Surface behaviour of human pancreatic and gastric lipases

The kinetics of the adsorption of human gastric lipase (HGL) and human pancreatic lipase (HPL) were studied by recording the changes in the surface pressure with time in the absence and presence of an egg phosphatidylcholine (PC) monomolecular film spread at the air/water interface. In the presence of PC film, the tensioactivtty of HGL increased considerably compared with its behaviour at the air/water interface, whereas HPL exhibited a comparable degree of tensioactivity whether or not a phospholipid monolayer was present at the interface. This difference in surface behaviour is consistent with the higher penetration capacity attributed to HGL. Procolipase considerably increased both the initial adsorption rate and the final surface pressure reached by HPL compared with its adsorption without colipase.

The kinetics of the hydrolysis of 1,2-didecanoyl-sn-glycerol (dicaprin) monolayers by HGL and HPL were measured using a “zero-order” trough. The large differences between the calculated characteristic adsorption times and the measured lag times indicate that the partition of the lipase molecules between the subsurface and the interface was probably limited by an energy barrier. The amplitude of this energy barrier can be partly attributed to the drastic conformational change in the enzyme, associated with the interfacial activation.

The area per dicaprin molecule (56 Ų) corresponding to the maximal activity of HPL was compared with the dimension of the hydrophobic cleft surrounding the serine (Ser 152) of the catalytic triad of HPL, as recently demonstrated by H. Van Tilbeurgh and co-workers (Nature, 359 (1992) 159; 362 (1993) 814) in their studies on the “open” and “closed” forms of the respective three-dimensional crystalline structures. The catalytic triad was not accessible to a sphere 8.4 Å in diameter, mimicking the van der Waals envelope of the dicaprin molecule, due to the steric hindrance of the side chains of aromatic and cyclic residues F 215, F 77, Y 114 and H 263. It can be concluded that the substrate molecule must also undergo some conformational changes at the contact of the enzyme to be accommodated in the active site.     

Bioartificial Organs I: Silica Gel Encapsulated Pancreatic Islets for the Treatment of Diabetes Mellitus

The primary objective of this work is to evaluate the potential of silica gel encapsulated pancreatic islets of Langerhans, or islet tissue, as a means by which insulin secretory capacity might be restored to individuals with insulin dependent, type 1, diabetes mellitus. The encapsulation material under investigation is comprised of sol-gel derived silica ceramic that hardens under conditions of pH, salinity, and temperature that are not harmful to living cells and organisms. Preliminary efficacy has been demonstrated by measurement of insulin secretory response of silica gel encapsulated pancreatic isletsin vitro and blood sugar levels of nonobese diabetic micein vivo.     

Antimycobacterial and anticancer activity of newly designed cinnamic acid hydrazides with favorable toxicity profile

A series of twelve novel hybrids of cinnamic acid and thiocarbohydrazones were designed, synthesized in high yield using a simple coupling strategy via acid chlorides, and evaluated for their impact against Mycobacterium tuberculosis (Mtb) and cancer cells survival. Among them, compound 3 demonstrated strong anti-Mtb activity by reducing bacilli survival for>90 % in all three treated Mtb isolates, whereas isoniazid and rifampicin did not. Moreover, compound 3 didn’t affect vitality of HepG-2 cells, implying on advantageous hepatotoxicity profile compared to current therapeutic options for tuberculosis. Compounds 2a and 3b displayed as strong inducers of apoptosis in A549 cells, both activating intrinsic caspase pathway and cell cycle arrest at the G0/G1 phase. Subsequent analyses disclosed differences in their activities, where 3b has ability to induce production of mitochondrial superoxide anions, while 2a significantly inhibited cellular mobility. More importantly, 3b considerably affected viability of HepG-2 and HaCaT cells, whereas 2a had moderate impact only on the later. Molecular modeling studies indicated high permeability and good absorption through the human intestine, and moderate aqueous solubility with poor blood–brain barrier permeability. In summary, our results reveal that novel compounds 3 and 2a represent promising agents for tuberculosis and cancer treatment, respectively, indicating that further investigation needs to be performed to clarify the mechanisms of their anti-Mtb and anticancer activity.     

Molecular modeling and dynamics of neuropeptide Y

Summary A combination of molecular modeling and molecular dynamics (MD) is used to determine a theoretical structure for neuropeptide Y (NPY). Starting with the X-ray structure for avian pancreatic polypeptide (APP), the substituted amino acids were mutated, the side chains oriented to local potential energy minima, and the entire structure minimized and subjected to an MD simulation. Comparison of the resulting NPY structure with APP X-ray and MD results showed secondary structural elements to be maintained and RMS fluctuations to be similar, although differences in both were observed. The approach presented offers a means to study the structure-function relationships of NPY and other similar polypeptides when combined with pharmacological measurements.Abbreviations NPY Neuropeptide Y - APP Avian pancreatic polypeptide - ABNR Adopted-basis Newton Raphson - MD Molecular dynamics     

人卵巢癌耐药细胞株的比较蛋白质组分析

本文采用高分辨二维凝胶电泳分离技术对人卵巢癌细胞株COC1及其耐药细胞株COC1/DDP中的蛋白质进行分离和差异表达分析, 应用基质辅助激光解吸电离-飞行时间质谱对酶解多肽进行测定[即测定蛋白质的肽质量指纹图(Peptide mass fingerprinting, PMF)], 并通过相应的数据库搜索来鉴定蛋白质. 为获得更准确的检索结果, 采用串联质谱技术对各肽段进行氨基酸测序, 并应用IPI-HUMAN数据库对上述检索结果进一步加以确认.      

Synthesis of poly(ether esters) from reaction of alpha-cyano-4-hydroxycinnamic acid and group IVB metallocenes

Poly(ether esters) are rapidly synthesized in moderate yield employing the interfacial polycondensation reaction system from the reaction of alpha-cyano-4-hydroxycinnamic acid and Group IVB metallocenes. The products are high polymers. Infrared spectroscopy shows the formation of new bands derived from the M-O and M-O(CO) linkages. It also shows that the products exist as alternating M-O and M-O(CO) linkages. The products show outstanding inhibition of a variety of cancer cell lines including two pancreatic cancer cell lines. EC₅₀ values for the polymers are in the nanogram/mL range. The ability to inhibit the cancer cell lines is generally Hf>Zr>Ti. Thus, future synthesis and testing might consider using compounds containing hafnocene and zirconocene in addition to the titanocene moiety.     

In vivo metabolic investigation of cetilistat in normal versus pseudo-germ-free rats using UPLC-QTOFMS/MS and in silico toxicological evaluation of its metabolites

Cetilistat (CET) is a pancreatic lipase inhibitor approved for management of obesity after the serious adverse effects exhibited by its analogue orlistat. Exhaustive literature review reveals lack of comprehensive reports on its biotransformation. With a view to study the same, the present study reports the identification and characterization of metabolites of CET in rats using UPLC–MS/MS. As the small intestine is the site of action for CET, it is important that the role of microbial flora in the metabolism of CET be explored. To achieve this, the metabolic profile of CET was compared between normal and pseudo-germ-free rats. The study involved the administration of a drug suspension to male Sprague–Dawley pseudo-germ-free and normal untreated rats followed by collection of urine, feces, and blood at specific intervals. Sample preparation was performed using liquid–liquid extraction and concentration of samples followed by analysis using LC–MS/MS. Finally, an in silico study was performed on the drug and metabolites to predict their toxicological properties using ADMET Predictor^TM software. Four metabolites of CET were observed in in vivo matrices. As expected, significant changes were observed both qualitatively and quantitatively, implying that formation of metabolites was both CYP enzymes and gut microflora mediated.     

Pressure-tuning of the secondary structures of proteins: Fourier transform infrared studies in the diamond anvil cell

Abstract

A self-deconvolution and fitting procedure is presented that allows the determination of pressure-induced changes in the secondary structure of proteins starting from the infrared spectrum. The method takes into account the elastic as well as the possible conformational shift of the spectral bands. Applications are presented on pressure-induced changes in bovine pancreatic trypsin inhibitor and in gramicidin incorporated into lipid bilayers.     

The impact of adsorption of bovine pancreatic trypsin inhibitor on CTAB‐protected gold nanoparticle arrays: a Raman spectroscopic comparison with solution denaturation

The influence of an array of cetyltrimethylammonium bromide (CTAB)‐protected gold nanoparticles on the structure of a model protein, bovine pancreatic trypsin inhibitor (BPTI) at pH 7.4, was studied by Raman spectroscopy. The structural consequences of array adsorption were compared with the effects of deposition of the protein directly onto a roughened gold substrate and with thermal and reductive treatment of BPTI in solution. Both thermal and reductive denaturation in solution result in loss of α‐helix structure, with an increase in random conformations of the protein in the case of reductive denaturation and β‐sheet conformation and random coil on thermal denaturation. For reductive denaturation in particular, extensive loss of secondary structure is evident. Deposition of the protein onto the array resulted in increased β‐sheet conformation similar to that observed on thermal treatment of the protein. However, unlike denaturation, which for both thermal and reductive process resulted in changes in the disulfide stretching wavenumber, this remains largely unchanged on application of the protein to the array. Furthermore, deposition of the protein onto bare gold results in significant heterogeneity in the S S stretching signal with appearance of ggt and nonequilibrium geometry of the CCSSCC dihedral angles. Thermal denaturation results in a red shift of the SS mode, whereas dithiothreitol (DTT) treatment, as expected, induces significant loss of the S S stretching signal, although a signal at 517 cm⁻¹ remains suggesting that the unreduced disulfide has changed to the ggt geometry. In addition, an SH mode is observed at 2570 cm⁻¹ in solution. The response of BPTI to thermal and DTT treatment while on the array is very different to its solution behavior, and suggests that adhesion to the array increases the stability of the protein. Copyright © 2009 John Wiley & Sons, Ltd.     

Extracellular matrix alterations in low‐grade lung adenocarcinoma compared with normal lung tissue by imaging mass spectrometry

Lung adenocarcinoma (LUAD) is the second most common cancer, affecting both men and women. Fibrosis is a hallmark of LUAD occurring throughout progression with excess production of extracellular matrix (ECM) components that lead to metastatic cell processes. Understanding the ECM cues that drive LUAD progression has been limited due to a lack of tools that can access and report on ECM components within the complex tumor microenvironment. Here, we test whether low‐grade LUAD can be distinguished from normal lung tissue using a novel ECM imaging mass spectrometry (ECM IMS) approach. ECM IMS analysis of a tissue microarray with 20 low‐grade LUAD tissues and 20 normal lung samples from 10 patients revealed 25 peptides that could discriminate between normal and low‐grade LUAD using area under the receiver‐operating curve (AUC) ≥0.7, P value ≤.001. Principal component analysis demonstrated that 62.4% of the variance could be explained by sample origin from normal or low‐grade tumor tissue. Additional work performed on a wedge resection with moderately differentiated LUAD demonstrated that the ECM IMS analytical approach could distinguish LUAD spectral features from spectral features of normal adjacent lung tissue. Conventional liquid chromatography with tandem mass spectrometry (LC‐MS/MS) proteomics demonstrated that specific sites of hydroxylation of proline (HYP) were a main collagen post translational modification that was readily detected in LUAD. A distinct peptide from collagen 3A1 modified by HYP was increased 3.5 fold in low‐grade LUAD compared with normal lung tissue (AUC 0.914, P value <.001). This suggests that regulation of collagen proline hydroxylation could be an important process during early LUAD fibrotic deposition. ECM IMS is a useful tool that may be used to define fibrotic deposition in low‐grade LUAD.