Will new diagnostic criteria for diabetes mellitus change phenotype of patients with diabetes? Reanalysis of European epidemiological data

Objective: To evaluate the impact of the revised diagnostic criteria for diabetes mellitus adopted by the American Diabetes Association on prevalence of diabetes and on classification of patients. For epidemiological purposes the American criteria use a fasting plasma glucose concentration ⩾7.0 mmol/l in contrast with the current World Health Organisation criteria of 2 hour glucose concentration ⩾11.1 mmol/l.

PDB-REPRDB: a database of representative protein chains from the Protein Data Bank (PDB)

PDB-REPRDB is a database of representative protein chains from the Protein Data Bank (PDB). The previous version of PDB-REPRDB provided 48 representative sets, whose similarity criteria were predetermined, on the WWW. The current version is designed so that the user may obtain a quick selection of representative chains from PDB. The selection of representative chains can be dynamically configured according to the user’s requirement. The WWW interface provides a large degree of freedom in setting parameters, such as cut-off scores of sequence and structural similarity. One can obtain a representative list and classification data of protein chains from the system. The current database includes 20 457 protein chains from PDB entries (August 6, 2000). The system for PDB-REPRDB is available at the Parallel Protein Information Analysis system (PAPIA) WWW server (http://www.rwcp.or.jp/papia/).

Role of the major histocompatibility complex class II Ea gene in lupus susceptibility in mice

The gene(s) encoded within major histocompatibility complex (MHC) act as one of the major genetic elements contributing to the susceptibility of murine systemic lupus erythematosus (SLE). We have recently demonstrated that lupus susceptibility is more closely linked to the I-E− H-2b haplotype than to the I-E+ H-2d haplotype in lupus-prone BXSB and (NZB × BXSB)F1 hybrid mice. To investigate whether the reduced susceptibility to SLE in H-2d mice is related to the expression of the MHC class II Ea gene (absent in H-2b mice), we determined the possible role of the Ea gene as a lupus protective gene in mice. Our results showed that (i) the development of SLE was almost completely prevented in BXSB (H-2b) mice expressing two copies of the Ead transgene at the homozygous level as well as in BXSB H-2k (I-E+) congenic mice as for H-2d BXSB mice, and (ii) the expression of two functional Ea (transgenic and endogenous) genes in either H-2d/b (NZB × BXSB)F1 or H-2k/b (MRL × BXSB)F1 mice provided protection from SLE at levels comparable to those conferred by the H-2d/d or H-2k/k haplotype. In addition, the level of the Ea gene-mediated protection appeared to be dependent on the genetic susceptibility to SLE in individual lupus-prone mice. Our results indicate that the reduced susceptibility associated with the I-E+ H-2d and H-2k haplotypes (versus the I-E− H-2b haplotype) is largely, if not all, contributed by the apparent autoimmune suppressive effect of the Ea gene, independently of the expression of the I-A or other MHC-linked genes.

Human aspartic protease memapsin 2 cleaves the β-secretase site of β-amyloid precursor protein

The cDNAs of two new human membrane-associated aspartic proteases, memapsin 1 and memapsin 2, have been cloned and sequenced. The deduced amino acid sequences show that each contains the typical pre, pro, and aspartic protease regions, but each also has a C-terminal extension of over 80 residues, which includes a single transmembrane domain and a C-terminal cytosolic domain. Memapsin 2 mRNA is abundant in human brain. The protease domain of memapsin 2 cDNA was expressed in Escherichia coli and was purified. Recombinant memapsin 2 specifically hydrolyzed peptides derived from the β-secretase site of both the wild-type and Swedish mutant β-amyloid precursor protein (APP) with over 60-fold increase of catalytic efficiency for the latter. Expression of APP and memapsin 2 in HeLa cells showed that memapsin 2 cleaved the β-secretase site of APP intracellularly. These and other results suggest that memapsin 2 fits all of the criteria of β-secretase, which catalyzes the rate-limiting step of the in vivo production of the β-amyloid (Aβ) peptide leading to the progression of Alzheimer's disease. Recombinant memapsin 2 also cleaved a peptide derived from the processing site of presenilin 1, albeit with poor kinetic efficiency. Alignment of cleavage site sequences of peptides indicates that the specificity of memapsin 2 resides mainly at the S1′ subsite, which prefers small side chains such as Ala, Ser, and Asp.

Trichostatin A reverses skewed expression of CD154, interleukin-10, and interferon-γ gene and protein expression in lupus T cells

In systemic lupus erythematosus (SLE), T helper cells exhibit increased and prolonged expression of cell-surface CD40 ligand (CD154), spontaneously overproduce interleukin-10 (IL-10), but underproduce interferon-gamma (IFN-γ). We tested the hypothesis that the imbalance of these gene products reflects skewed expression of CD154, IL-10, and IFN-γ genes. Here, we demonstrate that the histone deacetylase inhibitor, trichostatin A, significantly down-regulated CD154 and IL-10 and up-regulated IFN-γ gene expression in SLE T cells. This reversal corrected the aberrant expression of these gene products, thereby enhancing IFN-γ production and inhibiting IL-10 and CD154 expression. That trichostatin A can simultaneously reverse the skewed expression of multiple genes implicated in the immunopathogenesis of SLE suggests that this pharmacologic agent may be a candidate for the treatment of this autoimmune disease.

Selective binding of perfringolysin O derivative to cholesterol-rich membrane microdomains (rafts)

There is increasing evidence that sphingolipid- and cholesterol-rich microdomains (rafts) exist in the plasma membrane. Specific proteins assemble in these membrane domains and play a role in signal transduction and many other cellular events. Cholesterol depletion causes disassembly of the raft-associated proteins, suggesting an essential role of cholesterol in the structural maintenance and function of rafts. However, no tool has been available for the detection and monitoring of raft cholesterol in living cells. Here we show that a protease-nicked and biotinylated derivative (BCθ) of perfringolysin O (θ-toxin) binds selectively to cholesterol-rich microdomains of intact cells, the domains that fulfill the criteria of rafts. We fractionated the homogenates of nontreated and Triton X-100-treated platelets after incubation with BCθ on a sucrose gradient. BCθ was predominantly localized in the floating low-density fractions (FLDF) where cholesterol, sphingomyelin, and Src family kinases are enriched. Immunoelectron microscopy demonstrated that BCθ binds to a subpopulation of vesicles in FLDF. Depletion of 35% cholesterol from platelets with cyclodextrin, which accompanied 76% reduction in cholesterol from FLDF, almost completely abolished BCθ binding to FLDF. The staining patterns of BCθ and filipin in human epidermoid carcinoma A431 cells with and without cholesterol depletion suggest that BCθ binds to specific membrane domains on the cell surface, whereas filipin binding is indiscriminate to cell cholesterol. Furthermore, BCθ binding does not cause any damage to cell membranes, indicating that BCθ is a useful probe for the detection of membrane rafts in living cells.

Human autoantibody recognition of DNA.

Combinatorial IgG Fab phage display libraries prepared from a systemic lupus erythematosus (SLE) donor and a healthy donor were affinity selected against human placental DNA. Human monoclonal antibody Fab fragments specific for DNA were isolated from both libraries, although Fabs of the highest affinity were isolated only from the lupus library. Generally, apparent affinities of the Fabs for human placental DNA, purified double-stranded DNA, and denatured DNA were approximately equivalent. Surface plasmon resonance indicated Fab binding constants for a double-stranded oligodeoxynucleotide of 0.2-1.3 x 10(8) M-1. The higher-affinity Fabs, as ranked by binding to human placental DNA or to the oligonucleotide probe, tested positive in the Crithidia luciliae assay commonly used in the diagnosis of SLE, and interestingly the genes encoding the heavy-chain variable regions of these antibodies displayed evidence of only minimal somatic hypermutation. The heavy chains of the SLE Fabs were characterized by a predominance of basic residues toward the N terminus of complementarity-determining region 3 (CDR3). The crucial role of heavy-chain CDR3 (HCDR3) in high-affinity DNA recognition was suggested by the creation of DNA binding in an unrelated antibody by HCDR3 transplantation from SLE antibodies. We propose that high-affinity DNA-binding antibodies can arise in SLE without extensive somatic hypermutation in the variable-region genes because of the expression of inappropriate HCDR3s.