<Emphasis Type="Italic">Braf</Emphasis>, <Emphasis Type="Italic">Kras</Emphasis> and <Emphasis Type="Italic">Helicobacter pylori</Emphasis> epigenetic changes-associated chronic gastritis in Egyptian patients with and without gastric cancer

We aimed to study MLH1 and MGMT methylation status in Helicobacter pylori-associated chronic gastritis in Egyptian patients with and without gastric cancer. 39 patients were included in our study. They were divided into 2 groups; patients without (group I) and with gastric adenocarcinoma (group II). Patients were subjected to clinical examination, abdominal ultrasound and upper endoscopy for gastric biopsy. Biopsies were subjected to urease test, histological examination, and DNA purification. H. pylori, Braf, Kras, MLH1 and MGMT methylation were assessed by quantitative PCR. DNA sequencing was performed to assess Braf and Kras genes mutation. qPCR of H. pylori was significantly higher in patients with adenocarcinoma (group II) than those without adenocarcinoma (group I); with a p < 0.001 as well as in patients with age above 50 years with a p value = 0.008. By applying logistic regression analysis it was reported that the H. pylori qPCR is a significant predictor to the adenocarcinoma with OR = 1.025 (95 % CI: 1. 002–1.048), with sensitivity of 90 % and specificity of 100 %. Adenocarcinoma patients had a significantly higher mean age and levels of H. Pylori, Braf, K-ras, methylated MGMT and methylated MLH1 than those of gastritis patients. DNA sequence analysis of Braf (codon 12) and Kras (codon 600) had genes mutation in gastric adenocarcinoma versus chronic gastritis. Conclusion: H. pylori may cause epigenetic changes predisposing the patients to cancer stomach. Estimation of H. pylori by qPCR can be a good predictor to adenocarcinoma. Braf and Kras genes mutation were reveled in gastritis and adenocarcinoma patients.     

Protective effect of rosemary (Rosmarinus officinalis) against diethylnitrosamine-induced renal injury in rats

Abstract

Context: The kidney plays a central role in detoxification and excretion of toxic metabolites, and therefore, is susceptible to toxicity by xenobiotics.

Objective: To investigate the protective effect of Rosmarinus officinalis (rosemary) powder and its essential (volatile) oil against diethylnitrosamine (DEN)-induced renal injury in rats.

Materials and methods: Phenolic and flavonoid components were characterised in rosemary powder using HPLC-UV instrument while rosemary essential oil (E.O) was investigated via GC-MS technique. In rat model, rosemary was administrated orally (in diet) for two months. Lipid profile, antioxidant biomarkers, kidney functions and histopathological examinations were assessed.

Results: Hesperidin (4878.88?ppm) and ellagic acid (403.57?ppm) are among the major phenolic and flavonoid constituents in rosemary powder. Camphor (18.36%) and α-pinene (12.74%) represent the main E.O active ingredients. Rats treated with rosemary E.O showed a significant elevation in serum HDL (28.28%) accompanied by a decrease in LDL (115.47%). A significant decrease in serum creatinine and urea was also reported (69.72 and 109.89%, respectively). Moreover, serum glutathione peroxidise (GSH-Px) activity has been significantly increased. Kidney histopathological examinations confirmed the protective effect against DEN-induced abnormalities.

Conclusion: Rosemary (powder/E.O) was able to reduce or even prevent the severity of diethylnitrosamine-induced renal dysfunction.     

MORPHOLOGICAL CHANGES AND THE REQUIREMENTS FOR MACROMOLECULE SYNTHESIS DURING EXCYSTMENT OF ACANTHAMOEBA CASTELLANII

Light and phase-contrast microscopic observations of excystment in Acanthamoeba castellanii have been used to classify cells in excysting populations as free trophozoites, or mature, activated, or preemergent cysts. These categories have been used to describe the kinetics of excystment. A pH of 7 and a temperature of 30°C have been found to be optimal for the activation of mature cysts. Both activation and emergence are inhibited by cycloheximide and actinomycin D, but neither process is much affected by hydroxyurea. Cell-free extracts of high molecular weight components of cyst cytoplasm can support protein synthesis in vitro, although less efficiently than similar extracts from trophozoites. Evidence indicates that some of the functional RNA in the cyst extracts is synthesized before excystment.     

Potential therapeutic effects of induced pluripotent stem cells on induced salivary gland cancer in experimental rats

Salivary gland neoplasms exhibit complex histopathology in a variety of tumor types and treatment options depend largely on the stage of the cancer. Induced pluripotent stem cells (iPS) have been investigated for treating induced salivary gland cancer and for restoring salivary gland function. We investigated iPS treatment for salivary gland cancer both in vitro and in vivo. For our study in vitro, we re-programmed human skin fibroblasts to form iPS cells using a plasmid containing Oct4, Sox2, L-MYC and LIN28. For our study in vivo, we used 30 white male albino rats divided into the following groups of 10: group 1 (control): rats were injected with phosphate-buffered saline (PBS), group 2 induced squamous cell carcinoma (SCC): rat submandibular glands were injected with squamous carcinoma cells (SCC), group 3 (induced SCC/iPS): SCC treated rats treated with 5 × 106 iPS cells. Submandibular glands from rats of all groups were examined histologically and real time PCR was performed for amylase, and COX I and COX II gene expression. We confirmed that submandibular gland specimens included tumor tissue before starting treatment with iPS. iPS treated cases exhibited regeneration of salivary glands, although minor degenerative and vascularization changes remained. The acinar cells regained their proper organization, but continued to exhibit abnormal activity including hyperchromatism. iPS cells may be useful for treating salivary gland carcinomas.     

Regeneration and transformation of Egyptian maize inbred lines via immature embryo culture and a biolistic particle delivery system

Summary A regeneration system was developed for elite Egyptain maize inbred lines using immature embryos as explants. This system proved to be highly genotype-dependent. Line Gz 643 was identified as the best line, revealing the highest regeneration frequency (42.2%). Addition of l-proline and silver nitrate to culture media greatly enhanced the formation of embryogenic type II callus and the regenerability of some of the tested lines. Transformation of the scutellar tissue of immature embryos from inbred line Gz643 was performed with the particle delivery system using a single plasmid carrying both the GUS and Bar genes (pAB-6) or by co-transformation with two plasmids, pAct1-F (GUS) and pTW-a(Bar). Different transformation parameters were evaluated, i.e. ostomic treatment, acceleration pressure, and number of shots. Osmotic treatment (0.25 M sorbitol + 0.25 M mannitol) along with the use of either acceleration pressure 1300 psi and one shot per plate (for co-transformation with pAB-6) or 1100 psi and two shots per plate (for transformation with pAct1-F and pTW-a) gave the best results, as expressed by the number of blue spots in the β-glucuronidase (GUS) assay. Stable transformation was confirmed in Ro transformed plants by means of histochemical GUS assay and herbicide application. PCR and Southern blot analysis proved the integration of the full-length genes in some of the transgenics.     

Changes in the water status and osmotic solute contents in response to drought and salicylic acid treatments in four different cultivars of wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis>)

Salicylic acid (SA) controls growth and stress responses in plants. It also induces drought tolerance in plants. In this paper, four wheat (Triticum aestivum L.) cultivars with different drought responses were treated with SA in three levels of drain (90, 60, 30% of maximum field capacity) to examine its interactive effects on drought responses and contents of osmotic solutes that may be involved in growth and osmotic adjustment. Under drought condition, the cultivars Geza 164 and Sakha 69 had the plant biomass and leaf relative water content (LRWC) greater than the cultivars Gemaza 1 and Gemaza 3. In all cultivars, drought stress decreased the biomass, LRWC, and the contents of inorganic solutes (Ca, K, Mg) and largely increased the contents of organic solutes (soluble sugars and proline). By contrast, SA increased the biomass, LRWC and the inorganic and organic solute contents, except proline. Correlation analysis revealed that the LRWC correlated positively with the inorganic solute contents but negatively with proline in all cultivars. SA caused maximum accumulations of soluble sugars in roots under drought. These results indicated that SA-enhanced tolerance might involve solute accumulations but independently of proline biosynthesis. Drought-sensitive cultivars had a trait lowering Ca and K levels especially in shoots. Possible functions of the ions and different traits of cultivars were discussed.     

Trochelioid A and B,new cembranoid diterpenes from the Red Sea soft coral Sarcophyton trocheliophorum

Chemical investigations of the soft coral Sarcophyton trocheliophorum, has led to the isolation of six cembranoids, two of which are new, Trochelioid A (1) and B (2), and one, 16-oxosarcophytonin E (3) isolated from nature for the first time. Additionally, two have been isolated from S. trocheliophorum for the first time (4 and 6). Structures were elucidated by employing extensive NMR and HR-FAB-MS experimentation.     

Stable cytotoxic T cell escape mutation in hepatitis C virus is linked to maintenance of viral fitness

Mechanisms by which hepatitis C virus (HCV) evades cellular immunity to establish persistence in chronically infected individuals are not clear. Mutations in human leukocyte antigen (HLA) class I-restricted epitopes targeted by CD8(+) T cells are associated with persistence, but the extent to which these mutations affect viral fitness is not fully understood. Previous work showed that the HCV quasispecies in a persistently infected chimpanzee accumulated multiple mutations in numerous class I epitopes over a period of 7 years. During the acute phase of infection, one representative epitope in the C-terminal region of the NS3/4A helicase, NS3(1629-1637), displayed multiple serial amino acid substitutions in major histocompatibility complex (MHC) anchor and T cell receptor (TCR) contact residues. Only one of these amino acid substitutions at position 9 (P9) of the epitope was stable in the quasispecies. We therefore assessed the effect of each mutation observed during in vivo infection on viral fitness and T cell responses using an HCV subgenomic replicon system and a recently developed in vitro infectious virus cell culture model. Mutation of a position 7 (P7) TCR-contact residue, I1635T, expectedly ablated the T cell response without affecting viral RNA replication or virion production. In contrast, two mutations at the P9 MHC-anchor residue abrogated antigen-specific T cell responses, but additionally decreased viral RNA replication and virion production. The first escape mutation, L1637P, detected in vivo only transiently at 3 mo after infection, decreased viral production, and reverted to the parental sequence in vitro. The second P9 variant, L1637S, which was stable in vivo through 7 years of follow-up, evaded the antigen-specific T cell response and did not revert in vitro despite being less optimal in virion production compared to the parental virus. These studies suggest that HCV escape mutants emerging early in infection are not necessarily stable, but are eventually replaced with variants that achieve a balance between immune evasion and fitness for replication.     

Conserved hierarchy of helper T cell responses in a chimpanzee during primary and secondary hepatitis C virus infections

Control of hepatitis C virus (HCV) infection could be influenced by the timing and magnitude of CD4+ T cell responses against individual epitopes. We characterized CD4+ T cells targeting seven Pan troglodytes (Patr) class II-restricted epitopes during primary and secondary HCV infections of a chimpanzee. All Patr-DR-restricted HCV epitopes bound multiple human HLA-DR molecules, indicating the potential for overlap in epitopes targeted by both species. Some human MHC class II molecules efficiently stimulated IL-2 production by chimpanzee virus-specific T cell clones. Moreover, one conserved epitope designated NS3(1248) (GYKVLVLNPSV) overlapped a helper epitope that is presented by multiple HLA-DR molecules in humans who spontaneously resolved HCV infection. Resolution of primary infection in the chimpanzee was associated with an initial wave of CD4+ T cells targeting a limited set of dominant epitopes including NS3(1248.) A second wave of low-frequency CD4+ T cells targeting other subdominant epitopes appeared in blood several weeks later after virus replication was mostly contained. During a second infection 7 years later, CD4+ T cells against all epitopes appeared in blood sooner and at higher frequencies but the pattern of dominance was conserved. In summary, primary HCV infection in this individual was characterized by T cell populations targeting two groups of MHC class II-restricted epitopes that differed in frequency and kinetics of appearance in blood. The hierarchial nature of the CD4+ T cell response, if broadly applicable to other HCV-infected chimpanzees and humans, could be a factor governing the outcome of HCV infection.     

Influences on the sensitivity of real-time PCR for the detection of bovine DNA in heat-sterilised feedstuffs

The present study evaluated two previously developed methods for amplification of bovine mtDNA segments of 109 and 271 base pairs (bp) by real-time polymerase chain reaction (PCR). Beef samples were sterilised experimentally at different temperatures (126 degrees C, 129 degrees C, 132 degrees C and 135 degrees C). These experimentally sterilised beef samples and nine commercial meat and bone meals (MBM) were mixed to a reference plant concentrate in strengths of 50%, 10%, 5%, and 1%. The results of the following PCR showed that the Bos-109 real-time PCR assay was able to detect all the experimental beef samples with exception of the mixtures of beef heated experimentally to 135 degrees C. In mixtures of industrial MBM bovine DNA were always found. Comparatively, the beef sterilised at 135 degrees C and 132 degrees C (and their respective mixtures) and the mixture containing 1% of beef sterilised at 129 degrees C were not detectable with the PCR assay amplifying a target of 271 bp. Using this PCR mixtures of industrial MBM were only weakly detected. The low concentrated mixtures of the extremely processed MBM-1 and MBM-2 even reported negative. These results indicate that the detectability of bovine DNA is strongly influenced by the degree of the thermal treatment. Only the PCR assay amplifying relatively short fragments of a multi-copy mitochondrial target was reliable for the detection of correctly heated MBM in mixed feed.