Optimization of factors influencing enzyme activity and product selectivity and the role of proton transfer in the catalytic mechanism of patchoulol synthase

The patchoulol synthase (PTS) from Pogostemon cablin is a versatile sesquiterpene synthase and produces more than 20 valuable sesquiterpenes by conversion of the natural substrate farnesyl pyrophosphate (FPP). PTS has the potential to be used as a biocatalyst for the production of valuable sesquiterpenes such as (−)-patchoulol. The objective of the present study is to develop an efficient biotransformation and to characterize the biocatalytic mechanism of the PTS in detail. For this purpose, soluble PTS was prepared using an optimized cultivation protocol and continuous downstream process with a purity of 98%. The PTS biotransformation was then optimized regarding buffer composition, pH-value, and temperature for biotransformation as well as functional and kinetic properties to improve productivity. For the bioconversion of FPP, the highest enzyme activity was reached with the 2-(N-morphlino)ethanesulfonic acid (MES) buffer containing 10% (v/v) glycerol and 10 mM MgCl₂ at pH 6.4 and 34°C. The PTS showed an unusual substrate inhibition for sesquiterpene synthases indicating an intermediate sesquiterpene formed in the active center. Deuteration experiments were used to gain further insights into the biocatalytic mechanism described in literature. Thus it could be shown that a second substrate binding site must be responsible for substrate inhibition and that further protonation and deprotonation steps are involved in the reaction mechanism.     

In silico design and in vitro expression of novel multiepitope DNA constructs based on HIV-1 proteins and Hsp70 T-cell epitopes

Objectives

Epitope-driven vaccines carrying highly conserved and immunodominant epitopes have emerged as promising approaches to overcome human immunodeficiency virus-1 (HIV-1) infection.

Methods

Two multiepitope DNA constructs encoding T cell epitopes from HIV-1 Gag, Pol, Env, Nef and Rev proteins alone and/or linked to the immunogenic epitopes derived from heat shock protein 70 (Hsp70) as an immunostimulatory agent were designed. In silico analyses were applied including MHC-I and MHC-II binding, MHC-I immunogenicity and antigen processing, population coverage, conservancy, allergenicity, toxicity and hemotoxicity. The peptide-MHC-I/MHC-II molecular docking and cytokine production analyses were carried out for predicted epitopes. The selected highly immunogenic T-cell epitopes were then used to design two multiepitope fusion constructs. Next, prediction of the physicochemical and structural properties, B cell epitopes, and constructs-toll-like receptors (TLRs) molecular docking were performed for each construct. Finally, the eukaryotic expression plasmids harboring totally 12 cytotoxic T Lymphocyte (CTL) and 10 helper T lymphocytes (HTL) epitopes from HIV-1 proteins (i.e., pEGFP-N1-gag-pol-env-nef-rev), and linked to 2 CTL and 2 HTL epitopes from Hsp70 (i.e., pEGFP-N1-hsp70-gag-pol-env-nef-rev) were generated and transfected into HEK-293 T cells for evaluating the percentage of multiepitope peptides expression using flow cytometry and western blotting.

Results

The designed DNA constructs could be successfully expressed in mammalian cells. The expression rates of Gag-Pol-Env-Nef-Rev-GFP and Hsp70-Gag-Pol-Env-Nef-Rev-GFP were about 56–60% as the bands of?~?63 and?~?72 kDa confirmed in western blotting, respectively.

Conclusion

The combined in silico/in vitro methods indicated two multiepitope constructs can be produced and used as probable effective immunogens for HIV-1 vaccine development.

     

Cytokine gene polymorphism and graft-versus-host disease: a survey in Iranian bone marrow transplanted patients

Graft versus host disease (GVHD) is a major complication of bone marrow transplantation (BMT). Numerous studies have shown the potential role of cytokine genotypes in the occurrence of GVHD. In this retrospective, case–control study we aimed to investigate the association between 13 cytokine genes and acute GVHD (aGVHD) after HLA-identical sibling BMT in 91 Iranian subjects. Negative association was found between aGVHD and donor IL-10/GCC haplotype or donor IL-4Ra-A allele in the population study. When compared within the leukemia subgroup, we observed positive association between recipient IL-1α ?889/C allele and aGVHD. Also there were negative association between recipient IL-10/CAA haplotype and donor IL-4Ra/A allele and development of aGVHD. Among the different genotypes only donor IL-4Ra and donor IL-12 showed significant association. We conclude that several cytokine polymorphisms are positively and negatively associated with aGVHD in Iranian HLA matched siblings, of which IL-4Ra and IL-12 may play important roles.     

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.     

Découverte fortuite de fixations colorectales focales du FDG en TEP/TDM : corrélation aux données de la coloscopie

PurposeFDG-PET is an established tool for the diagnosis of recurrent or metastatic colorectal carcinoma. Several case series suggest that FDG-PET often detects incidental adenomatous polyps or colorectal adenocarcinomas. The aim of this study was to correlate unexpected colorectal foci of FDG uptake to pathology findings after systematic colonoscopy.Patients and methodsWe reviewed the records of 3541 patients who underwent FDG PET/CT in our institution over a 30-month period for the assessment of a known or suspected malignancy. In 85 of them, incidental, nodular shaped and well-circumscribed foci of abnormal uptake were identified in the area of the colon or rectum. Patients with segmental or diffuse abnormal colorectal uptake were excluded, as well as patients with known benign or malignant colorectal disease. Colonoscopy and complete pathology report was available in 29 patients. Maximal standardized uptake value (SUV_max) was measured in all lesions.ResultsUnexpected colorectal foci of FDG uptake were associated with colonoscopic abnormalities in 23 patients (true positive rate: 79 %). Adenocarcinomas were found in six patients (SUV_max = 7.3 ± 2.6), tubulous adenomas in four patients (SUV_max = 7.3 ± 4.9) and tubulovillous adenomas in 12 patients (SUV_max = 4.2 ± 1.1). Hyperplasic polyps with no sign of dysplasia were found in the last patient (SUV_max = 3.3). Concomitant CT abnormalities were found on PET/CT fusion in eight patients and consisted of wall thickening (n = 5) or nodular mass (n = 3). Conversely, PET was falsely positive in six patients (21 %), with no concomitant CT abnormalities and no abnormal findings at endoscopy (SUV_max = 6.2 ± 2.8, no significant difference with true positive lesions).ConclusionOur findings emphasize the need to perform a colonoscopy in front of incidental nodular colorectal foci of FDG uptake because malignant or pre-malignant neoplasms, which are not clinically apparent, are found in more than three-quarter of cases.     

Homozygosity mapping in consanguineous families reveals extreme heterogeneity of non-syndromic autosomal recessive mental retardation and identifies 8 novel gene loci

Autosomal recessive gene defects are arguably the most important, but least studied genetic causes of severe cognitive dysfunction. Homozygosity mapping in 78 consanguineous Iranian families with nonsyndromic autosomal recessive mental retardation (NS-ARMR) has enabled us to determine the chromosomal localization of at least 8 novel gene loci for this condition. Our data suggest that in the Iranian population NS-ARMR is very heterogeneous, and they argue against the existence of frequent gene defects that account for more than a few percent of the cases. Mohammad Mahdi Motazacker and Masoud Garshasbi have contributed equally to this work.     

Similarities and differences in the transcriptional regulation of the leptin gene promoter in gastric and adipose cells

The stomach was reported to synthesize and secrete leptin mainly in the gastric lumen. Gastric leptin release is markedly increased after food intake, by vagal cholinergic stimulation and by cholecystokinin and secretin. Here we show that human gastric MKN-74 cells produce leptin that increases upon challenge with cholecystokinin, insulin, glucocorticoids and all-trans retinoic acid through activation of the leptin gene promoter. In addition, we demonstrate that forskolin and BRL37344 which increased cAMP levels, fail to affect the activity of leptin gene promoter in MKN74 expressing beta(3)-adrenoceptor cells but, induce a 2-fold decrease in this activity in adipose 3T3-L1 cells. These data described for the first time, similarities and more interestingly, differences in the regulation of the leptin gene promoter in gastric cells as compared to adipocytes.     

Cytologic findings of a pleomorphic adenoma of the breast: a case report

BACKGROUND: Pleomorphic adenoma of the breast is a rare benign tumor. Only a few cases have been reported. The histologic features have been described well. However, the cytologic findings have been described in only a few papers. CASE: A 47-year-old female presented with a left breast mass of several months' duration. The clinical and mammographic findings were highly suspicious for malignancy. Following an aspiration biopsy diagnosis of "positive for malignancy," the mass was excised. The histologic diagnosis was pleomorphic adenoma (mixed tumor of salivary gland type) rather than carcinoma. CONCLUSION: The cytologic presentation of pleomorphic adenoma of the breast can masquerade as that of a malignant tumor, in this case colloid carcinoma. This case delineates the cytomorphologicfeatures of pleomorphic adenoma, which may mimic carcinoma.     

Design of a specific colonic mucus marker using a human commensal bacterium cell surface domain

Imaging living cells and organs requires innovative, specific, efficient, and well tolerated fluorescent markers targeting cellular components. Such tools will allow proceeding to the dynamic analysis of cells and the adaptation of tissues to environmental cues. In this study, we have identified and synthesized a novel non-toxic fluorescent marker allowing a specific fluorescent staining of the human colonic mucus. Our strategy to identify a molecule able to specifically bind to the human colonic mucus was on the basis of the mucus adhesion properties of commensal bacteria. We identified and characterized the mucus-binding property of a 70-amino acid domain (MUB(70)) expressed on the surface of Lactobacillus strains. The chemical synthesis of MUB(70) was achieved using the human commensal bacterium Lactobacillus reuteri AF120104 protein as a template. The synthesized Cy5-conjugated MUB(70) marker specifically stained the colonic mucus on fixed human, rabbit, and guinea pig tissues. Interestingly, murine tissue was not stained, suggesting significant differences in the composition of the murine colonic mucus. In addition, this marker stained the mucus of living cultured human colonic cells (HT29-MTX) and human colonic tissue explants. Using a biotinylated derivative of MUB(70), we demonstrated that this peptide binds specifically to Muc2, the most abundant secreted mucin, through its glycosylated moieties. Hence, Cy5-MUB(70) is a novel and specific fluorescent marker for mammalian colonic mucus. It may be used for live imaging analysis but also, as demonstrated in this study, as a marker for the diagnosis and the prognosis of colonic mucinous carcinomas.