A novel frameshift mutation in BLM gene associated with high sister chromatid exchanges (SCE) in heterozygous family members

The Bloom syndrome (BS) is an autosomic recessive disorder comprising a wide range of abnormalities, including stunted growth, immunodeficiency, sun sensitivity and increased frequency of various types of cancer. Bloom syndrome cells display a high level of genetic instability, including a 10-fold increase in the sister chromatid exchanges (SCE) level. Bloom syndrome arises through mutations in both alleles of the BLM gene, which was identified as a member of the RecQ helicase family. In this study, we screened a Tunisian family with three BS patients. Cytogenetic analysis showed several chromosomal aberrations, and an approximately 14-fold elevated SCE frequency in BS cells. A significant increase in SCE frequency was observed in some family members but not reaching the BS patients values, leading to suggest that this could be due to the heterozygous profile. Microsatellite genotyping using four fluorescent dye-labeled microsatellite markers revealed evidence of linkage to BLM locus and the healthy members, sharing higher SCE frequency, showed heterozygous haplotypes as expected. Additionally, the direct BLM gene sequencing identified a novel homozygous frameshift mutation c.3617–3619delAA (p.K1207fsX9) in BS patients and a heterozygous BLM mutation in the family members with higher SCE frequency. Our findings suggest that this latter mutation likely leads to a reduced BLM activity explaining the homologous recombination repair defect and, therefore, the increase in SCE. Based on the present data, the screening of this mutation could contribute to the rapid diagnosis of BS. The genetic confirmation of the mutation in BLM gene provides crucial information for genetic counseling and prenatal diagnosis.     

Entomopathogenic nematodes induce components of systemic resistance in plants: Biochemical and molecular evidence

Antagonism between entomopathogenic nematodes (EPNs) and plant-parasitic nematodes (PPNs) has been documented over the past two decades but its mechanism and ecological significance remain elusive. We investigated the effects of Steinernema carpocapsae and its symbiotic bacterium, Xenorhabdus nematophila applied to the potting medium on pyrogallol peroxidase (P-peroxidase), guaiacol peroxidase (G-peroxidase) and catalase activities in Hosta sp. and Arabidopsis thaliana leaves as components of induced systemic resistance. We found that P-peroxidase activity was significantly higher in the leaves from hosta plants treated with S. carpocapsae infective juveniles (IJs) and S. carpocapsae infected insect cadavers than in the leaves from the control plants 2 weeks after treatment. The G-peroxidase activity was significantly higher in S. carpocapsae infected cadaver and X. nematophila treatments 10 and 15 days after treatment (DAT) and in S. carpocapsae IJs treatment 5 and 15 DAT. The catalase activity in hosta leaves was significantly higher in S. carpocapsae infected cadaver and X. nematophilus treatments compared with the control 5 and 15 DAT and in S. carpocapsae IJs treatment 5 and 10 DAT. Further, the catalase activity in A. thaliana leaves was significantly higher in S. carpocapsae IJs treatment than in the control 7 DAT. We also determined the effects of S. carpocapsae infected cadavers and S. carpocapsae IJs on PR1-gene expression in transgenic A. thaliana leaves through GUS (β-glucuronidase) activity assay and found that the PR1-gene was expressed in leaves from all treatments except the control. Thus, we conclude that the EPNs and their symbiotic bacteria can induce systemic resistance in plants which may explain the elusive antagonistic effect of EPNs on PPNs.     

Influence of nitric oxide synthase inhibition on vasopressin and corticosterone secretion during water deprivation in rats

Nitric oxide (NO) is a short-lived radical that functions as a neurotransmitter in the central nervous system and plays a physiological role in the regulation of hypothalamic–pituitary–adrenal axis and vasopressinergic axis. In the present study, we aimed to investigate the interaction between the generation of NO and vasopressin (AVP) and corticosterone release after 3 days of water deprivation in rats. Animals were previously treated with intraperitoneal (i.p.) saline or l-nitro-arginine methyl ester (L-NAME) injection. l-NAME is a nonspecific inhibitor of nitric oxide synthases. In control rats given i.p. saline or l-NAME, hypothalamic, pituitary, and plasma AVP levels and plasma corticosterone did not change from baseline levels (p > 0.05). Three days of water deprivation increased significantly the corticosterone levels in plasma (p < 0.01) and AVP levels in hypothalamus and plasma (p < 0.01), but not in pituitary, which showed a significant decrease. These variations were concomitant with the elevation of nitrates/nitrates in plasma. l-NAME injection abolished significantly (p < 0.01) the elevation of plasma corticosterone and hypothalamic AVP levels induced by water deprivation. These findings showed that in water-deprived rats, nitric oxide synthase inhibition by l-NAME inhibits corticosterone and vasopressin release, suggesting a potent stimulatory role of NO.     

Maltodextrin: A Novel Excipient Used in Sugar-Based Orally Disintegrating Tablets and Phase Transition Process

The recent challenge in orally disintegrating tablets (ODT) manufacturing encompasses the compromise between instantaneous disintegration, sufficient hardness, and standard processing equipment. The current investigation constitutes one attempt to fulfill this challenge. Maltodextrin, in the present work, was utilized as a novel excipient to prepare ODT of meclizine. Tablets were prepared by both direct compression and wet granulation techniques. The effect of maltodextrin concentrations on ODT characteristics—manifested as hardness and disintegration time—was studied. The effect of conditioning (40°C and 75% relative humidity) as a post-compression treatment on ODT characteristics was also assessed. Furthermore, maltodextrin-pronounced hardening effect was investigated using differential scanning calorimetry (DSC) and X-ray analysis. Results revealed that in both techniques, rapid disintegration (30–40 s) would be achieved on the cost of tablet hardness (about 1 kg). Post-compression conditioning of tablets resulted in an increase in hardness (3 kg), while keeping rapid disintegration (30–40 s) according to guidance of the FDA for ODT. However, direct compression-conditioning technique exhibited drawbacks of long conditioning time and appearance of the so-called patch effect. These problems were, yet, absent in wet granulation-conditioning technique. DSC and X-ray analysis suggested involvement of glass-elastic deformation in maltodextrin hardening effect. High-performance liquid chromatography analysis of meclizine ODT suggested no degradation of the drug by the applied conditions of temperature and humidity. Overall results proposed that maltodextrin is a promising saccharide for production of ODT with accepted hardness-disintegration time compromise, utilizing standard processing equipment and phenomena of phase transition.     

The C-terminal half of Phytophthora infestans RXLR effector AVR3a is sufficient to trigger R3a-mediated hypersensitivity and suppress INF1-induced cell death in Nicotiana benthamiana

The RXLR cytoplasmic effector AVR3a of Phytophthora infestans confers avirulence on potato plants carrying the R3a gene. Two alleles of Avr3a encode secreted proteins that differ in only three amino acid residues, two of which are in the mature protein. Avirulent isolates carry the Avr3a allele, which encodes AVR3aKI (containing amino acids C19, K80 and I103), whereas virulent isolates express only the virulence allele avr3a, encoding AVR3aEM (S19, E80 and M103). Only the AVR3aKI protein is recognized inside the plant cytoplasm where it triggers R3a-mediated hypersensitivity. Similar to other oomycete avirulence proteins, AVR3aKI carries a signal peptide followed by a conserved motif centered on the consensus RXLR sequence that is functionally similar to a host cell-targeting signal of malaria parasites. The interaction between Avr3a and R3a can be reconstructed by their transient co-expression in Nicotiana benthamiana. We exploited the N. benthamiana experimental system to further characterize the Avr3a-R3a interaction. R3a activation by AVR3aKI is dependent on the ubiquitin ligase-associated protein SGT1 and heat-shock protein HSP90. The AVR3aKI and AVR3aEM proteins are equally stable in planta, suggesting that the difference in R3a-mediated death cannot be attributed to AVR3aEM protein instability. AVR3aKI is able to suppress cell death induced by the elicitin INF1 of P. infestans, suggesting a possible virulence function for this protein. Structure-function experiments indicated that the 75-amino acid C-terminal half of AVR3aKI, which excludes the RXLR region, is sufficient for avirulence and suppression functions, consistent with the view that the N-terminal region of AVR3aKI and other RXLR effectors is involved in secretion and targeting but is not required for effector activity. We also found that both polymorphic amino acids, K80 and I103, of mature AVR3a contribute to the effector functions.     

Loss of the surface antigen 3G11 characterizes a distinct population of anergic/regulatory T cells in experimental autoimmune encephalomyelitis

T cell anergy is an important mechanism in the induction of peripheral tolerance against autoimmune diseases, yet no surface marker unique to anergic T cells in these diseases has been identified. In this study we induced in vivo anergy by i.v. tolerance against experimental autoimmune encephalomyelitis in myelin basic protein TCR transgenic mice, and showed that the hyporesponsiveness of autoantigen-reactive T cells from tolerized mice was associated with a dramatic loss of 3G11, a cell surface molecule on the surface of CD4+ T cells. Purified 3G11-CD4+ T cells lost autoantigen-induced proliferation and IL-2 production, whereas 3G11+CD4+ T cells retained responsiveness. Furthermore, 3G11- T cells actively suppressed proliferation and Th1 cytokine production of 3G11+ T cells and splenocytes of nontolerized mice. Active suppression by 3G11- T cells was at least partially due to soluble immunoregulatory factors, including IL-10. The T regulatory property of 3G11- T cells was confirmed in vivo because the transfer of purified 3G11- T cells effectively suppressed clinical experimental autoimmune encephalomyelitis. We conclude that loss of the surface molecule 3G11 characterizes a distinct population of anergic/regulatory T cells. This is the first demonstration of the ability to identify and purify anergic T cells by a distinct cell surface marker in an autoimmune disease and paves the way for a better understanding of the mechanism of tolerance in autoimmune diseases.     

Mutations in CERS3 Cause Autosomal Recessive Congenital Ichthyosis in Humans

Autosomal recessive congenital ichthyosis (ARCI) is a rare genetic disorder of the skin characterized by abnormal desquamation over the whole body. In this study we report four patients from three consanguineous Tunisian families with skin, eye, heart, and skeletal anomalies, who harbor a homozygous contiguous gene deletion syndrome on chromosome 15q26.3. Genome-wide SNP-genotyping revealed a homozygous region in all affected individuals, including the same microdeletion that partially affects two coding genes (ADAMTS17, CERS3) and abolishes a sequence for a long non-coding RNA (FLJ42289). Whereas mutations in ADAMTS17 have recently been identified in autosomal recessive Weill-Marchesani-like syndrome in humans and dogs presenting with ophthalmologic, cardiac, and skeletal abnormalities, no disease associations have been described for CERS3 (ceramide synthase 3) and FLJ42289 so far. However, analysis of additional patients with non-syndromic ARCI revealed a splice site mutation in CERS3 indicating that a defect in ceramide synthesis is causative for the present skin phenotype of our patients. Functional analysis of patient skin and in vitro differentiated keratinocytes demonstrated that mutations in CERS3 lead to a disturbed sphingolipid profile with reduced levels of epidermis-specific very long-chain ceramides that interferes with epidermal differentiation. Taken together, these data present a novel pathway involved in ARCI development and, moreover, provide the first evidence that CERS3 plays an essential role in human sphingolipid metabolism for the maintenance of epidermal lipid homeostasis.