Synthesis of benzoyl phenyl benzoates as effective inhibitors for phospholipase A2 and hyaluronidase enzymes

Benzoylation of (hydroxy phenyl) phenyl methanone 2a-g to benzoyl phenyl benzoates 4a-g, a benzophenone analogue, was achieved in good yield. All the newly synthesized compounds were evaluated for their phospholipase A2 [E.C. 3.1.1.4] and hyaluronidase [E.C. 3.2.1.35] enzyme inhibitory activity in snake venom as source and their structure-activity relationship with respect to different groups is reported for the first time. The in vitro PLA2 enzyme inhibitory activity and in vivo anti-inflammatory activity studies of benzoyl phenyl benzoates are illustrated.     

Y-chromosome and mitochondrial DNA studies on the population structure of the Christmas Island community

Christmas Island is a remote Australian territory located close to the main Indonesian island of Java. Y-chromosome and mitochondrial DNA (mtDNA) markers were used to investigate the genetic structure of the population, which comprises communities of mixed ethnic origin. Analysis of 12 Y-chromosome biallelic polymorphisms revealed a high level of gene diversity and haplotype frequencies that were consistent with source populations in southern China and Southeast Asia. mtDNA hypervariable segment I (HVS-I) sequences displayed high levels of haplotype diversity and nucleotide diversity that were comparable to various Asian populations. Genetic distances revealed extremely low mtDNA differentiation among Christmas Islanders and Asian populations. This was supported by the relatively high proportion of sequence types shared among these populations. The most common mtDNA haplogroups were M* and B, followed by D and F, which are prevalent in East/Southeast Asia. Christmas Islanders of European descent were characterized by the Eurasian haplogroup R*, and a limited degree of admixture was observed. In general, analysis of the genetic data indicated population affinities to southern Chinese (in particular from the Yunnan Province) and Southeast Asia (Thailand, Malaysia, and Cambodia), which was consistent with historical records of settlement. The combined use of these different marker systems provides a useful and appropriate model for the study of contemporary populations derived from different ethnic origins.     

Co-populated conformational ensembles of beta2-microglobulin uncovered quantitatively by electrospray ionization mass spectrometry

Ordered assembly of monomeric human beta(2)-microglobulin (beta(2)m) into amyloid fibrils is associated with the disorder hemodialysis-related amyloidosis. Previously, we have shown that under acidic conditions (pH <5.0 at 37 degrees C), wild-type beta(2)m assembles spontaneously into fibrils with different morphologies. Under these conditions, beta(2)m populates a number of different conformational states in vitro. However, this equilibrium mixture of conformationally different species is difficult to resolve using ensemble techniques such as nuclear magnetic resonance or circular dichroism. Here we use electrospray ionization mass spectrometry to resolve different species of beta(2)m populated between pH 6.0 and 2.0. We show that by linear deconvolution of the charge state distributions, the extent to which each conformational ensemble is populated throughout the pH range can be determined and quantified. Thus, at pH 3.6, conditions under which short fibrils are produced, the conformational ensemble is dominated by a charge state distribution centered on the 9+ ions. By contrast, under more acidic conditions (pH 2.6), where long straight fibrils are formed, the charge state distribution is dominated by the 10+ and 11+ ions. The data are reinforced by investigations on two variants of beta(2)m (V9A and F30A) that have reduced stability to pH denaturation and show changes in the pH dependence of the charge state distribution that correlate with the decrease in stability measured by tryptophan fluorescence. The data highlight the potential of electrospray ionization mass spectrometry to resolve and quantify complex mixtures of different conformational species, one or more of which may be important in the formation of amyloid.     

Amyloid formation under physiological conditions proceeds via a native-like folding intermediate

Although most proteins can assemble into amyloid-like fibrils in vitro under extreme conditions, how proteins form amyloid fibrils in vivo remains unresolved. Identifying rare aggregation-prone species under physiologically relevant conditions and defining their structural properties is therefore an important challenge. By solving the folding mechanism of the naturally amyloidogenic protein beta-2-microglobulin at pH 7.0 and 37 degrees C and correlating the concentrations of different species with the rate of fibril elongation, we identify a specific folding intermediate, containing a non-native trans-proline isomer, as the direct precursor of fibril elongation. Structural analysis using NMR shows that this species is highly native-like but contains perturbation of the edge strands that normally protect beta-sandwich proteins from self-association. The results demonstrate that aggregation pathways can involve self-assembly of highly native-like folding intermediates, and have implications for the prevention of this, and other, amyloid disorders.     

Stacked Sets of Parallel, In-register ��-Strands of ��2-Microglobulin in Amyloid Fibrils Revealed by Site-directed Spin Labeling and Chemical Labeling

β₂-microglobulin (β₂m) is a 99-residue protein with an immunoglobulin fold that forms β-sheet-rich amyloid fibrils in dialysis-related amyloidosis. Here the environment and accessibility of side chains within amyloid fibrils formed in vitro from β₂m with a long straight morphology are probed by site-directed spin labeling and accessibility to modification with N-ethyl maleimide using 19 site-specific cysteine variants. Continuous wave electron paramagnetic resonance spectroscopy of these fibrils reveals a core predominantly organized in a parallel, in-register arrangement, by contrast with other β₂m aggregates. A continuous array of parallel, in-register β-strands involving most of the polypeptide sequence is inconsistent with the cryoelectron microscopy structure, which reveals an architecture based on subunit repeats. To reconcile these data, the number of spins in close proximity required to give rise to spin exchange was determined. Systematic studies of a model protein system indicated that juxtaposition of four spin labels is sufficient to generate exchange narrowing. Combined with information about side-chain mobility and accessibility, we propose that the amyloid fibrils of β₂m consist of about six β₂m monomers organized in stacks with a parallel, in-register array. The results suggest an organization more complex than the accordion-like β-sandwich structure commonly proposed for amyloid fibrils.     

The treatment of obstructive azoospermia by intracytoplasmic sperm injection

Intracytoplasmic sperm injection (ICSI) allows the treatment of virtually every type of male infertility. Unlike in vitro fertilization (IVF), its success does not depend on sperm concentration, motility or morphology and most of the physical barriers to fertilisation are by-passes. Since ICSI does not require strongly motile sperm, its use has now been expanded to incorporate immature sperm from the testes and epididymides. Successful fertilisation, pregnancies and healthy babies have all been reported. However, concerns about the safety of ICSI remain due to its short clinical history and the lack of testing on animal models. Male fertility potential for assisted reproduction by ICSI cannot be measured by conventional parameters. Sperm DNA integrity is increasingly recognised as a more useful indicator. Studies have shown that sperm with higher levels of DNA damage have lower fertilisation rates after IVF and ICSI. Sperm with DNA damage above a certain threshold are associated with a longer time to conceive in otherwise apparently fertile couples and a higher miscarriage rate. DNA damage has been shown to be associated with impaired embryo cleavage. Our group has shown that sperm DNA from testicular sperm is less fragmented than that from epididymal sperm and suggest its preferred use in ICSI. In addition to nuclear (n) DNA we also assessed the quality of mitochondrial (mt) DNA from testicular sperm from men with obstructive azoospermia undergoing ICSI. We observed that couples achieving a pregnancy had both less mtDNA deletions and less nDNA fragmentation. We found inverse relationships between pregnancy and sperm mtDNA deletion numbers, size and nDNA fragmentation. No relationships were observed with fertilisation rates. With this knowledge, we designed an algorithm for the prediction of pregnancy based on the quality of sperm nDNA and mtDNA. Each year 40,000 men have a vasectomy in the UK but every year 2500 request a reversal to begin a second family. For such men, vasectomy reversal has recently been replaced in part by testicular biopsy via fine-needle testicular sperm aspiration (TESA) or percutaneous epididymal sperm aspiration (PESA) performed at an outpatient clinic and subsequently used in ICSI. Since these were previously fertile men it has been assumed that they had ‘fertile’ sperm. However the assited conception success rates of these mens partners has not been assessed until recently. We have shown a significant reduction in the clinical pregnancy rates in the partners of men who had had a vasectomy ≥10yrs previously. There is also evidence to suggest that spermatogenesis is significantly impaired in vasectomised men. Marked decreases in spermatocytes, spermatids and spermatozoa have been observed. We have found this to be associated with concomitant increases in apoptotic markers, such as Fas, FasL and Bax. The quality of the remaining sperm is also compromised. Sperm DNA from vasectomized men shows substantial damage which increases with time after surgery. This new use of ICSI will be discussed.     

Viscoelastic study of the mechanical unfolding of a protein by AFM

We have applied a dynamic force modulation technique to the mechanical unfolding of a homopolymer of immunoglobulin (Ig) domains from titin, (C47S C63S I27)5, [(I27)5] to determine the viscoelastic response of single protein molecules as a function of extension. Both the stiffness and the friction of the homopolymer system show a sudden decrease when a protein domain unfolds. The decrease in measured friction suggests that the system is dominated by the internal friction of the (I27)5 molecule and not solvent friction. In the stiffness-extension spectrum we detected an abrupt feature before each unfolding event, the amplitude of which decreased with each consecutive unfolding event. We propose that these features are a clear indication of the formation of the known unfolding intermediate of I27, which has been observed previously in constant velocity unfolding experiments. This simple force modulation AFM technique promises to be a very useful addition to constant velocity experiments providing detailed viscoelastic characterization of single molecules under extension.