Biological evaluation of the bone healing process after application of two potentially osteogenic proteins: an animal experimental model

doi: 10.1111/j.1741‐2358.2011.00526.x
Biological evaluation of the bone healing process after application of two potentially osteogenic proteins: an animal experimental model Objective: The aim of this work was to analyse qualitatively and quantitatively the newly formed bone after insertion of rhBMP‐2 and protein extracted from Hevea brasiliensis (P‐1), associated or not with a carrier in critical bone defects created in Wistar rat calvarial bone, using histological and histomorphometrical analyses. Materials and methods: Eighty‐four male Wistar rats were used, divided into two groups, according to the period of time until the sacrifice (2 and 6 weeks). Each one of these groups was subdivided into six groups with seven animals each, according to the treatments: (1) 5 μg of pure rhBMP‐2, (2) 5 μg of rhBMP‐2/monoolein gel, (3) pure monoolein gel, (4) 5 μg of pure P‐1, (5) 5 μg of P‐1/monoolein gel and (6) critical bone defect controls. The animals were euthanised and the calvarial bone tissue removed for histological and histomorphometrical analyses. Result and conclusion: The results showed an improvement in the bone healing process using the rhBMP‐2 protein, associated or not with a material carrier in relation to the other groups, and this process demonstrated to be time dependent.     

Expression of ATP6V1C1 during oral carcinogenesis

We investigated the gene and protein expressions of V-type ATPase protein subunit C1 (ATP6V1C1) in cases of oral squamous cell carcinoma (OSCC) and contralateral normal mucosa in smokers, nonsmokers and former smokers. Subjects were separated into five groups of 15: group 1, smokers with OSCC; group 2, normal contralateral mucosa of OSCC patients; group 3, chronic smokers; group 4, former smokers who had stopped smoking 1 year earlier; group 5, individuals who had never smoked. Exfoliative cytology specimens from oral mucosa of smokers, former smokers and nonsmokers showed normal gene and protein expression. We found significantly greater gene expression in the OSCC group than in the nonsmoker groups. No difference in gene expression was observed between normal contralateral mucosa and nonsmoker groups, smoker and nonsmoker groups or former smoker and nonsmoker groups. We observed intense immunostaining for ATP6V1C1 protein in all cases of OSCC and weak or no staining in smoker, former smoker and nonsmoker groups. Significantly greater expression of ATP6V1C1 protein was observed in the OSCC group compared to the other groups, which supports the role of ATP6V1C1 in effecting changes associated with oral cancer. Analysis of the mucosae of chronic smokers, former smokers and the normal contralateral mucosa of patients with OSCC showed unaltered ATP6V1C1 gene and protein expression. Early stages of carcinogenesis, represented by altered epithelium of chronic smokers, had neither gene nor protein alterations as seen in OSCC. Therefore, we infer that the changes in ATP6V1C1 occur during later stages of carcinogenesis. Our preliminary study provides a basis for future studies of using ATP6V1C1 levels for detecting early stage OSCC.     

Novel 1,5-diphenyl-6-substituted 1H-pyrazolo[3,4-d]pyrimidin-4(5H)-ones induced apoptosis in RKO colon cancer cells

Novel 1,5-diphenyl-6-substituted-1H-pyrazolo[3,4-d]pyrimidin-4(5H)-ones were synthesized and characterized. All compounds were screened for their anti-proliferative activities in five different cancer cell lines. The results showed that compounds 7a and 7b comprising aminoguanidino or guanidino moiety at position 6 inhibited proliferation of RKO colon cancer cells with IC₅₀ of 8 and 4?μM, respectively. Compounds 7a and 7b induced apoptosis in RKO cells, which was confirmed by TUNEL and annexin V-FITC assays. Flow cytometric analysis indicated that compounds 7a and 7b arrested RKO cells in the G1 phase and the most active compound 7b increased levels of p53, p21, Bax, ERK1/2 and reduced levels of Bcl2 and Akt. Compound 7b also activates release of cytochrome c, which is consistent with activation of caspase-9. Additionally, compound 7b increased caspase-3 activity and cleaved PARP-1 in RKO cells. Collectively, these findings could establish a molecular basis for the development of new anti-cancer agents.     

SIRAC: Supervised Identification of Regions of Aberration in aCGH datasets

Background  

Array comparative genome hybridization (aCGH) provides information about genomic aberrations. Alterations in the DNA copy number may cause the cell to malfunction, leading to cancer. Therefore, the identification of DNA amplifications or deletions across tumors may reveal key genes involved in cancer and improve our understanding of the underlying biological processes associated with the disease.     

Cardiovascular and lung inflammatory effects induced by systemically administered diesel exhaust particles in rats

Pollution by particulates has consistently been associated with increased cardiorespiratory morbidity and mortality. It has been suggested that ultrafine particles, of which diesel exhaust particles (DEP) are significant contributors, are able to translocate from the airways into the bloodstream in vivo. In the present study, we assessed the effect of systemic administration of DEP on cardiovascular and respiratory parameters. DEP were administered into the tail vein of rats, and heart rate, blood pressure, blood platelet activation, and lung inflammation were studied 24 h later. Doses of 0.02, 0.1, or 0.5 mg DEP/kg (8, 42, or 212 microg DEP/rat) induced a significant decrease of heart rate and blood pressure compared with saline-treated rats. Although the number of platelets was not affected, all the doses of DEP caused a shortening of the bleeding time. Similarly, in addition to triggering lung edema, the bronchoalveolar lavage analysis revealed the presence of neutrophil influx in DEP-treated rats in a dose-dependent manner. We conclude that the presence of DEP in the systemic circulation leads not only to cardiovascular and haemostatic changes but it also triggers pulmonary inflammation.     

Genome-wide profiling of DNA methylation reveals a class of normally methylated CpG island promoters

The role of CpG island methylation in normal development and cell differentiation is of keen interest, but remains poorly understood. We performed comprehensive DNA methylation profiling of promoter regions in normal peripheral blood by methylated CpG island amplification in combination with microarrays. This technique allowed us to simultaneously determine the methylation status of 6,177 genes, 92% of which include dense CpG islands. Among these 5,549 autosomal genes with dense CpG island promoters, we have identified 4.0% genes that are nearly completely methylated in normal blood, providing another exception to the general rule that CpG island methylation in normal tissue is limited to X inactivation and imprinted genes. We examined seven genes in detail, including ANKRD30A, FLJ40201, INSL6, SOHLH2, FTMT, C12orf12, and DPPA5. Dense promoter CpG island methylation and gene silencing were found in normal tissues studied except testis and sperm. In both tissues, bisulfite cloning and sequencing identified cells carrying unmethylated alleles. Interestingly, hypomethylation of several genes was associated with gene activation in cancer. Furthermore, reactivation of silenced genes could be induced after treatment with a DNA demethylating agent or in a cell line lacking DNMT1 and/or DNMT3b. Sequence analysis identified five motifs significantly enriched in this class of genes, suggesting that cis-regulatory elements may facilitate preferential methylation at these promoter CpG islands. We have identified a group of non-X-linked bona fide promoter CpG islands that are densely methylated in normal somatic tissues, escape methylation in germline cells, and for which DNA methylation is a primary mechanism of tissue-specific gene silencing.     

Lipid composition of spermatozoa in normozoospermic and asthenozoospermic males

INTRODUCTION: Lipids play an important role in the structural and functional activity of spermatozoa. We investigated the phospholipids composition and fatty acid-bound phospholipid of spermatozoa from asthenozoospermic and normozoospermic men. PATIENTS AND METHODS: Semen samples were analyzed in 15 asthenozoospermic and eight normozoospermic subjects and the sperm phospholipids and fatty acids were determined using high performance thin layer chromatography and gas chromatography, respectively. RESULTS: The most abundant (mean+/-SE) phospholipids in normozoospermic and asthenozoospermic samples were phosphatidylethanolamine (70.9+/-11.5 and 44.2+/-8.5 nmol/10(8) spermatozoa, respectively) and phosphatidylcholine (58.6+/-9.5 and 34.6+/-3.2 nmol/10(8) spermatozoa, respectively). Compared to normozoospermic samples, asthenozoospermic samples showed lower levels of polyunsaturated fatty acids (PUFA; p<0.01) and higher levels of saturated fatty acids (SFA; p<0.05). DISCUSSION: Changes in content of phospholipids and its fatty acid composition of spermatozoa may be related to infertility in asthenozoospermic males.     

SA-hIL2双功能融合蛋白的研制及其生物学鉴定

目的：制备链亲和素标记的人白细胞介素-2(SA-hIL2)融合蛋白,并研究其生物学功能。方法：构建SA-L-IL2-pET24重组表达质粒,在大肠杆菌中表达SA-hIL2融合蛋白,对表达的SA-hIL2融合蛋白采用镍金属螯合（Ni-NTA）层析柱进行纯化,透析复性。CCK-8法检测SA-hIL2融合蛋白对PHA刺激的人外周血淋巴细胞的增值活性,流式细胞仪分析SA-hIL2融合蛋白对生物素化的B16.F10肿瘤细胞表面锚定修饰效率。结果：SA-hIL2在大肠杆菌中实现了高效表达,约占菌体总蛋白的20％,制备的SA-hIL2融合蛋白纯度达到95％,并具有双重活性,即hIL-2促进PHA刺激的人外周血淋巴细胞的增值活性和SA介导的高效结合至已生物素化的B16.F10肿瘤细胞表面的功能（表面锚定修饰效率约95％）。结论：研制的SA-hIL2融合蛋白具有双重活性,可为研制表面修饰的新型肿瘤细胞疫苗提供基础。     

Depression comorbidity among patients with tuberculosis in a university teaching hospital outpatient clinic in Nigeria

Background Tuberculosis (TB) remains a leading infectious cause of morbidity and mortality throughout the world. Medication non-compliance has been recognised as one of the drawbacks in the successful management of this disease. Hence, different approaches for ensuring medication compliance have been adopted; these include the Directly Observed Therapy Short course (DOTS). TB is associated with psychiatric morbidity, particularly depressive disorder, and this has been recognised as a cause of poor compliance and a cause of increased morbidity and mortality from the disease. Despite this recognition, little attention is paid to the identification of depression among TB patients, particularly in the DOTS clinics that most of these patients attend. This study was designed to determine the prevalence of depression in patients with TB attending the DOTS outpatient clinic in a university teaching hospital in Nigeria, and to find out the factors that may be associated with this.Method All consenting TB patients attending the clinic completed a socio-demographic questionnaire and nine-item Patient Health Questionnaire (PHQ-9) designed to screen for depression, especially in outpatient and primary care settings.Results Sixty-five patients participated in the study of whom 41 (63.1%) were males. The mean age of the respondents was 35.1 ± 14.4 (range 15-70 years). Eighteen (27.7%) of the patients had depression, comprising 14 (21.5%) with mild depression and four (6.2%) with moderate depression. Socio-demographic factors (age groups, P=0.024; and financial status, P=0.02) and a clinical factor (persistent cough, P=0.04) were significantly associated with depression.Conclusion Measures to reduce depression among patients with TB should include effective symptom control, particularly of coughing, and measures to improve the financial status of this group of patients. Financial empowerment of patients may reduce depression in them, improve the compliance rate to anti-TB medication, and could furthermore bring an improvement to their quality of life.