Rabip4' is an effector of rab5 and rab4 and regulates transport through early endosomes

We describe the characterization of an 80-kDa protein cross-reacting with a monoclonal antibody against the human La autoantigen. The 80-kDa protein is a variant of rabip4 with an N-terminal extension of 108 amino acids and is expressed in the same cells. For this reason, we named it rabip4'. rabip4' is a peripheral membrane protein, which colocalized with internalized transferrin and EEA1 on early endosomes. Membrane association required the presence of the FYVE domain and was perturbed by the phosphatidylinositol 3-kinase inhibitor wortmannin. Expression of a dominant negative rabip4' mutant reduced internalization and recycling of transferrin from early endosomes, suggesting that it may be functionally linked to rab4 and rab5. In agreement with this, we found that rabip4' colocalized with the two GTPases on early endosomes and bound specifically and simultaneously to the GTP form of both rab4 and rab5. We conclude that rabip4' may coordinate the activities of rab4 and rab5, regulating membrane dynamics in the early endosomal system.     

Prevention of autoimmune diabetes in NOD mice by troglitazone is associated with modulation of ICAM-1 expression on pancreatic islet cells and IFN-gamma expression in splenic T cells

Thiazolidinediones acting as PPAR-gamma agonists are a new generation of oral antidiabetics addressing insulin resistance as a main feature of type-2 diabetes. In accordance to our results, pre-clinical studies have demonstrated that the thiazolinedione troglitazone prevents the development of insulin-dependent autoimmune type-1 diabetes. To investigate whether TGZ acts by affecting the ICAM-1/LFA-1 pathway and/or the Th1/Th2 cytokine balance in NOD mice, we analysed the IL-1beta-induced ICAM-1 expression on islet-cells and the LFA-1, CD25, IL-2, IFN-gamma, IL-4, and IL-10 expression on splenocytes. After 200 days of oral TGZ administration, islet cells from TGZ-treated NOD mice showed a reduced ICAM-1 expression in response to the pro-inflammatory cytokine IL-1beta. The expression of the ligand LFA-1 on CD4(+) and CD8(+) T-cells was comparable to that of placebo- and untreated controls. Also, the expression of Th1/Th2 cytokines was comparable in groups receiving TGZ or Placebo. Nevertheless, the investigated NOD mice segregated into IFN-gamma low- and IFN-gamma high producers as revealed by cluster analysis. Interestingly, the majority of TGZ-treated mice belonged to the cluster of IFN-gamma low producers. Thus, the prevention of autoimmune diabetes in NOD mice by TGZ seems to be associated with suppression of IL-1beta-induced ICAM-1 expression leading to a reduced vulnerability of pancreatic beta-cells during the effector stage of beta-cell destruction. In addition, IFN-gamma production was modulated, implicating that alteration of the Th1/Th2 cytokine balance might have contributed to diabetes prevention. The findings of this study suggest that TGZ exerts its effects by influencing both the beta-cells as the target of autoimmune beta-cell destruction and the T-cells as major effectors of the autoimmune process.     

Structure-based improvement of the biophysical properties of immunoglobulin VH domains with a generalizable approach

In a systematic study of V gene families carried out with consensus V(H) and V(L) domains alone and in combinations in the scFv format, we found comparatively low expression yields and lower cooperativity in equilibrium unfolding in antibody fragments containing V(H) domains of human germline families 2, 4, and 6. From an analysis of the packing of the hydrophobic core, the completeness of charge clusters, the occurrence of unsatisfied hydrogen bonds, and residues with low beta-sheet propensities, positive Phi angles, and exposed hydrophobic side chains, we pinpointed residues potentially responsible for the unsatisfactory properties of these germline-encoded sequences. Several of those are in common between the domains of the even-numbered subgroups, but do not occur in the odd-numbered ones. In this study, we have systematically exchanged those residues alone and in combination in two different scFvs using the V(H)6 framework, and we describe their effect on equilibrium stability and folding yield. We improved the stability by 20.9 kJ/mol and the expression yield by a factor of 4 and can now use these data to rationally engineer antibodies derived from this and similar germline families for better biophysical properties. Furthermore, we provide an improved design for libraries exploiting the significant additional diversity provided by these frameworks. Both antibodies studied here completely retain their binding affinity, demonstrating that the CDR conformations were not affected.     

A microscope-based screening platform for large-scale functional protein analysis in intact cells

A modular microscope-based screening platform, with applications in large-scale analysis of protein function in intact cells is described. It includes automated sample preparation, image acquisition, data management and analysis, and the genome-wide automated retrieval of bioinformatic information. The modular nature of the system ensures that it is rapidly adaptable to new biological questions or sets of proteins. Two automated functional assays addressing protein secretion and the integrity of the Golgi complex were developed and tested. This shows the potential of the system in large-scale, cell-based functional proteomic projects.     

Identification of the bacteria-binding peptide domain on salivary agglutinin (gp-340/DMBT1), a member of the scavenger receptor cysteine-rich superfamily

Salivary agglutinin is encoded by DMBT1 and identical to gp-340, a member of the scavenger receptor cysteine-rich (SRCR) superfamily. Salivary agglutinin/DMBT1 is known for its Streptococcus mutans agglutinating properties. This 300-400 kDa glycoprotein is composed of conserved peptide motifs: 14 SRCR domains that are separated by SRCR-interspersed domains (SIDs), 2 CUB (C1r/C1s Uegf Bmp1) domains, and a zona pellucida domain. We have searched for the peptide domains of agglutinin/DMBT1 responsible for bacteria binding. Digestion with endoproteinase Lys-C resulted in a protein fragment containing exclusively SRCR and SID domains that binds to S. mutans. To define more closely the S. mutans-binding domain, consensus-based peptides of the SRCR domains and SIDs were designed and synthesized. Only one of the SRCR peptides, designated SRCRP2, and none of the SID peptides bound to S. mutans. Strikingly, this peptide was also able to induce agglutination of S. mutans and a number of other bacteria. The repeated presence of this peptide in the native molecule endows agglutinin/DMBT1 with a general bacterial binding feature with a multivalent character. Moreover, our studies demonstrate for the first time that the polymorphic SRCR domains of salivary agglutinin/DMBT1 mediate ligand interactions.     

Is it possible to replace stimulus animals by scent-filled cups in the social discrimination test?

A study in which the rat social discrimination test was refined is described. This test measures social memory by using, in general, juvenile rats as stimulus animals. Rats are offered a first juvenile to investigate (learning trial), and after a specified interval, the rats are offered the same rat and a second juvenile rat to investigate again (retrieval trial). When the rats sniff the second juvenile in the retrieval trial more than the first, social memory for the second juvenile is said to be present. This test is mainly based on scents from the juvenile. Attempts were made to refine the test to reduce the number of animals used, to enhance the scope of the test, and to improve its validity. Firstly, the stimulus animals were replaced by the scent of juveniles, in the form of cups filled with sawdust taken from cages of juvenile rats. Similar results to those in the original test were obtained when using these scents. Furthermore, male and female scents were tested, and showed the same results as for the juvenile scents. Secondly, rats were also given two cups (one scent-filled and one filled with plain sawdust) in the learning trial, to determine which allowed a more-precise delineation of motivational, discriminatory and memory components. Overall, it is possible to replace stimulus animals by scent-filled cups in the social discrimination test, to enhance the scope of the test, and to draw more-valid conclusions with respect to social memory.     

Solubility and diffusion of nitrogen in maltodextrin/protein tablets

The gas transport properties of compacted tablets consisting of an amorphous mixture of maltodextrin and sodium caseinate were studied by dissolving nitrogen gas in the tablets and then determining the gas release over time as a function of temperature and water activity. Gas was dissolved in the tablet matrix by heating the tablets under pressure, generally to temperatures above the glass transition temperature of the matrix, holding them at these conditions for a specified time and then rapidly cooling them while maintaining the external pressure. The solubility of nitrogen was found to be largely determined by the free volume of the matrix, which in turn can be influenced to some degree by thermal and pressure treatments during gas loading. At the levels of free volume studied, the dissolved nitrogen is densely packed in the free volume, the packing density being virtually independent of the externally applied pressure. Release of gas from the tablets at temperatures below the glass transition temperature is generally well described by Fickian diffusion. The effective diffusion coefficient of gas release is strongly dependent on the microstructure and porosity of the tablet matrix, and an approximate model describing the relationship between tablet structure and rate of gas release is formulated. The model is in semiquantitative agreement with the rates of gas diffusion obtained for tablets and dense granules. Owing to the structural heterogeneity and variability of the tablets and the history-dependent properties of the tablet matrix, the effective diffusion coefficients of gas release from the tablets showed a relatively large spread. The temperature dependence of diffusional release follows an Arrhenius relation below the glass transition temperature. This allows the prediction of the nitrogen retention in the tablets as function of time, temperature and pressure.     

Mutation analysis of the coding sequence of the MECP2 gene in infantile autism

Mutations in the coding region of the methyl-CpG-binding protein 2 ( MECP2) gene cause Rett syndrome and have also been reported in a number of X-linked mental retardation syndromes. Furthermore, such mutations have recently been described in a few autistic patients. In this study, a large sample of individuals with autism was screened in order to elucidate systematically whether specific mutations in MECP2 play a role in autism. The mutation analysis of the coding sequence of the gene was performed by denaturing high-pressure liquid chromatography and direct sequencing. Taken together, 14 sequence variants were identified in 152 autistic patients from 134 German families and 50 unrelated patients from the International Molecular Genetic Study of Autism Consortium affected relative-pair sample. Eleven of these variants were excluded for having an aetiological role as they were either silent mutations, did not cosegregate with autism in the pedigrees of the patients or represented known polymorphisms. The relevance of the three remaining mutations towards the aetiology of autism could not be ruled out, although they were not localised within functional domains of MeCP2 and may be rare polymorphisms. Taking into account the large size of our sample, we conclude that mutations in the coding region of MECP2 do not play a major role in autism susceptibility. Therefore, infantile autism and Rett syndrome probably represent two distinct entities at the molecular genetic level.