The primary structure of a chondrichthyan protamine: A new apparent contradiction in protamine evolution

We have determined the primary structure of protamine R3 from ratfish (Hydrolagus colliei), a species belonging to the order Chimaeriformes (an old phylogenetic line among the chondrichthyes). Protamine R3 contains 48 residues organized as follows: ARRRH SMKKK RKSVR RRKTR KNQRK RKNSL GRSFK (Q/A)HGFL KQPPR FRP. Comparison of this sequence with both protamine Z3 fromScyliorhinus canicula (a chondrichthyan) and typical protamines from bony fish generates an apparent contradiction: Two relatively close species (H. colhei andS. canicula, both chondichthyes) display different protamines, whereas species more distant in evolution (S. canicula and bony fish) contain very similar protamine molecules. We note that this is not an isolated case in the evolution of sperm nuclear basic proteins (SNBPs) and discuss the possible significance of this fact. Correspondence to: M. Chiva     

Rôle of phagostimulants of cotton leaves in the feeding behaviour of Spodoptera littoralis

Petroleum ether extract of cotton leaves was the most attractive to the hatched larvae of Spodoptera littoralis, and it remained so even after removal of the chlorophyll and other pigments. Steam distillation of this filtrate gave a volatile oil which also proved highly attractive to the larvae. In this fraction, 12 components were identified by thin-layer chromatography. Of these components, α-terpineol, citronellol and α-pinene were the most attractive, whereas humulene and linalool were less attractive. The remaining non-volatile lipid fraction was also attractive to the larva. In this fraction the unsaponifiable matter contained the attractive ingredients. This fraction proved to be a hydrocarbon. The infrared spectrum and nuclear magnetic resonance showed only typical hydrocarbon bonds.     

Macrocyclic diterpene esters from Euphorbia royleana

An ether soluble resin was prepared by extraction and partition of the latex of Euphorbia royleana. This resin demonstrated pronounced pro-inflammatory activity on mammalian skin and was separated into biologically active and inactive fractions by dry column chromatography. The inactive fraction was further separated by adsorption and partition thin-layer methods. The least polar zone consisted of an inseparable mixture of four tetra-esters of the macrocyclic diterpene ingol, whilst the more polar fraction consisted of a mixture of four tri-esters of the same diterpene.     

Streptococcal antigen I/II binds to extracellular proteins through intermolecular beta-sheets

One of the functions associated with the oral streptococcal surface protein I/II is to bind to human extracellular matrix molecules or blood components, which could act as opportunistic ligands in pathological circumstances. In order to understand the relative specificity of the binding repertoire of this bacterial adhesin, we examined by infrared measurements the mode of binding of the protein I/II from Streptococcus mutans OMZ175 (I/IIf) to fibronectin and fibrinogen. This approach revealed the beta-structure forming capacity of I/IIf upon interaction with both proteins. The forming of intermolecular beta-structures may provide a non-selective way of interaction between I/IIf and its possible targets.     

Interaction of microtubule-associated protein-2 and p63: a new link between microtubules and rough endoplasmic reticulum membranes in neurons

Neurons are polarized cells presenting two distinct compartments, dendrites and an axon. Dendrites can be distinguished from the axon by the presence of rough endoplasmic reticulum (RER). The mechanism by which the structure and distribution of the RER is maintained in these cells is poorly understood. In the present study, we investigated the role of the dendritic microtubule-associated protein-2 (MAP2) in the RER membrane positioning by comparing their distribution in brain subcellular fractions and in primary hippocampal cells and by examining the MAP2-microtubule interaction with RER membranes in vitro. Subcellular fractionation of rat brain revealed a high MAP2 content in a subfraction enriched with the endoplasmic reticulum markers ribophorin and p63. Electron microscope morphometry confirmed the enrichment of this subfraction with RER membranes. In cultured hippocampal neurons, MAP2 and p63 were found to concomitantly compartmentalize to the dendritic processes during neuronal differentiation. Protein blot overlays using purified MAP2c protein revealed its interaction with p63, and immunoprecipitation experiments performed in HeLa cells showed that this interaction involves the projection domain of MAP2. In an in vitro reconstitution assay, MAP2-containing microtubules were observed to bind to RER membranes in contrast to microtubules containing tau, the axonal MAP. This binding of MAP2c microtubules was reduced when an anti-p63 antibody was added to the assay. The present results suggest that MAP2 is involved in the association of RER membranes with microtubules and thereby could participate in the differential distribution of RER membranes within a neuron.     

Presentation of Integrins on Leukocyte Microvilli: A Role for the Extracellular Domain in Determining Membrane Localization

Adhesion of blood leukocytes to the endothelium involves multiple steps including initial attachment (tethering), rolling, and firm arrest. Presentation of adhesion molecules on leukocyte microvilli can substantially enhance tethering. Localization of L-selectin to microvilli and of CD44 to the planar cell body have been shown to depend upon their transmembrane and cytoplasmic domains. We investigated the role of leukocyte integrin transmembrane and cytoplasmic domains in initiating adhesion under flow and in microvillous localization. Integrins α4β7, αLβ2, and αMβ2 were heterologously expressed in K562 cells. α4β7 initiated adhesion under flow and localized to microvilli, whereas β2 integrins did not initiate adhesion and localized to the cell body. Chimeric integrins were produced by replacing the α4β7 cytoplasmic and/or transmembrane domains with the homologous domains of αLβ2 or αMβ2. Unexpectedly, these chimeras efficiently mediated adhesion to the α4β7 ligand mucosal addressin cell adhesion molecule–1 under flow and localized to microvilli. Therefore, differences between the transmembrane and cytoplasmic domains of α4 and β2 integrins do not account for differences in ability to support attachment under flow or in membrane localization. Integrins α4β1, α5β1, α6Aβ1, αvβ3, and αEβ7 also localized to microvilli. Transmembrane proteins known or suspected to associate with extracellular domains of microvillous integrins, including tetraspans and CD47, were concentrated on microvilli as well. These findings suggest that interactions between the extracellular domains of integrins and associated proteins could direct the assembly of multimolecular complexes on leukocyte microvilli.     

The roles of the hybrid cluster protein,Hcp and its reductase,Hcr, in high affinity nitric oxide reduction that protects anaerobic cultures of Escherichia coli against nitrosative stress

The hybrid cluster protein, Hcp, contains a 4Fe‐2S‐2O iron‐sulfur‐oxygen cluster that is currently considered to be unique in biology. It protects various bacteria from nitrosative stress, but the mechanism is unknown. We demonstrate that the Escherichia coli Hcp is a high affinity nitric oxide (NO) reductase that is the major enzyme for reducing NO stoichiometrically to N₂O under physiologically relevant conditions. Deletion of hcp results in extreme sensitivity to NO during anaerobic growth and inactivation of the iron‐sulfur proteins, aconitase and fumarase, by accumulated cytoplasmic NO. Site directed mutagenesis revealed an essential role in NO reduction for the conserved glutamate 492 that coordinates the hybrid cluster. The second gene of the hcp‐hcr operon encodes an NADH‐dependent reductase, Hcr. Tight interaction between Hcp and Hcr was demonstrated. Although Hcp and Hcr purified individually were inactive or when recombined, a co‐purified preparation reduced NO in vitro with a Km for NO of 500 nM. In an hcr mutant, Hcp is reversibly inactivated by NO concentrations above 200 nM, indicating that Hcr protects Hcp from nitrosylation by its substrate, NO.     

Association between SIRT1 Gene Polymorphisms and Breast Cancer in Egyptians

Background

Breast cancer is reported to cause the highest mortality among female cancer patients. Previous studies have explored the association of silent mating-type information regulator 2 homolog 1 (SIRT1) gene expression with prognosis in breast cancer. However, no studies exist, so far, on the role of SIRT1 gene polymorphism in breast cancer risk or prognosis. The present study aimed to assess the association between SIRT1 gene polymorphisms and breast cancer in Egyptians.

Methods

The study comprised 980 Egyptian females divided into a breast cancer group (541 patients) and a healthy control group (439 subjects). SIRT1 gene single nucleotide polymorphisms (SNPs) rs3758391, rs3740051 and rs12778366 were genotyped using real-time polymerase chain reaction (RT-PCR). Allelic and genotypic frequencies were determined in both groups and association with breast cancer and clinicopathological characteristics was assessed.

Results

Breast cancer patients exhibited elevated serum SIRT1 levels which varied among different tumor grades. SIRT1 rs3758391 and rs12778366 TT genotypes were more frequent, exhibited higher SIRT1 levels than CC and CT genotypes and were associated with histologic grade and lymph node status. SIRT1 rs12778366 TT genotype also correlated with negative estrogen receptor (ER) and progesterone receptor (PR) statuses. The T allele frequency for both SNPs was higher in breast cancer patients than in normal subjects. Combined GG and AG genotypes of rs3740051 were more frequent, showed higher serum SIRT1 levels than the AA genotype, and were associated with ER and PR expression. Furthermore, inheritance of the G allele was associated with breast cancer.

Conclusions

Our findings reveal that rs3758391 and rs12778366 polymorphisms of SIRT1 gene are associated with breast cancer risk and prognosis in the Egyptian population.