In silico investigation of heparanase-correlated genes in breast cancer subtypes / Investigação in silico dos genes correlacionados à heparanase nos diferentes subtipos de câncer de mama

ABSTRACT Objective To investigate the possible genes that may be related to the mechanisms that modulate heparanase-1. Methods The analysis was conducted at Universidade Federal de São Paulo, on the data provided by: The Cancer Genome Atlas, University of California Santa Cruz Genome Browser, Kyoto Encyclopedia of Genes and Genomes Pathway Database, Database for Annotation, Visualization and Integrated Discovery Bioinformatics Database and the softwares cBioPortal and Ingenuity Pathway Analysis. Results Using messenger RNA expression pattern of different molecular subtypes of breast cancer, we proposed that heparinase-1 was co-related with its progression. In addition, genes that were analyzed presented co-expression with heparanase-1. The results that showed that heparanase-1 co-expressed with phosphoinositide 3-kinase adapter protein 1, sialic acid-binding immunoglobulin-like lectin 7, and leukocyte-associated immunoglobulin-like receptor 1 are directed related with immune system evasion during breast cancer progression. Furthermore, cathepsin L was co-expressed with heparanase-1 and transformed inactive heparanase-1 form into active heparanase-1, triggering extracellular matrix remodeling, which contributes to enhanced tumor-host interaction of the tumor. Conclusion The signaling pathway analysis using bioinformatics tools gives supporting evidence of possible mechanisms related to breast cancer development. Evasion genes of the immune system co-expressed with heparanase-1, a enzyme related with tumor progression.

RESUMO Objetivo Investigar os genes que podem estar relacionados aos mecanismos que modulam a heparanase-1. Métodos A análise foi realizada na Universidade Federal de São Paulo, utilizando dados fornecidos por: The Cancer Genome Atlas, University of California Santa Cruz Genome Browser, Kyoto Encyclopedia of Genes and Genomes Pathway Database, Database for Annotation, Visualization and Integrated Discovery Bioinformatics Database e os softwares cBioPortal e Ingenuity Pathway Analysis. Resultados Usando o perfil de expressão de RNA mensageiro de diferentes subtipos moleculares de câncer de mama, propusemos que a heparanase-1 esteve correlacionada com a progressão tumoral. Além disso, os genes analisados apresentaram coexpressão com heparanase-1. Os resultados mostraram que a heparanase-1 coexpressa com proteína adaptadora 1 da fosfoinositídeo 3-quinase, lectina 7 tipo Ig de ligação ao ácido siálico e receptor 1 do tipo imunoglobulina associado a leucócitos, estes genes estão diretamente relacionados à evasão do sistema imune durante a progressão do câncer de mama. Além disso, a catepsina L foi coexpressa com a heparanase-1 e transformou a forma inativa da heparanase-1 em heparanase-1 ativa, desencadeando o remodelamento da matriz extracelular, o que contribuiu para a interação do tumor com o ambiente tumoral. Conclusão A análise utilizando bioinformática fornece evidências de possíveis mecanismos relacionados ao desenvolvimento do câncer de mama. Genes de evasão do sistema imune foram coexpressos com a heparanase-1, uma enzima relacionada à progressão tumoral.

Development and optimization of an intergeneric conjugation system and analysis of promoter activity in Streptomyces rimosus M527 / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

An efficient genetic transformation system and suitable promoters are essential prerequisites for gene expression studies and genetic engineering in streptomycetes. In this study, firstly, a genetic transformation system based on intergeneric conjugation was developed in Streptomyces rimosus M527, a bacterial strain which exhibits strong antagonistic activity against a broad range of plant-pathogenic fungi. Some experimental parameters involved in this procedure were optimized, including the conjugative media, ratio of donor to recipient, heat shock temperature, and incubation time of mixed culture. Under the optimal conditions, a maximal conjugation frequency of 3.05×10-5 per recipient was obtained. Subsequently, based on the above developed and optimized transformation system, the synthetic promoters SPL-21 and SPL-57, a native promoter potrB, and a constitutive promoter permE* commonly used for gene expression in streptomycetes were selected and their activity was analyzed using gusA as a reporter gene in S. rimosus M527. Among the four tested promoters, SPL-21 exhibited the strongest expression activity and gave rise to a 2.2-fold increase in β-glucuronidase (GUS) activity compared with the control promoter permE*. Promoter SPL-57 showed activity comparable to that of permE*. Promoter potrB, which showed the lowest activity, showed a 50% decrease in GUS activity compared with the control permE*. The transformation system developed in this study and the tested promotors provide a basis for the further modification of S. rimosus M527.

Serum Klotho (but not haplotypes) associate with the post-myocardial infarction status of older adults

OBJECTIVES: The number of deaths from vascular diseases is incredibly high worldwide, and reliable markers for major events are still needed. The current cross-sectional study investigated the association of Klotho haplotypes and Klotho serum levels with classic risk factors and a clinical history of vascular events. METHODS: Clinical, anthropometric, biochemical and nutritional assessments were conducted with 168 older adults, complemented by genotyping (rs9536314 and rs9527025) and the detection of serum Klotho (ELISA). RESULTS: Klotho levels and haplotypes did not associate with most classic risk factors for vascular events, including markers such as C-reactive protein and homocysteine. A positive association was only found between Klotho levels and the previous occurrence of a myocardial infarction by both correlational (p=0.006) and variance analyses (p<0.001), and these associations were independent of the context. CONCLUSION: Our results suggest that serum Klotho is higher in individuals with a clinical history of myocardial infarction but not with a history of coronary artery disease or stroke. None of the Klotho haplotypes were associated with the variables investigated herein.

Expression of heparanase in basal cell carcinoma and squamous cell carcinoma

Abstract: Background: Heparanase is an enzyme that cleaves heparan sulfate chains. Oligosaccharides generated by heparanase induce tumor progression. Basal cell carcinoma and squamous cell carcinoma comprise types of nonmelanoma skin cancer. Objectives: Evaluate the glycosaminoglycans profile and expression of heparanase in two human cell lines established in culture, immortalized skin keratinocyte (HaCaT) and squamous cell carcinoma (A431) and also investigate the expression of heparanase in basal cell carcinoma, squamous cell carcinoma and eyelid skin of individuals not affected by the disease (control). Methods: Glycosaminoglycans were quantified by electrophoresis and indirect ELISA method. The heparanase expression was analyzed by quantitative RT-PCR (qRTPCR). Results: The A431 strain showed significant increase in the sulfated glycosaminoglycans, increased heparanase expression and decreased hyaluronic acid, comparing to the HaCaT lineage. The mRNA expression of heparanase was significantly higher in Basal cell carcinoma and squamous cell carcinoma compared with control skin samples. It was also observed increased heparanase expression in squamous cell carcinoma compared to the Basal cell carcinoma. Conclusion: The glycosaminoglycans profile, as well as heparanase expression are different between HaCaT and A431 cell lines. The increased expression of heparanase in Basal cell carcinoma and squamous cell carcinoma suggests that this enzyme could be a marker for the diagnosis of such types of non-melanoma cancers, and may be useful as a target molecule for future alternative treatment.

Expression of heparanase gene in Egyptian acute leukemia patient

Heparanase is an endoglycosidase that degrades heparin sulfate, the main polysaccharide constituent of the extracellular matrix and basement membrane. Expression of the heparanase gene is associated with the invasive, angiogenic, and metastatic potential of diverse malignant tumors and cell lines. to investigate possible relation/correlation between Heparanase gene expression and quantitation in pediatric Acute leukemia patients and clinicopathologic variables as well as patients outcome in an attempt to determine it's prognostic value and the possibility of using it as a new target for treatment. Forty pediatric acute leukemia patients [20 acute myeloid leukemia [AML] and 20 acute lymphoblastic leukemia [ALL] as well as 11 normal volunteers were analyzed for the expression and level of Heparanase gene using real time quantitative reverse transcriptase polymerase chain reaction [RTQ-PCR] to investigate a possible relation, association, or correlation with the clinical and laboratory features of patients at diagnosis, and patient outcome after treatment and follow up. Comparing the 3 groups as regards the Heparanase gene level there was high statistical significant difference [p<0.001] being maximum in AML and minimum in controls, with mean Relative quantitation [RQ] level 2336.2 +/- 10405.2 in AML ,median 8.0 and range [3.1-46543.0], while mean RQ in ALL was 1.7 +/- 1.0 ,median 1.7 and range [0.1-3.1] and in controls mean was 0.8+/-0.3, median 0.8 and range [0.4-1.4].Comparison between each 2 groups as regards heparanase level was of high statistically significant difference, p value being [p<0.001] when comparing AML/ALL and AML/controls and [p=0.035] when comparing ALL/controls. Cut off value for heparanase gene was calculated using Roc curve and was found to be 1.413 with 80% sensitivity and 100% specificity. According to this cut off level, 20/20 [100%] AML cases were heparanase positive, 12/20 [60%] [[ALL] cases were heparanase positive and 8/20 ALL patients were negative, while all controls [100%] were negative. This was of high statistical significance [p<0.001]. Comparing the overall survival [OS] of AML/ALL there was no statistically significant difference [p=0.2916], while comparing the disease free survival [DPS] of AML/ALL was of statistical significant difference [0.0312]. Comparing the final status of the disease [complete remission [CR]/ progressive disease [PD] or death] as regards the heparanase gene level RQ, showed a high statistical significant difference [p<0.005] with the level being higher in patients with PD/death. There was no significant correlation between all group and heparanase gene level as regards age, TLC, hemoglobin, platelets and peripheral blood blasts [p=0.353,0.704,0.844,0.54 and 0.097] respectively, while there was significant negative correlation on comparing bone marrow blast% and heparanase gene level [r=-0.408 and p=0.09]. Heparanse gene is expressed in acute leukemia being higher in AML than ALL and controls. Patients with higher heparanase gene showed poorer outcome. These findings suggest that heparanase gene may be a novel significant therapeutic target for acute leukemia

The renin-angiotensin system and aging in the kidney

Aging is associated with progressive functional deterioration and structural changes in the kidney. Changes in the activity or responsiveness of the renin-angiotensin system (RAS) occur with aging. RAS changes predispose the elderly to various fluid and electrolyte imbalances as well as acute kidney injury and chronic kidney disease. Among the multiple pathways involved in renal aging, the RAS plays a central role. This review summarizes the association of the RAS with structural and functional changes in the aging kidney and age-related renal injury, and describes the underlying mechanisms of RAS-related renal aging. An improved understanding of the renal aging process may lead to better individualized care of the elderly and improved renal survival in age-related diseases.

Acute UV Irradiation Increases Heparan Sulfate Proteoglycan Levels in Human Skin

Glycosaminoglycans are important structural components in the skin and exist as various proteoglycan forms, except hyaluronic acid. Heparan sulfate (HS), one of the glycosaminoglycans, is composed of repeated disaccharide units, which are glucuronic acids linked to an N-acetyl-glucosamine or its sulfated forms. To investigate acute ultraviolet (UV)-induced changes of HS and HS proteoglycans (HSPGs), changes in levels of HS and several HSPGs in male human buttock skin were examined by immunohistochemistry and real-time quantitative polymerase chain reaction (qPCR) after 2 minimal erythema doses (MED) of UV irradiation (each n = 4-7). HS staining revealed that 2 MED of UV irradiation increased its expression, and staining for perlecan, syndecan-1, syndecan-4, CD44v3, and CD44 showed that UV irradiation increased their protein levels. However, analysis by real-time qPCR showed that UV irradiation did not change mRNA levels of CD44 and agrin, and decreased perlecan and syndecan-4 mRNA levels, while increased syndecan-1 mRNA level. As HS-synthesizing or -degrading enzymes, exostosin-1 and heparanase mRNA levels were increased, but exostosin-2 was decreased by UV irradiation. UV-induced matrix metalloproteinase-1 expression was confirmed for proper experimental conditions. Acute UV irradiation increases HS and HSPG levels in human skin, but their increase may not be mediated through their transcriptional regulation.

A choline monooxygenase gene promoter from Salicornia europaea increases expression of the -glucuronidase gene under abiotic stresses in tobacco (Nicotiana tabacum L.).

A 1312 bp 5' flanking region of Salicornia europaea choline monooxygenase (SeCMO) gene was isolated using the anchored PCR. To investigate the mechanism of regulation for this stress-induced gene, the SeCMO promoter--glucuronidase (GUS) chimeric gene constructs containing five deletions F1, F2, F3, F4 and F5 were introduced into tobacco (Nicotiana tabacum L.) by Agrobacterium-mediated transformation. The functional properties of each promoter fragment were examined by assaying GUS activity in the leaves of transgenic tobacco treated with abiotic stresses (NaCl, PEG6000 and low temperature). The GUS activity in transgenic tobacco with F2 (-1056 to +8) construct showed highest increase under all the three abiotic stresses. Thus, the study provided a potential promoter induced by the salt, dehydration and cold for the plant genetic manipulation.

Klotho and the Aging Process

The klotho gene was originally identified as a putative age-suppressing gene in mice that extends life span when overexpressed. It induces complex phenotypes resembling human premature aging syndromes when disrupted. The gene was named after a Greek goddess Klotho who spun the thread of life. Since then, various functional aspects of the klotho gene have been investigated, leading to the identification of multiple novel endocrine axes that regulate various metabolic processes and an unexpected link between mineral metabolism and aging. The purposes of this review were to overview recent progress on Klotho research and to discuss a novel aging mechanism.

A comparative analysis of green fluorescent protein and beta-glucuronidase protein-encoding genes as a reporter system for studying the temporal expression profiles of promoters.

The assessment of activity of promoters has been greatly facilitated by the use of reporter genes. However, the activity as assessed by reporter gene is a reflection of not only promoter strength, but also that of the stability of the mRNA and the protein encoded by the reporter gene. While a stable reporter gene product is an advantage in analysing activities of weak promoters, it becomes a major limitation for understanding temporal expression patterns of a promoter, as the reporter product persists even after the activity of the promoter ceases. In the present study we undertook a comparative analysis of two reporter genes, beta-glucuronidase (gus) and green fluorescent protein (sgfp), for studying the temporal expression pattern of tapetum-specific promoters A9 (Arabidopsis thaliana) and TA29 (Nicotiana tabacum). The activity of A9 and TA29 promoters as assessed by transcript profiles of the reporter genes (gus or sgfp ) remained the same irrespective of the reporter gene used. However, while the deduced promoter activity using gus was extended temporally beyond the actual activity of the promoter, sgfp as recorded through its fluorescence correlated better with the transcription profile. Our results thus demonstrate that sgfp is a better reporter gene compared to gus for assessment of temporal activity of promoters. Although several earlier reports have commented on the possible errors in deducing temporal activities of promoters using GUS as a reporter protein, we experimentally demonstrate the advantage of using reporter genes such as gfp for analysis of temporal expression patterns.

Cloning of an ovule specific promoter from Arabidopsis thaliana and expression of beta-glucuronidase.

Tissue specific expression of transgenes in plant species has several advantages over constitutive expression. Identification of ovule specific promoters would be useful in genetic engineering of plants with a variety of desirable traits such as genetically engineered parthenocarpy, female sterile plants or seedless fruits. Relative inaccessibility and difficulty in harvesting adequate amounts of tissue at known developmental stages has impeded the progress in cloning of promoters involved in ovule development. In the present study an ovule specific promoter was cloned from Arabidopsis AGL11 gene and used to express GUS (beta-glucuronidase) gene in transgenic Arabidopsis. Histochemical staining of GUS appeared in the center of young ovary (ovules), but no detectable GUS activity was observed in vegetative plant tissues, sepals, petals and androecium. AGL11 gene promoter can be useful to modify the developmental path of plants by expressing either plant hormones or lethal genes for agronomic purpose.

Polymorphism in the promoter region of the klotho gene (G-395A) is associated with early dysfunction in vascular access in hemodialysis patients

BACKGROUND/AIMS: Vascular access dysfunction is an important cause of morbidity and mortality in hemodialysis (HD) patients. Recent studies have shown that a klotho gene mutation is related to endothelial dysfunction, thrombosis, and arteriosclerosis, which are regarded as causes of vascular access dysfunction. We investigated the relationship between the klotho G-395A polymorphism and early dysfunction in vascular access in HD patients. METHODS: Patients who underwent vascular access operations between 1999 and 2002 were enrolled (n=126). Genotyping was performed by allelic discrimination using a 5'-nuclease polymerase chain reaction assay. Clinical data that could be relevant to access dysfunction were obtained from medical records. Early dysfunction of vascular access was defined as the need for any angioplastic or surgical intervention to correct or replace a poorly or nonfunctioning vascular access within 1 year and at least 8 weeks after initial access placement. RESULTS: Of the 126 patients, the genotype frequency of G-395A was 72.2% for GG (n=91), 24.6% for GA (n=31), and 3.2% for AA (n=4), and the frequency of minor allele was 0.155. Clinical data were similar between the two groups, divided according to the status of the A allele. Early dysfunction occurred in 34 (27.0%) of patients, but it occurred at a significantly higher rate in A allele carriers (45.7%, 16/35) than in noncarriers (19.8%, 18/91; p=0.003). CONCLUSIONS: Our results suggest that the klotho G-395A polymorphism could be a risk factor for early dysfunction of vascular access in HD patients.

Genetic transformation of peanut (Arachis hypogaea L.) using cotyledonary node as explant and a promoterless gus::nptII fusion gene based vector.

We have generated putative promoter tagged transgenic lines in Arachis hypogaea cv JL-24 using cotyledonary node (CN) as an explant and a promoterless gus::nptII bifunctional fusion gene mediated by Agrobacterium transformation. MS medium fortified with 6-benzylaminopurine (BAP) at 4mg/l in combination with 0.1 mg/l alpha -napthaleneacetic acid (NAA) was the most effective out of the various BAP and NAA combinations tested in multiple shoot bud formation. Parameters enhancing genetic transformation viz. seedling age, Agrobacterium genetic background and co-cultivation periods were studied by using the binary vector p35SGUSINT. Genetic transformation with CN explants from 6-day-old seedlings co-cultivated with Agrobacterium GV2260 strain for 3 days resulted in high kanamycin resistant shoot induction percentage (45%); approximately 31% transformation frequency was achieved with p35S GUSINT in beta-glucuronidase (GUS) assays. Among the in vivo GUS fusions studied with promoterless gus::nptII construct, GUS-positive sectors occupied 38% of the total transient GUS percentage. We have generated over 141 putative T 0 plants by using the promoterless construct and transferred them to the field. Among these, 82 plants survived well in the green house and 5 plants corresponding to 3.54% showed stable integration of the fusion gene as evidenced by GUS, polymerase chain reaction (PCR) and Southern blot analyses. Twenty-four plants were positive for GUS showing either tissue-specific expression or blue spots in at least one plant part. The progeny of 15 T 0 plants indicated Mendelian inheritance pattern of segregation for single-copy integration. The tissue-specific GUS expression patterns were more or less similar in both T 0 and corresponding T 1 progeny plants. We present the differential patterns of GUS expression identified in the putative promoter-tagged transgenic lines in the present communication.

Genetic transformation of Robinia pseudoacacia by Agrobacterium tumefaciens.

Transgenic Robinia pseudoacacia plants were obtained by Agrobacterium tumefaciens mediated gene transfer. Agrobacterium strain LBA4404 harbouring a binary vector that contained the chimeric neomycin phosphotransferase II (NPTII) and beta-glucuronidase (GUS) genes was co-cultivated with hypocotyl segments of in vitro raised seedlings of Robinia. Parameters important for high efficiency regeneration and transformation rates included type of explant, pre-conditioning of explants and appropriate length of co-cultivation period with Agrobacterium. A transformation frequency 16.67% was obtained by 48 hr of pre-conditioning followed by 48 hr of co-cultivation. Transformed tissue was selected by the ability to grow on kanamycin containing medium. Successful regeneration was followed after histochemical GUS assay for the detection of transgenic tissue. This transformation procedure has the potential to expand the range of genetic variation in Robinia.

Regeneration from mature and immature embryos and transient gene expression via Agrobacterium-mediated transformation in emmer wheat (Triticum dicoccum Schuble).

The present study establishes a regeneration protocol and optimizes conditions for Agrobacterium-mediated transformation of the tetraploid emmer wheat, Triticum dicoccum. Regeneration from mature and immature embryos was accomplished as a two-step process involving callus induction in the presence of 2,4-D followed by regeneration on a 2,4-D free, cytokinin-containing medium (RM1). Higher concentrations of 2,4-D (4 mg/l) though conducive for callusing (89.39% in mature embryos and 96% in immature embryos) proved detrimental for further regeneration. At lower 2,4-D (1 mg/ml) although callusing was suboptimal, (56.8% and 84% from mature and immature embryos, respectively) the regeneration response was the highest on RM1 medium (64.4% and 56.6% from mature and immature embryos, respectively). Overall, the regeneration response of immature embryos was lower than the mature embryos by 10-12%. Due to the ease of availability of mature embryos the mature embryo-derived calli were chosen as the target tissue for Agrobacterium-mediated transformation in the two Indian varieties DDK1001 and DDK1009. Histochemical GUS expression revealed the suitability of the mature embryo-derived calli for such investigations. Of the CaMV35S and Act1 promoters employed, the monocot promoter Act1 displayed higher GUS gene activity in the mature embryo derived calli when co-cultivated with LBA4404 (pBI101::Act1).

Transformation and regeneration of mung bean (Vigna radiata).

The procedure relied on a protocol in which shoot organogenesis was induced on cotyledons of mung bean genotypes selected for susceptibility to agrobacterium seems to work reproducibly if not efficiently. Approximately 4-5% of the shoots produced on the kanamycin selected cotyledons are transgenic based on assays on kanamycin resistance and GUS activity. This demonstrated that transformation and regeneration in mung bean are possible. However, raising the transformed plants in field condition is yet to be perfected.