[Oral hypoglycaemic agents in 2009]

Various new oral hypoglycaemic agents have been developed recently and have changed the therapy of type 2 diabetes mellitus. Six different classes of agents are available: Biguanides, sulfonylureas, glinides, glitazones, alpha-glucosidase inhibitors and dipeptidyl peptidase-4 inhibitors. The increasing number of these drugs does not facilitate the choice of the best medication for an individual patient. In the article we describe the specific mechanisms of action, side effects, advantages and disadvantages of the different agents. Every drug therapy should be supported by lifestyle changes. Despite all the new drugs type 2 diabetes is still a chronic and slowly progressive disease without chance of cure. Therefore, it is important to prevent type 2 diabetes by normalizing body weight and increasing physical exercise.

Phenotypic and genotypic identification of streptococci and related bacteria isolated from bovine intramammary infections.

BACKGROUND Streptococcus spp. and other Gram-positive, catalase-negative cocci (PNC) form a large group of microorganisms which can be found in the milk of cows with intramammary infection. The most frequently observed PNC mastitis pathogens (major pathogens) are Streptococcus uberis, Strep. dysgalactiae, and Strep. agalactiae. The remaining PNC include a few minor pathogens and a large nonpathogenic group. Improved methods are needed for the accurate identification and differentiation of PNC. A total of 151 PNC were collected from cows with intramammary infection and conclusively identified by 16S rRNA sequencing as reference method. Nine phenotypic microbiological tests (alpha-hemolysis, CAMP reaction, esculin hydrolysis, growth on kanamycin esculin azide agar and on sodium chloride agar, inulin fermentation, hippurate hydrolysis, leucine aminopeptidase and pyrrolidonyl peptidase activity), multiplex PCR for the three major pathogens (target genes for Strep. uberis, Strep. dysgalactiae and Strep. agalactiae: pauA, 16S rRNA, and sklA3, respectively), and mass spectroscopy using the matrix-assisted laser desorption ionization-time of flight (MALDI-TOF MS) were evaluated for the diagnosis and discrimination of the three clinically most relevant PNC. RESULTS The probability that a strain of Strep. uberis, Strep. dysgalactiae and Strep. agalactiae was correctly identified by combining the results of the 9 phenotypic tests was 92%, 90%, and 100%, respectively. Applying the multiplex PCR, all strains of the three major pathogens were correctly identified and no false positive results occurred. Correct identification was observed for all strains of Strep. uberis and Strep. agalactiae using MALDI-TOF MS. In the case of Strep. dysgalactiae, some variability was observed at the subspecies level, but all strains were allocated to one single cluster. CONCLUSIONS The results of the present study show that reliable identification of the clinically most relevant PNC (Strep. uberis, Strep. agalactiae and Strep. dysgalactiae) can be obtained by use of a combination of colony morphology, hemolysis type and catalase reaction, and a multiplex PCR with specific primers restricted to these 3 pathogens. The MALDI-TOF MS is a fast method that shows promising results, although identification of Strep. dysgalactiae at the subspecies level is not yet satisfactory.

Dynamic Remodeling of the Stressed Heart: Role of Protein Degradation Pathways

The heart is a remarkable organ. In order to maintain its function, it remodels in response to a variety of environmental stresses, including pressure overload, volume overload, mechanical or pharmacological unloading and hormonal or metabolic disturbances. All these responses are linked to the inherent capacity of the heart to rebuild itself. Particularly, cardiac pressure overload activates signaling pathways of both protein synthesis and degradation. While much is known about regulators of protein synthesis, little is known about regulators of protein degradation in hypertrophy. The ubiquitin-proteasome system (UPS) selectively degrades unused and abnormal intracellular proteins. I speculated that the UPS may play an important role in both qualitative and quantitative changes in the composition of heart muscle during hypertrophic remodeling. My study hypothesized that cardiac remodeling in response to hypertrophic stimuli is a dynamic process that requires activation of highly regulated mechanisms of protein degradation as much as it requires protein synthesis. My first aim was to adopt a model of left ventricular hypertrophy and determine its gene expression and structural changes. Male Sprague-Dawley rats were submitted to ascending aortic banding and sacrificed at 7 and 14 days after surgery. Sham operated animals served as controls. Effective aortic banding was confirmed by hemodynamic assessment by Doppler flow measurements in vivo. Banded rats showed a four-fold increase in peak stenotic jet velocities. Histomorphometric analysis revealed a significant increase in myocyte size as well as fibrosis in the banded animals. Transcript analysis showed that banded animals had reverted to the fetal gene program. My second aim was to assess if the UPS is increased and transcriptionally regulated in hypertrophic left ventricular remodeling. Protein extracts from the left ventricles of the banded and control animals were used to perform an in vitro peptidase assay to assess the overall catalytic activity of the UPS. The results showed no difference between hypertrophied and control animals. Transcript analysis revealed decreases in transcript levels of candidate UPS genes in the hypertrophied hearts at 7 days post-banding but not at 14 days. However, protein expression analysis showed no difference at either time point compared to controls. These findings indicate that elements of the UPS are downregulated in the early phase of hypertrophic remodeling and normalizes in a later phase. The results provide evidence in support of a dynamic transcriptional regulation of a major pathway of intracellular protein degradation in the heart. The discrepancy between transcript levels on the one hand and protein levels on the other hand supports post-transcriptional regulation of the UPS pathway in the hypertrophied heart. The exact mechanisms and the functional consequences remain to be elucidated.

Characterization of the gene for an immunodominant 72 kDa lipoprotein of Mycoplasma mycoides subsp. mycoides small colony type.

With the aim of characterizing specific immunogenic proteins of Mycoplasma mycoides subsp. mycoides small colony (SC) type, the aetiological agent of contagious bovine pleuropneumonia, a gene encoding a major immunogenic protein of 72 kDa named P72 was cloned and expressed in Escherichia coli. The expressed protein was of the same apparent molecular mass as that produced by the parent strain. The predicted molecular mass of P72, based on the DNA-deduced amino acid sequence, was 61.118 kDa, significantly lower than the apparent molecular mass of endogenous or recombinant P72 on SDS-PAGE. Analysis of the amino acid sequence revealed a typical prokaryotic signal peptidase II-membrane lipoprotein lipid attachment site and a transmembrane structure domain in the leader sequence at the amino-terminal end of the protein. P72 was shown to be a lipoprotein and its surface location was confirmed by trypsin treatment of whole cells. An unassigned gene encoding a peptide with some similarity to P72 was found on the genome sequence of M. capricolum subsp. capricolum but not on that of Mycoplasma genitalium. The P72 gene was detected in 11/11 M. mycoides subsp. mycoides SC strains. Antiserum against recombinant P72 reacted strongly with 12/12 strains of M. mycoides subsp. mycoides SC, weakly with Mycoplasma bovine group 7 strain PG50, but not with other members of the 'mycoides cluster' or closely related mycoplasmas. Cows experimentally contact-infected with M. mycoides subsp. mycoides SC developed a humoral response against P72 within 35 d. P72 is a specific antigenic membrane lipoprotein of M. mycoides subsp. mycoides SC with potential for use in development of diagnostic reagents. It seems to belong to a family of lipoproteins of the "mycoides cluster'.

Cloning and characterization of an Actinobacillus pleuropneumoniae outer membrane protein belonging to the family of PAL lipoproteins

A 14-kDa outer membrane protein (OMP) was purified from Actinobacillus pleuro-pneumoniae serotype 2. The protein strongly reacts with sera from pigs experimentally or naturally infected with any of the 12 serotypes of A. pleuropneumoniae. The gene encoding this protein was isolated from a gene library of A. pleuropneumoniae serotype 2 reference strain by immunoscreening. Expression of the cloned gene in Escherichia coli revealed that the protein is also located in the outer membrane fraction of the recombinant host. DNA sequence analysis of the gene reveals high similarity of the protein's amino acid sequence to that of the E. coli peptidoglycan-associated lipoprotein PAL, to the Haemophilus influenzae OMP P6 and to related proteins of several other Gram-negative bacteria. We have therefore named the 14-kDa protein PalA, and its corresponding gene, palA. The 20 amino-terminal amino acid residues of PalA constitute a signal sequence characteristic of membrane lipoproteins of prokaryotes with a recognition site for the signal sequence peptidase II and a sorting signal for the final localization of the mature protein in the outer membrane. The DNA sequence upstream of palA contains an open reading frame which is highly similar to the E. coli tolB gene, indicating a gene cluster in A. pleuropneumoniae which is very similar to the E. coli tol locus. The palA gene is conserved and expressed in all A. pleuropneumoniae serotypes and in A. lignieresii. A very similar palA gene is present in A. suis and A. equuli.

The Glucose-Dependent Insulinotropic Polypeptide Receptor: A Novel Target for Neuroendocrine Tumor Imaging-First Preclinical Studies

A new family of peptide receptors, the incretin receptor family, overexpressed on many neuroendocrine tumors (NETs) is of great importance because it may enable the in vivo peptide-based receptor targeting of a category of NETs that does not express the somatostatin receptor. Impressive in vivo diagnostic data were published for glucagonlike peptide 1 receptor-targeting radiopeptides. Recently, promising in vitro data have appeared for the second member of the incretin family, the glucose-dependent insulinotropic polypeptide (GIP) receptor. This prompted us to develop and evaluate a new class of radioligands with the potential to be used for the in vivo targeting of GIP receptor-positive tumors. METHODS GIP(1-42) was modified C-terminally, and the truncated peptides [Lys(30)(aminohexanoic acid [Ahx]-DOTA)]GIP(1-30)NH2 (EG1), [Lys(16)(Ahx-DOTA)]GIP(1-30)NH2 (EG2), and [Nle(14), Lys(30)(Ahx-DOTA)]GIP(1-30)NH2 (EG4) were conjugated with Ahx-DOTA via the Lys(16) and Lys(30) side chains. Their inhibitory concentration of 50% (IC50) was determined using [(125)I-Tyr(10)]GIP(1-30) as radioligand and GIP(1-30) as control peptide. The DOTA conjugates were labeled with (111)In and (68)Ga. In vitro evaluation included saturation and internalization studies using the pancreatic endocrine cell line INR1G9 transfected with the human GIP receptor (INR1G9-hGIPr). The in vivo evaluation consisted of biodistribution and PET imaging studies on nude mice bearing INR1G9-hGIPr tumors. RESULTS Binding studies (IC50 and saturation studies) showed high affinity toward GIP receptor for the GIP conjugates. Specific in vitro internalization was found, and almost the entire cell-associated activity was internalized (>90% of the cell-bound activity), supporting the agonist potency of the (111)In-vectors. (111)In-EG4 and (68)Ga-EG4 were shown to specifically target INR1G9-hGIPr xenografts, with tumor uptake of 10.4% ± 2.2% and 17.0% ± 4.4% injected activity/g, 1 h after injection, respectively. Kidneys showed the highest uptake, which could be reduced by approximately 40%-50% with a modified-fluid-gelatin plasma substitute or an inhibitor of the serine protease dipeptidyl peptidase 4. The PET images clearly visualized the tumor. CONCLUSION The evaluation of EG4 as a proof-of-principle radioligand indicated the feasibility of imaging GIP receptor-positive tumors. These results prompt us to continue the development of this family of radioligands for imaging of a broad spectrum of NETs.

ARRANGEMENT OF GENE LOCI IN EUPLOID CHINESE HAMSTER CELLS AND GENETIC CONSEQUENCES OF REARRANGEMENT IN CHO CELLS

In both euploid Chinese hamster (Cricetulus griseus) cells and pseudodiploid Chinese hamster ovary (CHO) cells, gene assignments were accomplished by G band chromosome and isozyme analysis (32 isozymes) of interspecific somatic cell hybrids obtained after HAT selection of mouse CL 1D (TK('-)) cells which were PEG-fused with either euploid Chinese hamster cells or HPRT('-) CHO cells. Hybrids slowly segregated hamster chromosomes. Clone panels consisting of independent hybrid clones and subclones containing different combinations of Chinese hamster chromosomes and isozymes were established from each type of fusion.^ These clone panels enabled us to provisionally assign the loci for: nucleoside phosphorylase (NP), glyoxalase (GLO), glutathione reductase (GSR), adenosine kinase (ADK), esterase D (ESD), peptidases B and S (PEPB and -S) and phosphoglucomutase 2 (PGM2, human nomenclature) to chromosome 1; adenylate kinase 1 (AK1), adenosine deaminase (ADA) and inosine triosephosphatase (ITP) to chromosome 6; triosephosphate isomerase (TPI) to chromosome 8; and glucose phosphate isomerse (GPI) and peptidase D (PEPD) to chromosome 9.^ We also confirm the assignments of 6-phosphogluconate dehydrogenase (PGD), PGM1, enolase 1 (ENO1) and diptheria toxin sensitivity (DTS) to chromosome 2 as well as provisionally assign galactose-1-phosphate uridyl transferase (GALT) and AK2 to chromosome 2. Selection in either HAT or BrdU for hybrids that had retained or lost the chromosome carrying the locus for TK enabled us to assign the loci for TK, galactokinase (GALK) and acid phosphatase 1 (ACP1) to Chinese hamster chromosome 7.^ These results are discussed in relation to current theories on the basis for high frequency of drug resistant autosomal recessive mutants in CHO cells and conservation of mammalian autosomal linkage groups. ^