNA and gastrin. Thus, while gastrin immunofluorescence predominates in secretory granules present in the basolateral region of the cells, gastrin mRNA is virtually restricted to the supranuclear region of the cells. Here it may be preferentially associated with granular endoplasmic reticulum. The strict subcellular localization of gastrin mRNA differs from that of general polyadenylated RNA in the G cells and raises questions whether specific transport routes or sites of accumulation for defined mRNA species exist.     

Particle-Induced Pulmonary Acute Phase Response Correlates with Neutrophil Influx Linking Inhaled Particles and Cardiovascular Risk

Background

Particulate air pollution is associated with cardiovascular disease. Acute phase response is causally linked to cardiovascular disease. Here, we propose that particle-induced pulmonary acute phase response provides an underlying mechanism for particle-induced cardiovascular risk.

Methods

We analysed the mRNA expression of Serum Amyloid A (Saa3) in lung tissue from female C57BL/6J mice exposed to different particles including nanomaterials (carbon black and titanium dioxide nanoparticles, multi- and single walled carbon nanotubes), diesel exhaust particles and airborne dust collected at a biofuel plant. Mice were exposed to single or multiple doses of particles by inhalation or intratracheal instillation and pulmonary mRNA expression of Saa3 was determined at different time points of up to 4 weeks after exposure. Also hepatic mRNA expression of Saa3, SAA3 protein levels in broncheoalveolar lavage fluid and in plasma and high density lipoprotein levels in plasma were determined in mice exposed to multiwalled carbon nanotubes.

Results

Pulmonary exposure to particles strongly increased Saa3 mRNA levels in lung tissue and elevated SAA3 protein levels in broncheoalveolar lavage fluid and plasma, whereas hepatic Saa3 levels were much less affected. Pulmonary Saa3 expression correlated with the number of neutrophils in BAL across different dosing regimens, doses and time points.

Conclusions

Pulmonary acute phase response may constitute a direct link between particle inhalation and risk of cardiovascular disease. We propose that the particle-induced pulmonary acute phase response may predict risk for cardiovascular disease.     

Amniotic Fluid Protein Profiles of Intraamniotic Inflammatory Response to Ureaplasma spp. and Other Bacteria

Objective

This study aimed to evaluate the amniotic fluid protein profiles and the intensity of intraamniotic inflammatory response to Ureaplasma spp. and other bacteria, using the multiplex xMAP technology.

Methods

A retrospective cohort study was undertaken in the Department of Obstetrics and Gynecology, University Hospital Hradec Kralove, Czech Republic. A total of 145 pregnant women with preterm prelabor rupture of membranes between gestational age 24+0 and 36+6 weeks were included in the study. Amniocenteses were performed. The presence of Ureaplasma spp. and other bacteria was evaluated using 16S rRNA gene sequencing. The levels of specific proteins were determined using multiplex xMAP technology.

Results

The presence of Ureaplasma spp. and other bacteria in the amniotic fluid was associated with increased levels of interleukin (IL)-6, IL-8, IL-10, brain-derived neurotropic factor, granulocyte macrophage colony stimulating factor, monocyte chemotactic protein-1, macrophage inflammatory protein-1, and matrix metalloproteinasis-9. Ureaplasma spp. were also associated with increased levels of neurotropin-3 and triggering receptor expressed on myeloid cells-1.

Conclusions

The presence of Ureaplasma spp. in the amniotic fluid is associated with a slightly different protein profile of inflammatory response, but the intensity of inflammatory response to Ureaplasma spp. is comparable with the inflammatory response to other bacteria.