Phase variations inBifidobacterium animalis

Strains isolated from rabbit, chicken, and rat feces and from sewage and fermented milk products, all identified asBifidobacterium animalis, were found to show phase variations in colony appearance and in cellular morphology. The rate of transition in a switching system from opaque to transparent colonies and vice versa was determined. Differences in protein components and in penicillin-binding proteins (PBPs) of the cells from different colony types are shown.     

Impact of Diffusion Barriers to Small Cytotoxic Molecules on the Efficacy of Immunotherapy in Breast Cancer

Molecular-focused cancer therapies, e.g., molecularly targeted therapy and immunotherapy, so far demonstrate only limited efficacy in cancer patients. We hypothesize that underestimating the role of biophysical factors that impact the delivery of drugs or cytotoxic cells to the target sites (for associated preferential cytotoxicity or cell signaling modulation) may be responsible for the poor clinical outcome. Therefore, instead of focusing exclusively on the investigation of molecular mechanisms in cancer cells, convection-diffusion of cytotoxic molecules and migration of cancer-killing cells within tumor tissue should be taken into account to improve therapeutic effectiveness. To test this hypothesis, we have developed a mathematical model of the interstitial diffusion and uptake of small cytotoxic molecules secreted by T-cells, which is capable of predicting breast cancer growth inhibition as measured both in vitro and in vivo. Our analysis shows that diffusion barriers of cytotoxic molecules conspire with γδ T-cell scarcity in tissue to limit the inhibitory effects of γδ T-cells on cancer cells. This may increase the necessary ratios of γδ T-cells to cancer cells within tissue to unrealistic values for having an intended therapeutic effect, and decrease the effectiveness of the immunotherapeutic treatment.     

Protein phosphorylation in rat liver mitochondria

Summary Incubation of rat liver mitochondria in the presence of either [³²P] Pi or ₃₂ ^y -P] ATP resulted in a phosphorylation of four proteins with Mr 50, 47, 44 and 36 kDa, respectively. The endogenous phosphorylation of these proteins in the presence of [³²P] Pi was markedly influenced by the osmolarity of the incubation medium and differentially affected by various effectors of mitochondrial functions, such as Ca²⁺, oligomycin, FCCP, arsenite and dichloroacetate. In particular, the 36 kDa protein, unlike the other proteins, appears to be phosphorylated also by direct incorporation of [³²P], independently of respiratory chain-linked ATP synthesis. The four proteins, located in the mitoplasts, seem to be phosphorylated by diiferent protein kinases, as suggested by the observation that the endogenous phosphorylation of 36 kDa protein resulted selectively increased by addition of exogenous protein kinases, such as casein kinases S and TS. A tentative identification of these phosphorylatable protein is discussed.     

Molecular insights into cell toxicity of a novel familial amyloidogenic variant of β2‐microglobulin

The first genetic variant of β₂‐microglobulin (b2M) associated with a familial form of systemic amyloidosis has been recently described. The mutated protein, carrying a substitution of Asp at position 76 with an Asn (D76N b2M), exhibits a strongly enhanced amyloidogenic tendency to aggregate with respect to the wild‐type protein. In this study, we characterized the D76N b2M aggregation path and performed an unprecedented analysis of the biochemical mechanisms underlying aggregate cytotoxicity. We showed that, contrarily to what expected from other amyloid studies, early aggregates of the mutant are not the most toxic species, despite their higher surface hydrophobicity. By modulating ganglioside GM1 content in cell membrane or synthetic lipid bilayers, we confirmed the pivotal role of this lipid as aggregate recruiter favouring their cytotoxicity. We finally observed that the aggregates bind to the cell membrane inducing an alteration of its elasticity (with possible functional unbalance and cytotoxicity) in GM1‐enriched domains only, thus establishing a link between aggregate‐membrane contact and cell damage.     

An ultrastructural study of oogenesis in a marine triclad

The ultrastructural features of oocyte differentiation were studied in the marine triclad Cercyra hastata. Oocytes at several stages of maturation, each surrounded by follicle cell projections, are present within each of the two ovaries. A pre-vitellogenic and a vitellogenic stage have been detected in the oogenesis of C. hastata. The pre-vitellogenic stage is mainly characterized by an increase in the nuclear and nucleolar volume and activity, and the appearance and development of cortical granule precursors which are elaborated by the Golgi complex. In early phases of the vitellogenic stage, intense delamination and blebbing of the nuclear envelope occurs which probably contributes to an increase in number of cytoplasmic membranes and to transfer of nuclear material to the cytoplasm. The rough endoplasmic reticulum is extensively developed and often assumes a ‘whorl’ array. Several areas of yolk precursor formation appear in the whorls. Numerous 2–5 μm protein yolk globules are subsequently formed which appear surrounded by a double membrane (cisternae of the smooth endoplasmic reticulum) and become randomly distributed throughout the cytoplasm of mature oocytes. The peripheral ooplasm is occupied by a monolayer of electron-dense cortical granules. Finally, the evolutionary significance of the autosynthetic mechanism of yolk production is discussed.     

Structure and polymorphism of bipolar isopranyl ether lipids from archaebacteria

We describe in this work the structure and polymorphism of a variety of lipids extracted from Sulfolobus solfataricus, an extreme thermoacidophilic archaebacterium growing at about 85 °C and pH 2. These lipids are quite different from the usual fatty acid lipids of eukaryotes and prokaryotes: each molecule consists of two C₄₀ ω-ω′ biphytanyl residues (with 0 to 4 cyclopentane groups per residue), ether linked at both ends to two (variably substituted) glycerol or nonitol groups. Four lipid preparations were studied; the total and the polar lipid extracts, and two hydrolytic fractions, the symmetric glycerol dialkyl glycerol tetraether and the asymmetric glycerol dialkyl nonitol tetraether, as a function of water content and temperature, using X-ray scattering techniques. The main conclusions from the study of the four lipid preparations can be summarized as follows. (1) As with other lipids, a remarkable number and variety of phases are observed over a temperature-concentration range close to “physiological” conditions. The possibility is discussed that this polymorphism reflects a fundamental property of lipids, closely related to their physiological rôle. (2) As in other lipids, two types of chain conformations are observed: a disordered one (type α) at high temperature; at lower temperature, a more ordered packing of stiff chains, all parallel to each other (type β′). At temperatures and degrees of hydration approaching the conditions prevailing in the living cell, the conformation is of type α. (3) In all the phases with chains in the α conformation, the unsubstituted glycerol headgroups, whose concentration is high in these lipids, segregate in the hydrocarbon matrix, away from the other polar groups. This property may have interesting biological consequences: for example, the chains of a fraction of the bipolar lipid molecules can span hydrocarbon gaps as wide as 75 Å. (4) Two cubic phases are observed in the total and the polar lipid extracts, which display a remarkable degree of metastability, most unusual in lipid phase transitions involving structures with chains in the α conformation. This phenomenon can be explained by the interplay of the physical structure of the cubic phases (the two contain two intertwined and unconnected three-dimensional networks of rods) and the chemical structure of the lipid molecules: the two headgroups of most molecules being anchored on each of the two networks of rods, the migration of the lipid molecules is hindered by the two independent diffusion processes and by the entanglement of the chains. The possibility is discussed that this phenomenon may reflect an evolutionary response to a challenge of the natural habitat of these archaebacteria.     

Phosphorylation of cytosolic proteins by casein kinases in human erythrocytes. Response to ionic strength and to 2,3-bisphosphoglycerate

The endogenous phosphorylation of human erythrocyte cytosolic proteins is markedly increased when the crude cytosol, prior to incubation in the presence of [y-³²P] ATP, is submitted to DEAE-cellulose chromatography. Some proteins, including 22 and 23 kDa proteins, are preferentially phosphorylated by cytosolic casein kinase CS, whereas other proteins, including 42 kDa protein, are preferentially phosphorylated by casein kinase CTS. The CS-catalyzed phosphorylation is strongly inhibited by physiological ionic strength (150 mM KCl or NaCl) and by physiological levels (3 mM) of 2,3-bisphosphoglycerate, while CTS-catalyzed phosphorylation is unaffected. The very poor endogenous phosphorylation of these proteins in the crude cytosol may be due to the presence of other cytosolic inhibitors which are removed by DEAE-cellulose chromatography.     

Chemical synthesis and characterization of wild‐type and biotinylated N‐terminal domain 1–64 of β2‐glycoprotein I

The antiphospholipid syndrome (APS) is a severe autoimmune disease associated with recurrent thrombosis and fetal loss and characterized by the presence of circulating autoantibodies (aAbs) mainly recognizing the N‐terminal domain (DmI) of β2‐glycoprotein I (β2GpI). To possibly block anti‐β2GpI Abs activity, we synthesized the entire DmI comprising residues 1–64 of β2GpI by chemical methods. Oxidative disulfide renaturation of DmI was achieved in the presence of reduced and oxidized glutathione. The folded DmI (N‐DmI) was purified by RP‐HPLC, and its chemical identity and correct disulfide pairing (Cys4‐Cys47 and Cys32‐Cys60) were established by enzymatic peptide mass fingerprint analysis. The results of the conformational characterization, conducted by far‐ and near‐UV CD and fluorescence spectroscopy, provided strong evidence for the native‐like structure of DmI, which is also quite resistant to both Gdn‐HCl and thermal denaturation. However, the thermodynamic stability of N‐DmI at 37°C was remarkably low, in agreement with the unfolding energetics of small proteins. Of note, aAbs failed to bind to plates coated with N‐DmI in direct binding experiments. From ELISA competition experiments with plate‐immobilized β2GpI, a mean IC₅₀ value of 8.8 μM could be estimated for N‐DmI, similar to that of the full‐length protein, IC₅₀(β2GpI) = 6.4 μM, whereas the cysteine‐reduced and carboxamidomethylated DmI, RC‐DmI, failed to bind to anti‐β2GpI Abs. The versatility of chemical synthesis was also exploited to produce an N‐terminally biotin‐(PEG)₂‐derivative of N‐DmI (Biotin‐N‐DmI) to be possibly used as a new tool in APS diagnosis. Strikingly, Biotin‐N‐DmI loaded onto a streptavidin‐coated plate selectively recognized aAbs from APS patients.     

Amino acid composition of zein molecular components

Zein extracted from maize endosperm has been fractionated into four polypeptide chains, having the following MWs 23 000, 21 000, 13 500 and 9600. By amino acid analysis the two smaller MW chains (representing 30% of total zeins) have been found to be zein-type molecules. These two chains are thought to be responsible for zein granule formation via -S-S- bridges. Zein is also highly heterogeneous in charge, and is resolved into at least 15 components, with pI's in the pH range 5–9. As demonstrated by amino acid analysis, part of this heterogeneity is due to spot mutations in some of the genes responsible for zein synthesis.