Quantification of oxygenated volatile organic compounds in seawater by membrane inlet-proton transfer reaction/mass spectrometry

The role of the ocean in the cycling of oxygenated volatile organic compounds (OVOCs) remains largely unanswered due to a paucity of datasets. We describe the method development of a membrane inlet-proton transfer reaction/mass spectrometer (MI-PTR/MS) as an efficient method of analysing methanol, acetaldehyde and acetone in seawater. Validation of the technique with water standards shows that the optimised responses are linear and reproducible. Limits of detection are 27 nM for methanol, 0.7 nM for acetaldehyde and 0.3 nM for acetone. Acetone and acetaldehyde concentrations generated by MI-PTR/MS are compared to a second, independent method based on purge and trap-gas chromatography/flame ionisation detection (P&T-GC/FID) and show excellent agreement. Chromatographic separation of isomeric species acetone and propanal permits correction to mass 59 signal generated by the PTR/MS and overcomes a known uncertainty in reporting acetone concentrations via mass spectrometry. A third bioassay technique using radiolabelled acetone further supported the result generated by this method. We present the development and optimisation of the MI-PTR/MS technique as a reliable and convenient tool for analysing seawater samples for these trace gases. We compare this method with other analytical techniques and discuss its potential use in improving the current understanding of the cycling of oceanic OVOCs.

THE IDENTIFICATION AND CHARACTERIZATION OF AN INNER ACROSOMAL MEMBRANE ASSOCIATED PROTEIN, IAM38, RESPONSIBLE FOR SECONDARY SPERM-ZONA BINDING DURING FERTILIZATION

During mammalian fertilization, the exposure of the inner acrosomal membrane (IAM) after acrosomal exocytosis is essential for the secondary binding between sperm and zona pellucida (ZP) of the oocyte, a prerequisite for sperm penetration through the ZP. The identification of the sperm protein(s) responsible for secondary binding has posed a challenge for researchers. We were able to isolate a sperm head fraction in which the IAM was exposed. Attached to the IAM was an electon dense layer, which we termed the IAM extracellular coat (IAMC). The IAMC was also observable in acrosome reacted sperm. High salt extraction removed the IAMC including a prominent 38 kDa polypeptide, referred to as IAM38. Antibodies raised against IAM38 confirmed its presence in the IAMC of intact, sonicated, and acrosome-reacted sperm. Sequencing of IAM38 revealed it as the ortholog of porcine SP38, a protein that was found to bind specifically to ZP2 but whose intra-acrosomal location was not known. We showed that IAM38 occupied the leading edge of sperm contact with the zona pellucida during fertilization, and that secondary binding and fertilization were inhibited in vitro by antibodies directed against IAM38. As for the mechanism of secondary sperm-zona binding by IAM38, we provided evidence that the synthetic peptide derived from the ZP2-binding motif of IAM38 had a competitive inhibitory effect on both sperm-zona binding and fertilization while its mutant form was ineffective. In summary, our study provides a novel approach to obtain direct information on the peripheral and integral protein composition of the IAM and consolidates IAM38 as a genuine secondary sperm-zona binding protein. In addition, our investigation also provides an ultrastructural description of the origin, expression and assembly of IAM38 during spermatogenesis. It shows that IAM38 is originally secreted by the Golgi apparatus as part of the dense contents of the proacrosomic granules but later, during acrosome capping phase of spermiogenesis, is redistributed to the inner periphery of the acrosomal membrane. This relocation occurs at the time of acrosomal compaction, an obligatory structural change that fails to occur in Zpbp1-/- knockout mice, which do not express IAM38 and are infertile.

Amyloid B-peptide 1-40 increases neuronal membrane fluidity: role of cholesterol and brain region

There is increasing evidence of an interaction between cholesterol dynamics and Alzheimer's disease (AD), and amyloid ß-peptide may play an important role in this interaction. Aß destabilizes brain membranes and this action of Aß may be dependent on the amount of membrane cholesterol. We tested this hypothesis by examining effects of Aß1-40 on the annular fluidity (i.e., lipid environment adjacent to proteins) and bulk fluidity of rat synaptic plasma membranes (SPM) of the cerebral cortex, cerebellum, and hippocampus using the fluorescent probe pyrene and energy transfer. Amounts of cholesterol and phospholipid of SPM from each brain region were determined. SPM of the cerebellum were significantly more fluid as compared with SPM of the cerebral cortex and hippocampus. Aß significantly increased (P 0.01) annular and bulk fluidity in SPM of cerebral cortex and hippocampus. In contrast, Aß had no effect on annular fluidity and bulk fluidity of SPM of cerebellum. The amounts of cholesterol in SPM of cerebral cortex and hippocampus were significantly higher (P 0.05) than amount of cholesterol in SPM of cerebellum. There was significantly less (P 0.05) total phospholipid in cerebellar SPM as compared with SPM of cerebral cortex. Neuronal membranes enriched in cholesterol may promote accumulation of Aß by hydrophobic interaction, and such an interpretation is consistent with recent studies showing that soluble Aß can act as a seed for fibrillogenesis in the presence of cholesterol.