Molten Globule of Hemoglobin Proceeds into Aggregates and Advanced Glycated End Products

Conformational alterations of bovine hemoglobin (Hb) upon sequential addition of glyoxal over a range of 0–90% v/v were investigated. At 20% v/v glyoxal, molten globule (MG) state of Hb was observed by altered tryptophan fluorescence, high ANS binding, existence of intact heme, native-like secondary structure as depicted by far-UV circular dichroism (CD) and ATR-FTIR spectra as well as loss in tertiary structure as confirmed by near-UV CD spectra. In addition, size exclusion chromatography analysis depicted that MG state at 20% v/v glyoxal corresponded to expanded pre-dissociated dimers. Aggregates of Hb were detected at 70% v/v glyoxal. These aggregates of Hb had altered tryptophan environment, low ANS binding, exposed heme, increased β-sheet secondary structure, loss in tertiary structure, enhanced thioflavin T (ThT) fluorescence and red shifted Congo Red (CR) absorbance. On incubating Hb with 30% v/v glyoxal for 0–20 days, advanced glycation end products (AGEs) were detected on day 20. These AGEs were characterised by enhanced tryptophan fluorescence at 450 nm, exposure of heme, increase in intermolecular β-sheets, enhanced ThT fluorescence and red shift in CR absorbance. Comet assay revealed aggregates and AGEs to be genotoxic in nature. Scanning electron microscopy confirmed the amorphous structure of aggregates and branched fibrils of AGEs. The transformation of α-helix to β-sheet usually alters the normal protein to amyloidogenic resulting in a variety of protein conformational disorders such as diabetes, prion and Huntington''s.     

Lumican is a major proteoglycan component of the bone matrix.

MC3T3-E1 mouse calvaria cells are a clonal population of committed osteoprogenitors that in the presence of appropriate supplements form a mineralized bone matrix. The development of the MC3T3-E1 cells can be divided into three major stages, namely, proliferation, differentiation, and mineralization. Recently, using the cDNA microarray technology we found lumican to be abundantly expressed during the mineralization and differentiation stages of the MC3T3-E1 development and not during the proliferation stage. Lumican has been shown to play essential roles in regulating collagen fibril formation in different extracellular matrices but its expression in the developing bone matrix remains elusive. By examining the expression profile of this gene during the different stages of MC3T3-E1 development, utilizing the 'real-time' PCR technology, we observed that the expression of lumican increases as the osteoblast culture differentiates and matures, suggesting that lumican may be involved in regulating collagen fibrillogenesis in bone matrices. Using immunostaining, we observed that during the early embryonic development of mouse (E11 to E13), lumican is mainly expressed in the cartilaginous matrices. However, in the older embryos (E14 to E16), the expression of lumican is more prominent in the developing bone matrices. Our data suggest that lumican is a significant proteoglycan component of bone matrix, which is secreted by differentiating and mature osteoblasts only and therefore it can be used as a marker to distinguish proliferating pre-osteoblasts from the differentiating osteoblasts.     

Characterisation of Ilomastat for Prolonged Ocular Drug Release

We are developing tablet dosage forms for implantation directly into the subconjunctival space of the eye. The matrix metalloproteinase inhibitor, ilomastat, has previously been shown to be efficacious at suppressing scarring following glaucoma filtration surgery (GFS). We report on the physical characterisation of ilomastat which is being developed for ocular implantation. Since ilomastat is being considered for implantation it is necessary to examine its polymorphs and their influence on aspects of the in vitro drug release profile. X-ray powder diffraction identified two polymorphs of ilomastat from different commercial batches of the compound. Tablets were prepared from the two different polymorphs. Isothermal perfusion calorimetry was used to show that amorphous content is not increased during tablet formulation. The melting points of the two polymorphs are 188 and 208°C as determined by differential scanning calorimetry. Utilising single crystal X-ray diffraction, the structural conformations and packing arrangements of the different polymorphs were determined. The orthorhombic crystal crystallised as a monohydrate while the second monoclinic crystal form is non-solvated. Ilomastat tablets prepared from the two different solid forms exhibited similar drug release profiles in vitro under conditions mimicking the aqueous composition, volume and flow of the subconjunctival space after GFS. This suggests that a reproducible dose at each time point during release after implantation should be achievable in vivo with ilomastat tablets prepared from the two polymorphs identified.     

Trifluoroethanol and acetonitrile induced formation of the molten globule states and aggregates of cellulase

A systematic investigation on the effects of trifluoroethanol and acetonitrile at various concentrations on cellulase (EC 3.2.1.4) was studied by enzyme assay, intrinsic fluorescence, ANS binding, circular dichroism and ATR-Fourier transform infra red spectroscopy. The results show the presence of molten globule states at 3% (v/v) TFE and 80% (v/v) ACN. Cellulase aggregates at 25% (v/v) TFE and 90% (v/v) ACN, as detected by decrease in intrinsic and ANS fluorescence, loss in tertiary structure and enzyme activity, increase non-native β-sheet structure as evident from far-UV CD and FTIR spectra, increase in Thioflavin T fluorescence and shift in Congo red assay.     

Synthesis and evaluation of a selective molecularly imprinted polymer for the contraceptive drug levonorgestrel

A molecularly imprinted polymer (MIP) has been prepared using levonorgestrel (LEV) as template. The polymer was synthesised in a non-covalent approach using methacrylic acid (MAA) as functional monomer and ethylene glycol dimethacrylate (EGDMA) as cross-linking monomer via a free radical polymerization. An equivalent blank polymer was also synthesised in the absence of the template compound. Batch adsorption experiments were used to evaluate the binding affinity of the imprinted polymer. After packing MIP into a stainless steel column (150 mm x 4.6 mm i.d.), retention and elution of the template and related compounds were evaluated by high-performance liquid chromatography (HPLC). This LEV imprinted polymer was further applied for selective solid phase extraction (SPE) of LEV from human serum. It was confirmed that the binding ability of the prepared MIP for LEV was essentially sufficient in the presence of other compounds coexisting in serum sample. Therefore, as a selective and efficient solid phase material, LEV imprinted polymer has a high potential application in analysis of this steroidal hormone in clinical purposes.     

Narrow genetic basis for the Australian dingo confirmed through analysis of paternal ancestry

The dingo (Canis lupus dingo) is an iconic animal in the native culture of Australia, but archaeological and molecular records indicate a relatively recent history on the continent. Studies of mitochondrial DNA (mtDNA) imply that the current dingo population was founded by a small population of already tamed dogs from Southeast Asia. However, the maternal genetic data might give a unilateral picture, and the gene pool has yet to be screened for paternal ancestry. We sequenced 14,437?bp of the Y-chromosome (Y-chr) from two dingoes and one New Guinea Singing Dog (NGSD). This positioned dingo and NGSD within the domestic dog Y-chr phylogeny, and produced one haplotype not detected before. With this data, we characterized 47 male dingoes in 30 Y-chr single-nucleotide polymorphism sites using protease-mediated allele-specific extension technology. Only two haplotypes, H3 and H60, were found among the dingoes, at frequencies of 68.1 and 31.9?%, respectively, compared to 27 haplotypes previously established in the domestic dog. While H3 is common among Southeast Asian dogs, H60 was specifically found in dingoes and the NGSD, but was related to Southeast Asian dog Y-chr haplotypes. H3 and H60 were observed exclusively in the western and eastern parts of Australia, respectively, but had a common range in Southeast. Thus, the Y-chr diversity was very low, similar to previous observations for d-loop mtDNA. Overall genetic evidence suggests a very restricted introduction of the first dingoes into Australia, possibly from New Guinea. This study further confirms the dingo as an isolated feral dog.     

Copper association with iron sulfide magnetosomes in a magnetotactic bacterium

Greigite (Fe₃S₄) and pyrite (FeS₂) particles in the magnetosomes of a many-celled, magnetotactic prokaryote (MMP), common in brackish-to-marine, sulfidic, aquatic habitats, contained relatively high concentrations of copper which ranged from about 0.1 to 10 atomic per cent relative to iron. In contrast, the greigite particles in the magnetosomes of a curved magnetotactic bacterium collected from the same sampling site did not contain significant levels of copper. The ability of the MMP to biomineralize copper within its magnetosomes appeared to be limited to that organism and dependent upon the site from which it was collected. Although the chemical mechanism and physiological function of copper accumulation in the magnetosomes of the MMP is unclear, the presence of copper is the first evidence that another transition metal ion could be incorporated in the mineral phase of the magnetosomes of a magnetotactic bacterium.Abbreviation MMP many-celled magnetotactic prokaryote     

Conceptual Method of Temperature Sensation in Bionic Hand by Extraordinary Perceptual Phenomenon

Lack of temperature sensation of myoelectric prosthetic hand limits the daily activities of amputees.To this end,a non-invasive temperature sensation method is proposed to train amputees to sense temperature with psychophysical sensory substitution.In this study,22 healthy participants took part besides 5 amputee participants.The duration time of the study was 31 days with five test steps according to the Leitner technique.An adjustable temperature mug and a Peltier were used to change the temperature of the water/phantom digits to induce temperature to participants.Also,to isolate the surround-ings and show colors,a Virtual Reality(VR)glass was employed.The statistical results conducted are based on the response of participants with questionnaire method.Using Chi-square tests,it is concluded that participants answer the experiment significantly correctly using the Leitner technique(P value＜0.05).Also,by applying the"Repeated Measures ANOVA",it is noticed that the time of numbness felt by participants had significant(P value＜0.001)difference.Participants could remember lowest and highest temperatures significantly better than other temperatures(P value＜0.001);furthermore,the well-trained amputee participant practically using the prosthesis with 72.58％could identify object's temperature with only once time experimenting the color temperature.