Altered B:9-23 insulin, when administered intranasally with cholera toxin adjuvant, suppresses the expression of insulin autoantibodies and prevents diabetes

Insulin peptide B:9-23 is a major autoantigen in type 1 diabetes that contains two distinct CD4 epitopes (B:9-16 and B:13-23). One of the two epitopes, B:13-23, overlaps with a CTL epitope (B:15-23). In this study, we report that the elimination of the CTL epitope from the B:9-23 peptide by amino acid substitution (with alanine) at positions B:16 and 19 (A16,19 altered peptide ligand) or truncation of the C-terminal amino acids from the peptide (B:9-21), neither of which stimulated the proliferation of insulin B:15-23 reactive CD8 T cells, provided significant intranasally induced suppression of diabetes when coadministered with a potent mucosal adjuvant cholera toxin (CT). Intranasal treatment with A16,19 resulted in the elimination of spontaneous insulin autoantibodies, significant inhibition of insulitis and remission from hyperglycemia, and prevented the progression to diabetes. Intranasal administration of native B:9-23/CT or B:11-23/CT resulted in a significant enhancement of insulin autoantibody expression and severity of insulitis and failed to prevent diabetes. Our present study indicates that elimination of the CTL epitope from the B:9-23 peptide was critically important for mucosally induced diabetes prevention. The A16,19 altered peptide ligand, but not other native insulin peptides, suppresses insulin autoantibodies associated with protection from and remission of diabetes.     

Effects of acid exposure on the conformation, stability, and aggregation of monoclonal antibodies

Exposure of antibodies to low pH is often unavoidable for purification and viral clearance. The conformation and stability of two humanized monoclonal antibodies (hIgG4-A and -B) directed against different antigens and a mouse monoclonal antibody (mIgG1) in 0.1M citrate at acidic pH were studied using circular dichroism (CD), differential scanning calorimetry (DSC), and sedimentation velocity. Near- and far-UV CD spectra showed that exposure of these antibodies to pH 2.7-3.9 induced only limited conformational changes, although the changes were greater at the lower pH. However, the acid conformation is far from unfolded or so-called molten globule structure. Incubation of hIgG4-A at pH 2.7 and 3.5 at 4 degrees C over the course of 24 h caused little change in the near-UV CD spectra, indicating that the acid conformation is stable. Sedimentation velocity showed that the hIgG4-A is largely monomeric at pH 2.7 and 3.5 as well as at pH 6.0. No time-dependent changes in sedimentation profile occurred upon incubation at these low pHs, consistent with the conformational stability observed by CD. The sedimentation coefficient of the monomer at pH 2.7 or 3.5 again suggested that no gross conformational changes occur at these pHs. DSC analysis of the antibodies showed thermal unfolding at pH 2.7-3.9 as well as at pH 6.0, but with decreased melting temperatures at the lower pH. These results are consistent with the view that the antibodies undergo limited conformational change, and that incubation at 4 degrees C at low pH results in no time-dependent conformational changes. Titration of hIgG4-A from pH 3.5 to 6.0 resulted in recovery of native monomeric proteins whose CD and DSC profiles resembled those of the original sample. However, titration from pH 2.7 resulted in lower recovery of monomeric antibody, indicating that the greater conformational changes observed at this pH cannot be fully reversed to the native structure by a simple pH titration.     

Metabolism of glutamine and glutathione via gamma-glutamyltranspeptidase and glutamate transport in Helicobacter pylori: possible significance in the pathophysiology of the organism

gamma-Glutamyltranspeptidase (GGT) is a periplasmic enzyme of Helicobacter pylori implicated in its pathogenesis towards mammalian cells. We have cloned and expressed the H. pylori strain 26695 recombinant GGT protein in Escherichia coli and purified it to homogeneity. The purified protein exhibited hydrolysis activity with very high affinities for glutamine and glutathione shown by apparent K(m) values lower than 1 muM. H. pylori cells were unable to take up extracellular glutamine and glutathione directly. Instead, these substances were hydrolysed to glutamate by the action of GGT outside the cells. The glutamate produced was then transported by a Na(+)-dependent reaction into H. pylori cells, where it was mainly incorporated into the TCA cycle and partially utilized as a substrate for glutamine synthesis. These observations show that one of the principle physiological functions of H. pylori GGT is to enable H. pylori cells to utilize extracellular glutamine and glutathione as a source of glutamate. As glutamine and glutathione are important nutrients for maintenance of healthy gastrointestinal tissue, their depletion by the GGT enzyme is hypothesized to account for the damaging of mammalian cells and the pathophysiology of H. pylori.     

pSLA2-M of Streptomyces rochei is a composite linear plasmid characterized by self-defense genes and homology with pSLA2-L

The 113,463-bp nucleotide sequence of the linear plasmid pSLA2-M of Streptomyces rochei 7434AN4 was determined. pSLA2-M had a 69.7% overall GC content, 352-bp terminal inverted repeats with 91% (321/352) identity at both ends, and 121 open reading frames. The rightmost 14.6-kb sequence was almost (14,550/14,555) identical to that of the coexisting 211-kb linear plasmid pSLA2-L. Adjacent to this homologous region an 11.8-kb CRISPR cluster was identified, which is known to function against phage infection in prokaryotes. This cluster region as well as another one containing two large membrane protein genes (orf78 and orf79) were flanked by direct repeats of 194 and 566 bp respectively. Hence the insertion of circular DNAs containing each cluster by homologous recombination was suggested. In addition, the orf71 encoded a Ku70/Ku80-like protein, known to function in the repair of double-strand DNA breaks in eukaryotes, but disruption of it did not affect the radiation sensitivity of the mutant. A pair of replication initiation genes (orf1-orf2) were identified at the extreme left end. Thus, pSLA2-M proved to be a composite linear plasmid characterized by self-defense genes and homology with pSLA2-L that might have been generated by multiple recombination events.     

Mieap, a p53-inducible protein, controls mitochondrial quality by repairing or eliminating unhealthy mitochondria

Maintenance of healthy mitochondria prevents aging, cancer, and a variety of degenerative diseases that are due to the result of defective mitochondrial quality control (MQC). Recently, we discovered a novel mechanism for MQC, in which Mieap induces intramitochondrial lysosome-like organella that plays a critical role in the elimination of oxidized mitochondrial proteins (designated MALM for Mieap-induced accumulation of lysosome-like organelles within mitochondria). However, a large part of the mechanisms for MQC remains unknown. Here, we report additional mechanisms for Mieap-regulated MQC. Reactive oxygen species (ROS) scavengers completely inhibited MALM. A mitochondrial outer membrane protein NIX interacted with Mieap in a ROS-dependent manner via the BH3 domain of NIX and the coiled-coil domain of Mieap. Deficiency of NIX also completely impaired MALM. When MALM was inhibited, Mieap induced vacuole-like structures (designated as MIV for Mieap-induced vacuole), which engulfed and degraded the unhealthy mitochondria by accumulating lysosomes. The inactivation of p53 severely impaired both MALM and MIV generation, leading to accumulation of unhealthy mitochondria. These results suggest that (1) mitochondrial ROS and NIX are essential factors for MALM, (2) MIV is a novel mechanism for lysosomal degradation of mitochondria, and (3) the p53-Mieap pathway plays a pivotal role in MQC by repairing or eliminating unhealthy mitochondria via MALM or MIV generation, respectively.     

Long-term treatment with the Na+-glucose cotransporter inhibitor T-1095 causes sustained improvement in hyperglycemia and prevents diabetic neuropathy in Goto-Kakizaki Rats

We examined the effects of T-1095, an orally active inhibitor of Na(+)-glucose cotransporter (SGLT), on the development and severity of diabetes in Goto-Kakizaki (GK) rat, a spontaneous, non-obese model of type 2 diabetes. T-1095 was administered as dietary admixture (0.1% w/w) beginning at 7 weeks of age for 32 weeks. Untreated male GK rats were hyperglycemic compared with Wistar rats. Throughout the study, T-1095 treatment significantly decreased both blood glucose and hemoglobin A(1C) levels in the GK rats. The concomitant increase of urinary glucose excretion indicated that the hypoglycemic action of T-1095 is derived from the enhancement of urinary glucose disposal. Although food intake was not changed in the T-1095-treated rats, the body weight gain was retarded. T-1095 treatment partially ameliorated oral glucose tolerance but not the impaired glucose-induced insulin secretion. Homeostasis model assessment (HOMA) indicated the existence of insulin resistance in GK rats and a significant restoration by T-1095-treatment. There was a reduction of the thermal response in tail-flick testing following long-term hyperglycemia (diabetic neuropathy). Treatment of T-1095 significantly prevented the development of diabetic neuropathy in male GK rats. Sustained improvement of hyperglycemia and prevention of diabetic neuropathy by the T-1095-treatment provide further support the use of SGLT inhibitors for the treatment of diabetes.     

Molecular cloning and functional expression of hemolysin from the sea anemone Actineria villosa

The full-length cDNA that encodes the hemolytic toxin Avt-I, with 226 amino acids, from the venomous sea anemone Actineria villosa has been cloned using the oligo-capping method. The cDNA contains 681bp open reading frame and its predicted amino acid sequences revealed that Avt-I was basic polypeptides without cysteine residues and Arg-Gly-Asp (RGD) motif sequence. The mature Avt-I has a predicted molecular weight of 19.6 kDa and its theoretical isoelectric point is 9.3. The Avt-I revealed 99, 61, 57, and 57% amino acid similarity with hemolytic toxins Pstx20, EqtII, StII, and HmT from Phyllodiscus semoni, Actinia Equina, Stichodactyla helianthus, and Heteractis magnifica, respectively. The characteristic amphiphilic alpha-helix structure was found at the N-terminal region of the mature Avt-I. Recombinant Avt-I (rAvt-I) was expressed in Escherichia coli BL21 (DE3) strain as a biologically active form and purified rAvt-I caused 50% hemolytic activity against 1% sheep erythrocytes at a concentration of 6.3 ng/ml (0.32 nM). M9Y medium led to more than 2-fold increase in rAvt-I yield than cultivation in Luria-Bertani medium.     

Predicted and measured disorder in peripherin/rds, a retinal tetraspanin

Vertebrate photoreceptor outer segment (OS) morphogenesis requires peripherin/rds (P/rds). We have characterized this protein's C-terminus and present evidence that suggests it is intrinsically disordered. We propose that structural flexibility may underlie the multifunctionality proposed for this domain previously. The extremely short C-termini present in other tetraspanin family members suggest that intrinsic disorder may also play a role for those proteins.