Kinetic analysis of chloride efflux from normal and cystic fibrosis fibroblasts

Chloride permeability in 9 cystic fibrosis- and 11 normal-skin fibroblast lines has been investigated. Chloride efflux, under steady-state conditions, involves two intracellular compartments characterized by slow- and fast-rate constants of efflux. We show here that the fast rate constant in cystic fibrosis cells is reduced by 25% in comparison with controls. The data presented support recent studies indicating that isolated sweat glands and respiratory epithelia of patients suffering from cystic fibrosis have an unusual low permeability to chloride ions compared to control epithelia. It is concluded that variation in chloride transport can successfully be studied in cultured fibroblasts, which are not directly involved in the pathology of the disease.     

Internal lipid synthesis and vesicle growth as a step toward self-reproduction of the minimal cell

One of the major properties of the semi-synthetic minimal cell, as a model for early living cells, is the ability to self-reproduce itself, and the reproduction of the boundary layer or vesicle compartment is part of this process. A minimal bio-molecular mechanism based on the activity of one single enzyme, the FAS-B (Fatty Acid Synthase) Type I enzyme from Brevibacterium ammoniagenes, is encapsulated in 1-palmitoyl-2oleoyl-sn-glycero-3-phosphatidylcholine (POPC) liposomes to control lipid synthesis. Consequently molecules of palmitic acid released from the FAS catalysis, within the internal lumen, move toward the membrane compartment and become incorporated into the phospholipid bilayer. As a result the vesicle membranes change in lipid composition and liposome growth can be monitored. Here we report the first experiments showing vesicles growth by catalysis of one enzyme only that produces cell boundary from within. This is the prototype of the simplest autopoietic minimal cell.     

Identification of the coding region for the putidaredoxin reductase gene from the plasmid of Pseudomonas putida

The first 12 NH₂-terminal amino acids of the Pseudomonas putida putidaredoxin reductase were shown to be Met-Asn-Ala-Asn-Asp-Asn-Val-Val-Ile-Val-Gly-Thr. Comparison of these data with the DNA sequence of the BamHI-HindIII 197-base fragment derived from the PstI 2.2-kb fragment obtained from the P. putida plasmid showed that the putidaredoxin reductase gene was downstream from the cytochrome P-450 gene and the intergenic region had the 24-nucleotide sequence TAAACACATGGGAGTGCGTGCTAA. The Shine-Dalgarno sequence GGAG was detected in this region. The initiating triplet for the reductase gene was GTG, which normally codes for valine, but in the initiating codon position codes for methionine. From the amino acid sequence and X-ray data comparisons with other flavoproteins, what appears to be the AMP binding region of the FAD can be recognized in the NH₂-terminal portion of the reductase involving residues 5–35.This article was presented during the proceedings of the International Conference on Macromolecular Structure and Function, held at the National Defence Medical College, Tokorozawa, Japan, December 1985.     

The "Alzheimer's disease signature": potential perspectives for novel biomarkers

Alzheimer's disease is a progressive and neurodegenerative disorder which involves multiple molecular mechanisms. Intense research during the last years has accumulated a large body of data and the search for sensitive and specific biomarkers has undergone a rapid evolution. However, the diagnosis remains problematic and the current tests do not accurately detect the process leading to neurodegeneration. Biomarkers discovery and validation are considered the key aspects to support clinical diagnosis and provide discriminatory power between different stages of the disorder. A considerable challenge is to integrate different types of data from new potent approach to reach a common interpretation and replicate the findings across studies and populations. Furthermore, long-term clinical follow-up and combined analysis of several biomarkers are among the most promising perspectives to diagnose and manage the disease. The present review will focus on the recent published data providing an updated overview of the main achievements in the genetic and biochemical research of the Alzheimer's disease. We also discuss the latest and most significant results that will help to define a specific disease signature whose validity might be clinically relevant for future AD diagnosis.     

Membrane excitability expressed in human neuroblastoma cell hybrids

The voltage-sensitive Na+ channel is responsible for the action potential of membrane electrical excitability in neuronal tissue. Three methods were used to demonstrate the presence of neurotoxin-responsive Na+ channels in two hybrid cell lines resulting from the fusion of excitable human neuroblastoma cells with mouse fibroblasts. Only one of the two electrically active hybrid cell lines maintained the sensitivity of the neuroblastoma parent to tetrodotoxin (TTX). The other hybrid, although electrically active, was not responsive to TTX or scorpion venom. Comparisons of the patterns of expression of membrane excitability and of chromosome complements in these human neuroblastoma cell hybrids suggest that the phenotype of membrane excitability is composed of genetically distinct elements.