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.     

Proteomic analysis of rice leaves during drought stress and recovery

Three-week old plants of rice (Oryza sativa L. cv CT9993 and cv IR62266) developed gradual water stress over 23 days of transpiration without watering, during which period the mid-day leaf water potential declined to approximately -2.4 MPa, compared with approximately -1.0 MPa in well-watered controls. More than 1000 protein spots that were detected in leaf extracts by proteomic analysis showed reproducible abundance within replications. Of these proteins, 42 spots showed a significant change in abundance under stress, with 27 of them exhibiting a different response pattern in the two cultivars. However, only one protein (chloroplast Cu-Zn superoxide dismutase) changed significantly in opposite directions in the two cultivars in response to drought. The most common difference was for proteins to be up-regulated by drought in CT9993 and unaffected in IR62266; or down-regulated by drought in IR62266 and unaffected in CT9993. By 10 days after rewatering, all proteins had returned completely or largely to the abundance of the well-watered control. Mass spectrometry helped to identify 16 of the drought-responsive proteins, including an actin depolymerizing factor, which was one of three proteins detectable under stress in both cultivars but undetectable in well-watered plants or in plants 10 days after rewatering. The most abundant protein up-regulated by drought in CT9993 and IR62266 was identified only after cloning of the corresponding cDNA. It was found to be an S-like RNase homologue but it lacked the two active site histidines required for RNase activity. Four novel drought-responsive mechanisms were revealed by this work: up-regulation of S-like RNase homologue, actin depolymerizing factor and rubisco activase, and down-regulation of isoflavone reductase-like protein.     

Host preference by Allothrombium pulvinum (Acari: Trombidiidae) larvae on aphids: Macrosiphum rosae,Aphis gossypii and Hyalopterus amygdali (Homoptera: Aphididae)

In this study aphid-plant association and its effect on host preference of parasitic Allothrombium pulvinum larvae was examined with multiple-choice tests. Host species selection, host size selection and superparasitism with mite larvae were studied with two-choice tests. Three aphid species were used: Macrosiphum rosae, Aphis gossypii and Hyalopterus amygdali. In multiple-choice tests, larvae of A. pulvinum showed no significant preference for any aphid-plant association when given M. rosae on rose, A. gossypii on cucumber and H. amygdali on apricot simultaneously. Two-choice tests showed that larval mites preferred H. amygdali to A. gossypii, but had no preference when offered a choice between A. gossypii and M. rosae or between H. amygdali and M. rosae. In host size selection and superparasitism tests, significantly more mites selected the larger host (M. rosae). Furthermore, parasitised H. amygdali were preferred to unparasitised ones. This revised version was published online in July 2006 with corrections to the Cover Date.     

Interleukin-4 therapy of psoriasis induces Th2 responses and improves human autoimmune disease

Selective skewing of autoreactive interferon-gamma (IFN-gamma)-producing T helper cells (Th1) toward an interleukin-4 (IL-4)-producing (Th2) phenotype can in experimental animals alleviate autoimmune disease without inducing general immunosuppression. In a prospective dose escalation study, we assessed treatment with human IL-4 (rhuIL-4) in 20 patients with severe psoriasis. The therapy was well tolerated, and within six weeks all patients showed decreased clinical scores and 15 improved more than 68%. Stable reduction of clinical scores was significantly better at 0.2-0.5 microg rhuIL-4 than at < or =0.1 microg rhuIL-4 (P = 0.009). In psoriatic lesions, treatment with 0.2-0.5 microg/kg rhuIL-4 reduced the concentrations of IL-8 and IL-19, two cytokines directly involved in psoriasis; the number of chemokine receptor CCR5+ Th1 cells; and the IFN-gamma/IL-4 ratio. In the circulation, 0.2-0.5 microg/kg rhuIL-4 increased the number of IL-4+CD4+ T cells two- to three-fold. Thus, IL-4 therapy can induce Th2 differentiation in human CD4+ T cells and has promise as a potential treatment for psoriasis, a prototypic Th1-associated autoimmune disease.     

Fluid phase endocytosis of [125I]iodixanol in rat liver parenchymal, endothelial and Kupffer cells

Endocytosis of [125I]iodixanol was studied in vivo and in vitro in rat liver cells to determine fluid phase endocytic activity in different liver cells (hepatocytes, Kupffer cells and endothelial cells). The Kupffer cells were more active in the uptake of [l25I]iodixanol than parenchymal cells or endothelial cells. Inhibition of endocytic uptake via clathrin-coated pits (by potassium depletion and hypertonic medium) reduced uptake of [125I]iodixanol much more in Kupffer cells and endothelial cells than in hepatocytes. To gain further information about the importance of clathrin-mediated fluid phase endocytosis, the expression of proteins known to be components of the endocytic machinery was investigated. Using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting, endothelial cells and Kupffer cells were found to express approximately fourfold more rab4, rab5 and rab7 than parenchymal cells, while clathrin was expressed at a higher level in endothelial cells than in Kupffer cells and hepatocytes. Using electron microscopy it was shown that liver endothelial cells contained approximately twice as many coated pits per membrane unit than the parenchymal and Kupffer cells, thus confirming the immunoblotting results concerning clathrin expression. Electron microscopy on isolated liver cells following fluid phase uptake of horseradish peroxidase (HRP) showed that HRP-containing organelles had a different morphology in the different cell types: In the liver endothelial cells HRP was in small, tubular endosomes, while in Kupffer cells HRP was mainly found in larger structures, reminiscent of macropinosomes. Parenchymal cells contained HRP in small vacuolar endosomes with a punctuated distribution. In conclusion, we find that the Kupffer cells and the endothelial cells have a higher pinocytic activity than the hepatocytes. The hepatocytes do, however, account for most of the total hepatic uptake. The fluid phase endocytosis in liver endothelial cells depends mainly on clathrin-mediated endocytosis, while the parenchymal cells have additional clathrin-independent mechanisms that may play an important role in the uptake of plasma membrane components. In the Kupffer cells the major uptake of fluid phase markers seems to take place via a macropinocytic mechanism.     

The Role of a Novel TRMT1 Gene Mutation and Rare GRM1 Gene Defect in Intellectual Disability in Two Azeri Families

Cognitive impairment or intellectual disability (ID) is a widespread neurodevelopmental disorder characterized by low IQ (below 70). ID is genetically heterogeneous and is estimated to affect 1–3% of the world’s population. In affected children from consanguineous families, autosomal recessive inheritance is common, and identifying the underlying genetic cause is an important issue in clinical genetics. In the framework of a larger project, aimed at identifying candidate genes for autosomal recessive intellectual disorder (ARID), we recently carried out single nucleotide polymorphism-based genome-wide linkage analysis in several families from Ardabil province in Iran. The identification of homozygosity-by-descent loci in these families, in combination with whole exome sequencing, led us to identify possible causative homozygous changes in two families. In the first family, a missense variant was found in GRM1 gene, while in the second family, a frameshift alteration was identified in TRMT1, both of which were found to co-segregate with the disease. GRM1, a known causal gene for autosomal recessive spinocerebellar ataxia (SCAR13, MIM#614831), encodes the metabotropic glutamate receptor1 (mGluR1). This gene plays an important role in synaptic plasticity and cerebellar development. Conversely, the TRMT1 gene encodes a tRNA methyltransferase that dimethylates a single guanine residue at position 26 of most tRNAs using S-adenosyl methionine as the methyl group donor. We recently presented TRMT1 as a candidate gene for ARID in a consanguineous Iranian family (Najmabadi et al., 2011). We believe that this second Iranian family with a biallelic loss-of-function mutation in TRMT1 gene supports the idea that this gene likely has function in development of the disorder.     

Retinal Microvascular Abnormalities and Risk of Renal Failure in Asian Populations

BackgroundRetinal microvascular signs may provide insights into the structure and function of small vessels that are associated with renal disease. We examined the relationship of retinal microvascular signs with both prevalent and incident end-stage renal disease (ESRD) in a multi-ethnic Asian population.MethodsA total of 5763 subjects (aged ≥40 years) from two prospective population-based studies (the Singapore Malay Eye Study and the Singapore Prospective Study) were included for the current analysis. Retinopathy was graded using the modified Airlie House classification system. Retinal vascular parameters were measured using computer-assisted programs to quantify the retinal vessel widths (arteriolar and venular caliber) and retinal vascular network (fractal dimension). Data on ESRD was obtained by record linkage with the ESRD cases registered by National Registry of Diseases Office, Singapore. Multi-variable adjusted regression analyses were performed to assess the associations of baseline retinal vascular parameters and prevalent and incident ESRD.ResultsAt baseline, 21(0.36%) persons had prevalent ESRD. During a median follow-up of 4.3 years, 33 (0.57%) subjects developed ESRD. In our analyses, retinopathy was associated with prevalent ESRD (multi-variable adjusted odds ratio [OR], 3.21, 95% confidence interval [CI]: 1.28–8.05) and incident ESRD (multi-variable adjusted hazard ratio [HR], 2.51, 95%CI: 1.14–5.54). This association was largely seen in person with diabetes (HR, 2.60, 95%CI: 1.01–6.66) and not present in persons without diabetes (HR, 1.65, 95%CI: 0.14–18.98). Retinal arteriolar caliber, retinal venular caliber and retinal vascular fractal dimension were not associated with ESRD.ConclusionRetinopathy signs in persons with diabetes are related to an increased risk of ESRD; however, other microvascular changes in the retina are not associated with ESRD.     

Characterization of Lung Fibroblasts More than Two Decades after Mustard Gas Exposure

Purpose

In patients with short-term exposure to the sulfur mustard gas, the delayed cellular effects on lungs have not been well understood yet. The lung pathology shows a dominant feature consistent with obliterative bronchiolitis, in which fibroblasts play a central role. This study aims to characterize alterations to lung fibroblasts, at the cellular level, in patients with delayed respiratory complications after short-term exposure to the sulfur mustard gas.

Methods

Fibroblasts were isolated from the transbronchial biopsies of patients with documented history of exposure to single high-dose sulfur mustard during 1985–7 and compared with the fibroblasts of control subjects.

Results

Compared with controls, patients’ fibroblasts were thinner and shorter, and showed a higher population doubling level, migration capacity and number of filopodia. Sulfur mustard decreased the in vitro viability of fibroblasts and increased their sensitivity to induction of apoptosis, but did not change the rate of spontaneous apoptosis. In addition, higher expression of alpha smooth muscle actin showed that the lung''s microenvironment in these patients is permissive for myofibroblastic differentiation.

Conclusions

These findings suggest that in patients under the study, the delayed pulmonary complications of sulfur mustard should be considered as a unique pathology, which might need a specific management by manipulation of cellular components.