Other Publications (10)
- The FEBS Journal
- The Journal of Biological Chemistry
- Proceedings of the National Academy of Sciences of the United States of America
- International Journal of Cancer. Journal International Du Cancer
- Acta Neuropathologica
- Journal of Neuroscience Methods
- Neoplasia (New York, N.Y.)
- Journal of Clinical Microbiology
- Brain : a Journal of Neurology
- Current Microbiology
Articles by Mohammad R. Hasan in JoVE
Perfusão intravenosa de tinta Carbon Black Permite visualização dos vasos cerebrais Mohammad R. Hasan1, Josephine Herz1, Dirk M. Hermann1, Thorsten R. Doeppner1 1Department of Neurology, University of Duisburg-Essen Medical School Análise da anatomia cerebrovascular roedor desempenha um papel importante na investigação do acidente vascular cerebral experimental. Neste contexto, a perfusão intravascular com látex corado tem sido considerada como um instrumento padrão para vários anos. No entanto, esta técnica implica limitações técnicas distintas, que minam a sua reprodutibilidade. Aqui, nós descrevemos um método simples para visualizar os vasos cerebrais, de uma forma reprodutível. A injecção de uma mistura de duas tintas pretas de carbono disponíveis comercialmente, através dos resultados do ventrículo esquerdo do miocárdio, em enchimento adequado de vasos cerebrais com visualização de alto contraste. Nós temos aplicado com sucesso esta técnica para identificar os pontos de anastomose entre territórios vasculares cerebrais de ratos com diferentes origens genéticas. Finalmente dar provas de que este método novo e simples para a coloração de navio podem ser combinados com cloreto de trifeniltetrazólio (TTC) de coloração - uma ferramenta amplamente utilizada para observar e analisar volumes do enfarte em ratos.
Other articles by Mohammad R. Hasan on PubMed
Identification of a 250 KDa Putative Microtubule-associated Protein As Bovine Ferritin. Evidence for a Ferritin-microtubule Interaction The FEBS Journal. Feb, 2005 | Pubmed ID: 15670162 We reported previously on the purification and partial characterization of a putative microtubule-associated protein (MAP) from bovine adrenal cortex with an approximate molecular mass of 250 kDa. The protein was expressed ubiquitously in mammalian tissues, and bound to microtubules in vitro and in vivo, but failed to promote tubulin polymerization into microtubules. In the present study, partial amino acid sequencing revealed that the protein shares an identical primary structure with the widely distributed iron storage protein, ferritin. We also found that the putative MAP and ferritin are indistinguishable from each other by electrophoretic mobility, immunological properties and morphological appearance. Moreover, the putative MAP conserves the iron storage and incorporation properties of ferritin, confirming that the two are structurally and functionally the same protein. This fact led us to investigate the interaction of ferritin with microtubules by direct electron microscopic observations. Ferritin was bound to microtubules either singly or in the form of large intermolecular aggregates. We suggest that the formation of intermolecular aggregates contributes to the intracellular stability of ferritin. The interactions between ferritin and microtubules observed in this study, in conjunction with the previous report that the administration of microtubule depolymerizing drugs increases the serum release of ferritin in rats [Ramm GA, Powell LW & Halliday JW (1996) J Gastroenterol Hepatol11, 1072-1078], support the probable role of microtubules in regulating the intracellular concentration and release of ferritin under different physiological circumstances.
Ferritin Contains Less Iron (59Fe) in Cells when the Protein Pores Are Unfolded by Mutation The Journal of Biological Chemistry. Nov, 2008 | Pubmed ID: 18805796 Ferric minerals in ferritins are protected from cytoplasmic reductants and Fe2+ release by the protein nanocage until iron need is signaled. Deletion of ferritin genes is lethal; two critical ferritin functions are concentrating iron and oxidant protection (consuming cytoplasmic iron and oxygen in the mineral). In solution, opening/closing (gating) of eight ferritin protein pores controls reactions between external reductant and the ferritin mineral; pore gating is altered by mutation, low heat, and physiological urea (1 mm) and monitored by CD spectroscopy, protein crystallography, and Fe2+ release rates. To study the effects of a ferritin pore gating mutation in living cells, we cloned/expressed human ferritin H and H L138P, homologous to the frog open pore model that was unexpressable in human cells. Human ferritin H L138P behaved like the open pore ferritin model in vitro as follows: (i) normal protein cage assembly and mineralization, (ii) increased iron release (t1/2) decreased 17-fold), and (iii) decreased alpha-helix (8%). Overexpression (> 4-fold), in HeLa cells, showed for ferritin H L138P equal protein expression and total cell 59Fe but increased chelatable iron, 16%, p < 0.01 (59Fe in the deferoxamine-containing medium), and decreased 59Fe in ferritin, 28%, p < 0.01, compared with wild type. The coincidence of decreased 59Fe in open pore ferritin with increased chelatable 59Fe in cells expressing the ferritin open pore mutation suggests that ferritin pore gating influences to the amount of iron (59Fe) in ferritin in vivo.
The Ferritin Fe2 Site at the Diiron Catalytic Center Controls the Reaction with O2 in the Rapid Mineralization Pathway Proceedings of the National Academy of Sciences of the United States of America. Nov, 2008 | Pubmed ID: 19011101 Oxidoreduction in ferritin protein nanocages occurs at sites that bind two Fe(II) substrate ions and O(2), releasing Fe(III)(2)-O products, the biomineral precursors. Diferric peroxo intermediates form in ferritins and in the related diiron cofactor oxygenases. Cofactor iron is retained at diiron sites throughout catalysis, contrasting with ferritin. Four of the 6 active site residues are the same in ferritins and diiron oxygenases; ferritin-specific Gln(137) and variable Asp/Ser/Ala(140) substitute for Glu and His, respectively, in diiron cofactor active sites. To understand the selective functions of diiron substrate and diiron cofactor active site residues, we compared oxidoreductase activity in ferritin with diiron cofactor residues, Gln(137) --> Glu and Asp(140) --> His, to ferritin with natural diiron substrate site variations, Asp(140), Ser(140), or Ala(140). In Gln(137) --> Glu ferritin, diferric peroxo intermediates were undetectable; an altered Fe(III)-O product formed, DeltaA(350) = 50% of wild type. In Asp(140) --> His ferritin, diferric peroxo intermediates were also undetectable, and Fe(II) oxidation rates decreased 40-fold. Ferritin with Asp(140), Ser(140), or Ala(140) formed diferric peroxo intermediates with variable kinetic stabilities and rates: t(1/2) varied 1- to 10-fold; k(cat) varied approximately 2- to 3-fold. Thus, relatively small differences in diiron protein catalytic sites determine whether, and for how long, diferric peroxo intermediates form, and whether the Fe-active site bonds persist throughout the reaction cycle (diiron cofactors) or break to release Fe(III)(2)-O products (diiron substrates). The results and the coding similarities for cofactor and substrate site residues-e.g., Glu/Gln and His/Asp pairs share 2 of 3 nucleotides-illustrate the potential simplicity of evolving active sites for diiron cofactors or diiron substrates.
Inhibition of VEGF Induces Cellular Senescence in Colorectal Cancer Cells International Journal of Cancer. Journal International Du Cancer. Nov, 2011 | Pubmed ID: 21618508 Vascular endothelial growth factor (VEGF) inhibitors, such as bevacizumab, have improved outcomes in metastatic colorectal cancer (CRC). Recent studies have suggested that VEGF can delay the onset of cellular senescence in human endothelial cells. As VEGF receptors are known to be upregulated in CRC, we hypothesized that VEGF inhibition may directly influence cellular senescence in this disease. In our study, we observed that treatment with bevacizumab caused a significant increase (p < 0.05) in cellular senescence in vitro in several CRC cells, such as MIP101, RKO, SW620 and SW480 cells, compared to untreated or human IgG-treated control cells. Similar results were also obtained from cells treated with a VEGFR2 kinase inhibitor Ki8751. In vivo, cellular senescence was detected in MIP101 tumor xenografts from 75% of mice treated with bevacizumab, while cellular senescence was undetectable in xenografts from mice treated with saline or human IgG (p < 0.05). Interestingly, we also observed that the proportion of senescent cells in colon cancer tissues obtained from patients treated with bevacizumab was 4.4-fold higher (p < 0.01) than those of untreated patients. To understand how VEGF inhibitors may regulate cellular senescence, we noted that among the two important regulators of senescent growth arrest of tumor cells, bevacizumab-associated increase in cellular senescence coincided with an upregulation of p16 but appeared to be independent of p53. siRNA silencing of p16 gene in MIP101 cells suppressed bevacizumab-induced cellular senescence, while silencing of p53 had no effect. These findings demonstrate a novel antitumor activity of VEGF inhibitors in CRC, involving p16.
Enhancement of Endogenous Neurogenesis in Ephrin-B3 Deficient Mice After Transient Focal Cerebral Ischemia Acta Neuropathologica. Oct, 2011 | Pubmed ID: 21779764 Cerebral ischemia stimulates endogenous neurogenesis. However, the functional relevance of this phenomenon remains unclear because of poor survival and low neuronal differentiation rates of newborn cells. Therefore, further studies on mechanisms regulating neurogenesis under ischemic conditions are required, among which ephrin-ligands and ephrin-receptors (Eph) are an interesting target. Although Eph/ephrin proteins like ephrin-B3 are known to negatively regulate neurogenesis under physiological conditions, their role in cerebral ischemia is largely unknown. We therefore studied neurogenesis, brain injury and functional outcome in ephrin-B3(-/-) (knockout) and ephrin-B3(+/+) (wild-type) mice submitted to cerebral ischemia. Induction of stroke resulted in enhanced cell proliferation and neuronal differentiation around the lesion site of ephrin-B3(-/-) compared to ephrin-B3(+/+) mice. However, prominent post-ischemic neurogenesis in ephrin-B3(-/-) mice was accompanied by significantly increased ischemic injury and motor coordination deficits that persisted up to 4Â weeks. Ischemic injury in ephrin-B3(-/-) mice was associated with a caspase-3-dependent activation of the signal transducer and activator of transcription 1 (STAT1). Whereas inhibition of caspase-3 had no effect on brain injury in ephrin-B3(+/+) animals, infarct size in ephrin-B3(-/-) mice was strongly reduced, suggesting that aggravated brain injury in these animals might involve a caspase-3-dependent activation of STAT1. In conclusion, post-ischemic neurogenesis in ephrin-B3(-/-) mice is strongly enhanced, but fails to contribute to functional recovery because of caspase-3-mediated aggravation of ischemic injury in these animals. Our results suggest that ephrin-B3 might be an interesting target for overcoming some of the limitations of further cell-based therapies in stroke.
Visualization of Macroscopic Cerebral Vessel Anatomy--a New and Reliable Technique in Mice Journal of Neuroscience Methods. Mar, 2012 | Pubmed ID: 22155382 Visualizing rodent cerebral vasculature is an important tool in experimental stroke research. Intravascular perfusion with colored latex has been the method of choice until recently. However, latex perfusion has some technical limitations which compromise its reproducibility. We therefore describe a simple and reproducible method to visualize cerebral vessels in mice. A mixture of two commercially available carbon black inks is injected into the thoracic aorta resulting in efficient filling and high contrast visualization of cerebral vessels. Feasibility of this technique has been validated by identifying anastomotic points between anterior and middle cerebral arteries. Furthermore, perfusion with combined carbon inks allows visualization of significantly smaller vessel diameters at a higher vessel density in comparison to perfusion with diluted/undiluted latex. Thus, perfusion with combined carbon inks offers a simple, cost-effective and reproducible technique in order to visualize cerebral vasculature.
Inhibition of COX-2 in Colon Cancer Modulates Tumor Growth and MDR-1 Expression to Enhance Tumor Regression in Therapy-refractory Cancers in Vivo Neoplasia (New York, N.Y.). Jul, 2012 | Pubmed ID: 22904679 Higher cyclooxygenase 2 (COX-2) expression is often observed in aggressive colorectal cancers (CRCs). Here, we attempt to examine the association between COX-2 expression in therapy-refractory CRC, how it affects chemosensitivity, and whether, in primary tumors, it is predictive of clinical outcomes. Our results revealed higher COX-2 expression in chemoresistant CRC cells and tumor xenografts. In vitro, the combination of either aspirin or celecoxib with 5-fluorouracil (5-FU) was capable of improving chemosensitivity in chemorefractory CRC cells, but a synergistic effect with 5-FU could only be demonstrated with celecoxib. To examine the potential clinical significance of these observations, in vivo studies were undertaken, which also showed that the greatest tumor regression was achieved in chemoresistant xenografts after chemotherapy in combination with celecoxib, but not aspirin. We also noted that these chemoresistant tumors with higher COX-2 expression had a more aggressive growth rate. Given the dramatic response to a combination of celecoxib + 5-FU, the possibility that celecoxib may modulate chemosensitivity as a result of its ability to inhibit MDR-1 was examined. In addition, assessment of a tissue microarray consisting of 130 cases of CRCs revealed that, in humans, higher COX-2 expression was associated with poorer survival with a 68% increased risk of mortality, indicating that COX-2 expression is a marker of poor clinical outcome. The findings of this study point to a potential benefit of combining COX-2 inhibitors with current regimens to achieve better response in the treatment of therapy-refractory CRC and in using COX-2 expression as a prognostic marker to help identify individuals who would benefit the greatest from closer follow-up and more aggressive therapy.
Short-term Stability of Pathogen-specific Nucleic Acid Targets in Clinical Samples Journal of Clinical Microbiology. Dec, 2012 | Pubmed ID: 23052319 The stability of pathogen-specific DNA or RNA amplification targets in clinical samples following short-term storage at room temperature, 4Â°C, and -80Â°C was assessed by real-time PCR. In purified nucleic acid extracts, both DNA and RNA targets were stable for up to 30 days, irrespective of storage temperature. In unextracted samples, temperature-dependent loss of targets (P < 0.05) was observed in serum and cerebrospinal fluid specimens, while no changes were observed for EDTA blood samples.
The Novel Proteasome Inhibitor BSc2118 Protects Against Cerebral Ischaemia Through HIF1A Accumulation and Enhanced Angioneurogenesis Brain : a Journal of Neurology. Nov, 2012 | Pubmed ID: 23169919 Only a minority of stroke patients receive thrombolytic therapy. Therefore, new therapeutic strategies focusing on neuroprotection are under review, among which, inhibition of the proteasome is attractive, as it affects multiple cellular pathways. As proteasome inhibitors like bortezomib have severe side effects, we applied the novel proteasome inhibitor BSc2118, which is putatively better tolerated, and analysed its therapeutic potential in a mouse model of cerebral ischaemia. Stroke was induced in male C57BL/6 mice using the intraluminal middle cerebral artery occlusion model. BSc2118 was intrastriatally injected 12 h post-stroke in mice that had received normal saline or recombinant tissue-plasminogen activator injections during early reperfusion. Brain injury, behavioural tests, western blotting, MMP9 zymography and analysis of angioneurogenesis were performed for up to 3 months post-stroke. Single injections of BSc2118 induced long-term neuroprotection, reduced functional impairment, stabilized blood-brain barrier through decreased MMP9 activity and enhanced angioneurogenesis when given no later than 12 h post-stroke. On the contrary, recombinant tissue-plasminogen activator enhanced brain injury, which was reversed by BSc2118. Protein expression of the transcription factor HIF1A was significantly increased in saline-treated and recombinant tissue-plasminogen activator-treated mice after BSc2118 application. In contrast, knock-down of HIF1A using small interfering RNA constructs or application of the HIF1A inhibitor YC1 (now known as RNA-binding motif, single-stranded-interacting protein 1 (RBMS1)) reversed BSc2118-induced neuroprotection. Noteworthy, loss of neuroprotection after combined treatment with BSc2118 and YC1 in recombinant tissue-plasminogen activator-treated animals was in the same order as in saline-treated mice, i.e. reduction of recombinant tissue-plasminogen activator toxicity through BSc2118 did not solely depend on HIF1A. Thus, the proteasome inhibitor BSc2118 is a promising new candidate for stroke therapy, which may in addition alleviate recombinant tissue-plasminogen activator-induced brain toxicity.
Optimal Use of MRSASelect and PCR to Maximize Sensitivity and Specificity of MRSA Detection Current Microbiology. Jan, 2013 | Pubmed ID: 23053488 Suspected colonies of methicillin-resistant Staphylococcus aureus (MRSA) on chromogenic, MRSASelect (BioRad) medium were confirmed using routine microbiological methods, and a multiplex real-time PCR (nÂ =Â 108). Although the specificity of MRSASelect assessed at 24Â h of incubation was much higher than that of 48Â h (91.4 vs. 60Â %), extending the incubation time to 48Â h, along with PCR confirmation, increased the total number of true positive samples by 27.8Â %. These results provide a cost effective method for sensitive and specific detection of MRSA.