Effect of near physiologic insulin therapy on hypoglycemia counterregulation in type-1 diabetes

OBJECTIVES: The aim of this study was to examine hormonal counterregulation during insulin-induced hypoglycemia in type-1 diabetic patients during long-term near normoglycemic insulin therapy and intensive clinical care. METHODS: Type-1 diabetic patients (age 35.3 +/- 2 years, body mass index 22.8 +/- 1 kg x m(-2), mean diabetes duration 13.6 (11-17 years), mean HbA1c during the last year 6.6 +/- 0.1%) and nondiabetic subjects were studied during (0-120 min) and after (120-240 min) hypoglycemic (3.05 mmol/l) hyperinsulinemic (approximately 330 pmol/l) clamp tests. RESULTS: During hypoglycemia peak plasma concentrations of glucagon (199 +/- 16 vs. 155 +/- 11 ng/l, p < 0.05), epinephrine (4,514 +/- 644 vs. 1,676 +/- 513 pmol/l, p < 0.001), norepinephrine (2.21 +/- 0.14 vs. 1.35 +/- 0.19 nmol/l, p < 0.01) and cortisol (532 +/- 44 vs. 334 +/- 61 nmol/l) were reduced in the diabetic patients. Plasma lactate did not change from baseline values (0.51 +/- 0.06 mmol/l) in diabetic but doubled in healthy subjects (1.13 +/- 0.111 mmol/l, p < 0.001 vs. control). During the posthypoglycemic recovery period plasma concentrations of free fatty acids were higher in diabetic patients at 240 min (1.34 +/- 0.12 vs. 2.01 +/- 0.23 mmol/l, p < 0.05). CONCLUSION: Despite long-term near physiologic insulin substitution and the low incidence of hypoglycemia, hormonal hypoglycemia counterregulation was impaired in type-1 diabetic patients after a diabetes duration of more than 10 years.     

Mdm2 is a RING finger-dependent ubiquitin protein ligase for itself and p53

Mdm2 has been shown to regulate p53 stability by targeting the p53 protein for proteasomal degradation. We now report that Mdm2 is a ubiquitin protein ligase (E3) for p53 and that its activity is dependent on its RING finger. Furthermore, we show that Mdm2 mediates its own ubiquitination in a RING finger-dependent manner, which requires no eukaryotic proteins other than ubiquitin-activating enzyme (E1) and an ubiquitin-conjugating enzyme (E2). It is apparent, therefore, that Mdm2 manifests an intrinsic capacity to mediate ubiquitination. Mutation of putative zinc coordination residues abrogated this activity, as did chelation of divalent cations. After cation chelation, the full activity could be restored by addition of zinc. We further demonstrate that the degradation of p53 and Mdm2 in cells requires additional potential zinc-coordinating residues beyond those required for the intrinsic activity of Mdm2 in vitro. Replacement of the Mdm2 RING with that of another protein (Praja1) reconstituted ubiquitination and proteasomal degradation of Mdm2. However, this RING was ineffective in ubiquitination and proteasomal targeting of p53, suggesting that there may be specificity at the level of the RING in the recognition of heterologous substrates.     

Differences in Elasticity of Vinculin-Deficient F9 Cells Measured by Magnetometry and Atomic Force Microscopy

We have investigated a mouse F9 embryonic carcinoma cell line, in which both vinculin genes were inactivated by homologous recombination, that exhibits defective adhesion and spreading [Collet al.(1995)Proc. Natl. Acad. Sci. USA92, 9161–9165]. Using a magnetometer and RGD-coated magnetic microbeads, we measured the local effect of loss and replacement of vinculin on mechanical force transfer across integrins. Vinculin-deficient F9Vin(−/−) cells showed a 21% difference in relative stiffness compared to wild-type cells. This was restored to near wild-type levels after transfection and constitutive expression of increasing amounts of vinculin into F9Vin(−/−) cells. In contrast, the transfection of vinculin constructs deficient in amino acids 1–288 (containing the talin- and α-actinin-binding site) or substituting tyrosine for phenylalanine (phosphorylation site, amino acid 822) in F9Vin(−/−) cells resulted in partial restoration of stiffness. Using atomic force microscopy to map the relative elasticity of entire F9 cells by 128 × 128 (n= 16,384) force scans, we observed a correlation with magnetometer measurements. These findings suggest that vinculin may promote cell adhesion and spreading by stabilizing focal adhesions and transferring mechanical stresses that drive cytoskeletal remodeling, thereby affecting the elastic properties of the cell.     

Bacterial Community Dynamics during Start-Up of a Trickle-Bed Bioreactor Degrading Aromatic Compounds

This study was performed with a laboratory-scale fixed-bed bioreactor degrading a mixture of aromatic compounds (Solvesso100). The starter culture for the bioreactor was prepared in a fermentor with a wastewater sample of a car painting facility as the inoculum and Solvesso100 as the sole carbon source. The bacterial community dynamics in the fermentor and the bioreactor were examined by a conventional isolation procedure and in situ hybridization with fluorescently labeled rRNA-targeted oligonucleotides. Two significant shifts in the bacterial community structure could be demonstrated. The original inoculum from the wastewater of the car factory was rich in proteobacteria of the alpha and beta subclasses, while the final fermentor enrichment was dominated by bacteria closely related to Pseudomonas putida or Pseudomonas mendocina, which both belong to the gamma subclass of the class Proteobacteria. A second significant shift was observed when the fermentor culture was transferred as inoculum to the trickle-bed bioreactor. The community structure in the bioreactor gradually returned to a higher complexity, with the dominance of beta and alpha subclass proteobacteria, whereas the gamma subclass proteobacteria sharply declined. Obviously, the preceded pollutant adaptant did not lead to a significant enrichment of bacteria that finally dominated in the trickle-bed bioreactor. In the course of experiments, three new 16S as well as 23S rRNA-targeted probes for beta subclass proteobacteria were designed, probe SUBU1237 for the genera Burkholderia and Sutterella, probe ALBO34a for the genera Alcaligenes and Bordetella, and probe Bcv13b for Burkholderia cepacia and Burkholderia vietnamiensis. Bacteria hybridizing with the probe Bcv13b represented the main Solvesso100-degrading population in the reactor.Many branches of industry produce waste gases which contain odorous organic and inorganic components. Apart from the conventional thermal and physicochemical techniques for removal of pollutants from exhaust air, biological waste gas treatment is becoming more and more important. This kind of treatment is advantageous in cases in which the recovery of the components (e.g., absorption in liquids and adsorption in solids) or the utilization of a thermal process (thermal or catalytic combustion) is not economical. Today three different process variations for biological waste gas treatment are used: biofilters, bioscrubbers, and trickle-bed bioreactors. In biofilters and trickle-bed reactors, the pollutant-degrading microorganisms are immobilized on a carrier material, whereas in bioscrubbers the microorganisms are dispersed in the liquid phase. Biofilters and bioscrubbers are preferred in industry, while biofilters are common in compost production and sewage plants (10).Biological waste gas treatment has a long tradition. Already in 1953, a soil system was employed for the treatment of odorous sewer exhaust gases in Long Beach, Calif. (25), and although up to now a lot of efforts have been funneled into process engineering (14, 17, 18, 24), current knowledge of the involved microorganisms is still very limited. Diversity of the microbial communities in the bioreactors for the exhaust gas purification have mostly been analyzed by culture-dependent methods (9, 12, 28, 31). However, there is a large discrepancy between the total (direct) microscopic cell counts and viable plate counts in many ecosystems and every cultivation medium selects for certain microorganisms. Therefore, cultivation-based studies of bacterial populations may give wrong impressions of the actual community structure in an ecosystem (35). A possible means of avoiding qualitative and quantitative errors in the analysis of microbial community structure in complex ecosystems is the use of fluorescently labeled, rRNA-targeted oligonucleotides (5) for the in situ identification and enumeration of bacteria. This method has already been used successfully in complex microbial communities, such as multispecies biofilms (6, 22, 26), trickling filters (27), and activated sludge (37).The current study was performed with a laboratory-scale trickle-bed bioreactor degrading a mixture of aromatic compounds (Solvesso100). The starter culture for the inoculation of the bioreactor was an enrichment prepared in a fermentor which was itself started with a wastewater sample from a car painting factory as the inoculum and Solvesso100 as the sole carbon source. The goal of our study was to use for the first time fluorescent in situ hybridization (FISH) to investigate the microbial community structure and dynamics in the fermentor and the bioreactor during start-up. One of the open questions was whether the fermentor enrichment, which is done in suspension, indeed selects for those bacteria that later are immobilized in the bioreactor. In the course of this study, new 16S as well as 23S rRNA-targeted probes for phylogenetic groups within the beta subclass of the class Proteobacteria have been developed and applied in order to obtain a higher taxonomic resolution of the molecular techniques. The molecular data were compared to those obtained by traditional cultivation-dependent techniques.     

rRNA based identification and detection systems for rhizobia and other bacteria

Ribosomal ribonucleic acids are excellent marker molecules for the elucidation of bacterial phylogeny; they also provide useful target sites for identification and detection with nucleic acid probes. Based on the currently available 16S rRNA sequence data, bacteria of the rhizobial phenotype (plant nodulation, nitrogen fixation) are members of three moderately related phylogenetic sub-groups of the -subclass of the Proteobacteria: i.e. the rhizobia group, the bradyrhizobia group, and the azorhizobia group. All rhizobia, azo-, brady-, meso- and sinorhizobia are closely related to and in some cases phylogenetically intermixed with, non-symbiotic and/or non-nitrogen-fixing bacteria. Especially in the case of Bradyrhizobium japonicum strains, the 16S rRNA sequence data indicate substantial heterogeneity. Specific probe design and evaluation are discussed. A multiprobe concept for resolving specificity problems with group specific probes is presented. In situ identification with group specific probes of rhizobia in cultures as well as rhizobia and cyanobacteria within plant material is shown.     

The Tryptophan Fluorescence of Tetracarbidium conophorum Agglutinin II and a Solution-Based Assay for the Binding of a Biantennary Glycopeptide

The plant lectin Tetracarbidium conophorum agglutinin II binds to glycoproteins and glycopeptides in a structurally specific manner [Animashaun et al., (1994) Glycoconjugate J. 11, 299–303]. We have characterized the steady-state and time-resolved fluorescence of the tryptophan residues of this lectin. The fluorescence (λ_ex = 295 nm, λ_em = 350 nm) decay is complex and can be described by four decay times with the following values: τ₁ = 7.4nsec, α₁ = 0.22; τ₂ = 2.9 nsec, α₂ = 0.25; τ₃ = l.0 nsec, α₃ = 0.34; τ₄ = 0.2 nsec, α₄ = 0.18. The addition of a biantennary glycopeptide $\begin{array}{*{20}c} {Gal\beta (1 \to 4)GlcNAc\beta (1 \to 2)Man\alpha (1 \to 6)\neg } \\ {Man\beta (1 \to 4)GlcNAC\beta (1 \to 4)GlcAc\beta (1 \to )\begin{array}{*{20}c} {Glu - Nh_2 } \\ | \\ {Asn} \\ | \\ {COOH} \\ \end{array} } \\ {Gal\beta (1 \to 4)GlcNAc\beta (1 \to 2)Man\alpha (1 \to 3)} \\ \end{array} $ to the lectin results in a quench and an 8 nm blue shift of the emission spectrum. The effect is saturable, and is described by an association constant of 1.8×10⁵ M^?1. The tryptophan fluorescence of Tetracarbidium conophorum agglutinin II may therefore be utilized to characterize thermodynamically the binding interactions between this lectin and complex glycoprotein.