Purification of [I]-vasoactive intestinal peptide by reverse-phase HPLC

VIP was labeled with sodium ¹²⁵iodide, and ¹²⁵I-VIP was purified by reverse-phase high performance liquid chromatography. Optimal separations of ¹²⁵I-VIP and unlabeled VIP were obtained using two C₁₈-Novapak columns in series and a gradient of acetonitrile in triethylamine phosphate for elution. The specific activity of the ¹²⁵I-VIP was 1.99±0.21 Ci/μmole, approaching the maximum specific activity of monoiodinated VIP (2.26 Ci/μmole). Radioimmunoassay and radioreceptorassay for VIP were more sensitive (2.6-fold, and 2.5-fold, respectively) using ¹²⁵I-VIP purified by HPLC compared to ¹²⁵I-VIP obtained from an open-end cellulose column. These results demonstrate the advantage of preparing purified ¹²⁵I-VIP by HPLC for the accurate assay of VIP and VIP-receptors in tissues and biological fluids.     

Antioxidant Enzyme Level Response to Prooxidant Allelochemicals in Larvae of the Southern Armyworm Moth, Spodoptera Eridania

Larvae of the southern armyworm, Spodoplera eridania, are highly polyphagous feeders which frequently encounter and feed upon plants containing high levels of prooxidant allelochemicals. While ingestion of moderate quantities of prooxidants can be tolerated by these larvae, ingestion of larger quantities can result in toxicity. Studies were conducted to assess the role of the antioxidant enzymes, superoxide dismutase (SOD), catalase (CAT) and glutathione reductase (GR) in the protection of S. eridania against redox active prooxidant plant allelochemicals. Dietary exposure of mid-fifth-instar larvae to either quercetin (a flavonoid) or xanthotoxin (a photoactive furanocoumarin), which generate superoxide radical, and singlet oxygen, repsectively, resulted in an increase in SOD levels. CAT levels increased in all groups of S. eridania including control insects. This may have been due to the sudden exposure to food following an extended fast of 18 h (to insure that larvae would not reject the diet because of the prooxidants' bitter taste) with an eventual lowering of CAT values with time. GR activities did not significantly change except for a slight inhibition at the highest prooxidant concentrations used at 12-h post-ingestion. The data from these studies suggest that SOD responds to prooxidant challenges in these insects and together with CAT and GR contributes to the insect's defense against potentially toxic prooxidant compounds.     

A Highlights from MBoC Selection: NAADP and the two-pore channel protein 1 participate in the acrosome reaction in mammalian spermatozoa

The functional relationship between the formation of hundreds of fusion pores during the acrosome reaction in spermatozoa and the mobilization of calcium from the acrosome has been determined only partially. Hence, the second messenger NAADP, promoting efflux of calcium from lysosome-like compartments and one of its potential molecular targets, the two-pore channel 1 (TPC1), were analyzed for its involvement in triggering the acrosome reaction using a TPCN1 gene–deficient mouse strain. The present study documents that TPC1 and NAADP-binding sites showed a colocalization at the acrosomal region and that treatment of spermatozoa with NAADP resulted in a loss of the acrosomal vesicle that showed typical properties described for TPCs: Registered responses were not detectable for its chemical analogue NADP and were blocked by the NAADP antagonist trans-Ned-19. In addition, two narrow bell-shaped dose-response curves were identified with maxima in either the nanomolar or low micromolar NAADP concentration range, where TPC1 was found to be responsible for activating the low affinity pathway. Our finding that two convergent NAADP-dependent pathways are operative in driving acrosomal exocytosis supports the concept that both NAADP-gated cascades match local NAADP concentrations with the efflux of acrosomal calcium, thereby ensuring complete fusion of the large acrosomal vesicle.     

Changes in morphology of endocytotic vesicles in cardiac capillary endothelial cells and myocytes of rats after treatment with adenine and AMP

Isolated rat hearts were perfused with media containing no additions (sham), adenine, or AMP to determine if adenine or AMP would affect post-ischaemic myocyte ultrastructure. The most significant change induced by both adenine and AMP was a dramatic increase in the number and size of endocytotic vesicles apparent in capillary endothelia and myocytes. This observation suggests endocytosis as a possible transport mechanism for AMP or adenine, or other large polar molecules including purine nucleotide precursors, from the coronary circulation across capillary endothelia and into myocytes.     

An Improved Lead-Tetraacetate:Schiff Procedure

Sections from tissues fixed in a 10% solution of formalin in 90% alcohol were treated with lead tetraacetate (PbAc₄) in different solvents: glacial acetic acid, dilute acetic acid, methanol, ethanol, toluene and benzene. The excess reagent was removed with a 2% solution of ethylene diamine tetraacetate (EDTA) at pH 8. The sections were then stained with Schiff's reagent for 20 minutes. The chemical stability of PbAc₄ in different solvents, the effect of its concentration and the time of exposure on the intensity of the Schiff reaction, were studied. A 0.023 N solution of PbAc₄ in benzene with a reaction time of 5 minutes is recommended. The stability of PbAc₄ in benzene permitted such solutions to be used for 3-4 days. Satisfactory results were obtained with mammalian tissues, algae, bacteria, fungi and protozoa. Some of the preparations obtained by using the technics described are illustrated in an accompanying plate of photomicrographs.     

Prostate apoptosis response-4 and tumor suppression: it’s not just about apoptosis anymore

The tumor suppressor prostate apoptosis response-4 (Par-4) has recently turned ‘twenty-five’. Beyond its indisputable role as an apoptosis inducer, an increasing and sometimes bewildering, new roles for Par-4 are being reported. These roles include its ability to regulate autophagy, senescence, and metastasis. This growing range of responses to Par-4 is reflected by our increasing understanding of the various mechanisms through which Par-4 can function. In this review, we summarize the existing knowledge on Par-4 tumor suppressive mechanisms, and discuss how the interaction of Par-4 with different regulators influence cell fate. This review also highlights the new secretory pathway that has emerged and the likely discussion on its clinical implications.Subject terms: Tumour-suppressor proteins, Apoptosis, Stress signalling, Target identification     

The PsbS protein controls the macro-organisation of photosystem II complexes in the grana membranes of higher plant chloroplasts

The PsbS protein is a critical component in the regulation of non-photochemical quenching (NPQ) in higher plant photosynthesis. Electron microscopy and image analysis of grana membrane fragments from wild type and mutant Arabidopsis plants showed that the semi-crystalline domains of photosystem II supercomplexes were identical in the presence and absence of PsbS. However, the frequency of the domains containing crystalline arrays was increased in the absence of PsbS. Conversely, there was a complete absence of such arrays in the membranes of plants containing elevated amounts of this protein. It is proposed that PsbS controls the macro-organisation of the grana membrane, providing an explanation of its role in NPQ.