Protective effect of intracellular ice during freezing?

Injury results during freezing when cells are exposed to increasing concentrations of solutes or by the formation of intracellular ice. Methods to protect cells from the damaging effects of freezing have focused on the addition of cryoprotective chemicals and the determination of optimal cooling rates. Based on other studies of innocuous intracellular ice formation, this study investigates the potential for this ice to protect cells from injury during subsequent slow cooling. V-79W Chinese hamster fibroblasts and Madin-Darby Canine Kidney (MDCK) cells were cultured as single attached cells or confluent monolayers. The incidence of intracellular ice formation (IIF) in the cultures at the start of cooling was pre-determined using one of two different extracellular ice nucleation temperatures (-5 or -10 degrees C). Samples were then cooled at 1 degrees C/min to the experimental temperature (-5 to -40 degrees C) where samples were warmed rapidly and cell survival assessed using membrane integrity and metabolic activity. For single attached cells, the lower ice nucleation temperature, corresponding to increased incidence of IIF, resulted in decreased post-thaw cell recovery. In contrast, confluent monolayers in which IIF has been shown to be innocuous, show higher survival after cooling to temperatures as low as -40 degrees C, supporting the concept that intracellular ice confers cryoprotection by preventing cell dehydration during subsequent slow cooling.     

Phospholipase C-γ2 promotes intracellular survival of mycobacteria

Mycobacterium tuberculosis (Mtb) infects millions of people each year. These bacilli can survive inside macrophages. To favor their survival, pathogen alters various signal transduction pathways in host cells. Phospholipase C (PLC) signaling regulates various processes in mammalian cells but has never been investigated for their roles in regulating phagocytosis and killing of mycobacteria by macrophages. Here, we report that infection with Mtb but not Mycobacterium smegmatis (MS) induces phosphorylation of PLC-γ2 at tyrosine 1217 in J774A.1 cells. Small interfering RNA–mediated knockdown of PLC-γ2 expression leads to the enhanced killing of both MS and Mtb by these cells suggesting that Mtb activates PLC-γ2 to promote its intracellular survival within macrophages. Knockdown of PLC-γ2 also lead to increased uptake of Mtb but not MS by J774.A.1 cells. Further, we have observed that PLC-γ2 was required for Mtb-induced inhibition of expression of proinflammatory cytokine tumor necrosis factor-α, inducible nitric oxide synthase, and chemokine (C-C motif) ligand 5 (RANTES). Altogether, our results for the first time demonstrate that Mtb induces activation of macrophages PLC-γ2 to inhibit their mycobactericidal response.     

Regulation of stress responses by intracellular vesicle trafficking?

Being grounded to one place, plants are constantly exposed to unexpected changes in the surrounding environment. Often, the changes in environmental conditions can be very rapid, compelling the plants to continuously monitor the outside environment and to adjust their metabolism to new conditions. Many of the primary environmental stresses ensue the development of a secondary oxidative stress, resulting in tissue damage and necrosis. The acclimation process almost invariably involves changes in the pattern of expressed proteins and other molecules. This necessitates the removal of the existing molecules from their compartments and the delivery of new compounds to their target organelles. The trafficking of macromolecules is performed by a bi-directional intracellular vesicle trafficking system that delivers newly synthesized molecules to organelles and retrieves material from the organelles to cytosolic compartments, such as vacuoles or lysosomes. The plasma membrane is among the organelles that are most exposed to oxidative stress damage and therefore must be constantly recycled. Here I propose that, by adjusting the rate of trafficking to and from the plasma membrane, the cells can regulate the stress outcome. Since the vesicle trafficking is closely linked to general signal transduction pathways, such as the phosphoinositide kinase pathway, and is influenced by major plant hormones, such as abscisic acid and auxin, the vesicle trafficking machinery holds the potential to regulate the plant responses to different environmental stresses.     

Improvement of intracellular β-galactosidase production in fed-batch culture of Kluyveromyces fragilis

Four fed-batch control strategies were evaluated to improve the specific lactase activity of Kluyveromyces fragilis. Control strategies tested included DO-stat control, exponential feeding, exponential feeding with manual feedback control and corrected feed-forward control. Each was implemented with standard sensors (i.e., temperature, dissolved oxygen and pH sensors) commonly installed in fermenters. The highest specific activity was obtained using the corrected feed-forward control strategy, a strategy incorporating a novel method for on-line estimation of specific growth rate. The control strategy was able to operate effectively to a final cell density of 69 g dry wt l^–1 with a specific lactase activity of 2 U mg^–1 cell dry wt.     

Ubiquitination of intracellular bacteria: a new bacteria-sensing system?

Ubiquitination is a protein modification generally used by cells to tag proteins that are destined for proteasomal degradation. In a recent article, Perrin et al. reported that the ubiquitination system has a role in the recognition of bacterial pathogens in the cytosol of mammalian cells. They showed that polyubiquitinated proteins accumulate on the surface of cytosolic Salmonella typhimurium. In macrophages, but not epithelial cells, proteasomes become associated with the surface of cytosolic bacteria. The authors proposed that the ubiquitin-proteasome machinery might be implicated indirectly in bacterial clearance.     

Calcium: a fundamental regulator of intracellular membrane fusion?

For many years, it has been known that an increase in cytosolic calcium triggers the fusion of secretory granules and synaptic vesicles with the plasma membrane. However, the role of calcium in the intracellular membrane-fusion reactions that coordinate the secretory and endocytic pathways has been less clear. Initially, there was accumulating evidence to indicate that a focally localized and transient calcium signal is required to trigger even those fusion events formerly classified as 'constitutive'-that is, those that normally occur in the absence of global cytosolic calcium increases. Therefore, calcium seemed to be a required fundamental co-factor underlying all biological membrane-fusion steps, perhaps with a conserved mechanism of action. However, although such unification would be gratifying, new data indicate that several intracellular fusion events do not require calcium after all. In this review, the evidence for calcium requirements and its modes of action in constitutive trafficking are discussed. As a challenging perspective, I suggest that the specific absence of calcium requirements for some transport steps in fact expands the function of calcium in trafficking, because divergent luminal calcium concentrations and requirements for fusion might increase the specificity with which intracellular membrane-fusion partners are determined.     

Kinetic analysis of the active site of an intracellular β-glucosidase fromCellulomonas biazotea

Purified β-glucosidase fromCellulomonas biazotea had an apparentK _m andV for 2-nitrophenyl β-d-glucopyranoside (oNPG) of 0.416 mmol/L and 0.22 U/mg protein, respectively. The activation energy for the hydrolysis of pNPG of β-glucosidase was 65 kJ/mol. The inhibition by Mn²⁺ vs. oNPG of parental β-glucosidase was of mixed type with apparent inhibition constants of 0.19 and 0.60 μmol/L for the enzyme and enzyme-substrate complex, respectively. Ethanol at lower concentrations activated while at higher concentrations it inhibited the enzyme. The determination of apparent pK _a’s at different temperatures and in the presence of 30 % dioxane indicated two carboxyl groups which control theV value. The thermal stability of β-glucosidase decreased in the presence of 10 % ethanol. The half-life of β-glucosidase in 1.75 mol/L urea at 35 °C was 145 min, as determined by 0–9 mol/L transverse urea gradient-PAGE. This work was financed in part by a grant made by theUS Agency for International Development under PSTC proposal 6-163,USAID grant no. 9365542-G-00-89-42-00, and PAEC.     

Are respiratory enzymes the primary sources of intracellular hydrogen peroxide?

Endogenous H2O2 is believed to be a source of chronic damage in aerobic organisms. To quantify H2O2 formation, we have generated strains of Escherichia coli that lack intracellular scavenging enzymes. The H2O2 that is formed within these mutants diffuses out into the medium, where it can be measured. We sought to test the prevailing hypothesis that this H2O2 is primarily generated by the autoxidation of redox enzymes within the respiratory chain. The rate of H2O2 production increased when oxygen levels were raised, confirming that H2O2 is formed by an adventitious chemical process. However, mutants that lacked NADH dehydrogenase II and fumarate reductase, the most oxidizable components of the respiratory chain in vitro, continued to form H2O2 at normal rates. NADH dehydrogenase II did generate substantial H2O2 when it was when overproduced or quinones were absent, forcing electrons to accumulate on the enzyme. Mutants that lacked both NADH dehydrogenases respired very slowly, as expected; however, these mutants showed no diminution of H2O2 excretion, suggesting that H2O2 is primarily formed by a source outside the respiratory chain. That source has not yet been identified. In respiring cells the rate of H2O2 production was approximately 0.5% the rate of total oxygen consumption, with only modest changes when cells used different carbon sources.     

Partial purification and characterization of intracellular car☐ypeptidase ofCandida albicans

Intracellular car☐ypeptidase was partially purified from the yeast form ofCandida albicans H-317 by acid dialysis, ammonium sulfate fractionation, gel filtration, and ion-exchange chromatography. Peptidase activity was measured with an enzyme-coupled colorimetric assay. Fractionation by native polyacrylamide gel electrophoresis and Sephacryl S-200 gel filtration indicated the presence of a single peak of car☐ypeptidase, with the isoelectric point at pH 4.6 and a molecular weight of 100,000. The pH optimum and apparentK_m usingN-carbobenzoxy-l-phenylalanine-l-leucine (N-Cbz-l-Phe-l-Leu) as substrate were 6.5 and 2 × 10⁻⁴M, respectively. The best substrates for the enzyme wereN-Cbz-Ala-X peptides, withN-Cbz-Ala-Leu giving the highest rate of hydrolysis. Substrates that gave 7% or less of the control (N-Cbz-Phe-Leu) rate of hydrolysis were Gly-Leu, Gly-Phe, Ile-Phe, Ile-Met, Glu-Phe, Glu-Tyr, Val-Phe, and Pro-Phe. There was no detectable hydrolysis of the followingN-Cbz peptides; Gly-Met, Gly-Val, Gly-Tyr, Gly-Ile, or Ile-Val. Enzyme activity was inhibited by phenylmethylsulfonyl fluoride,p-chloromercuribenzoate, benzyloxy-carbonyl-l-phenylalanine chloromethyl ketone, and tosyl-l-phenylalanine chloromethyl ketone, but was not affected by EDTA, tosyl-l-lysine chloromethyl ketone, pepstatin A, leupeptin, bestatin, or antipain. Although secretory proteinases are thought to play a role in the pathogenesis of this organism, the role of this intracellular car☐ypeptidase has yet to be determined.     

Adaptive value of polymorphism in intracellular self/not-self discrimination?

A microbial pathogen species can adapt to its host species to the extent that members of the host species are uniform. Loss of this uniformity would make it difficult for a pathogen species to transfer, from one member of the host species to another, what it had "learned" through selection of its members with advantageous mutations. The existence of major histocompatibility complex (MHC) polymorphism indicates that non-uniformity within a species is an effective host defence strategy. By virtue of this molecular discontinuity among its members the host species can "present a moving target" to the pathogen. Many proteins other than MHC proteins show polymorphism - a phenomenon which has suggested that mutations in regions of protein molecules which do not affect overt function are neutral. However, in the context of the author's differential aggregation theory of intracellular self/not-self discrimination as previously applied to the problem of the antigenicity of cancer cells, such polymorphism should serve for the recruitment of subsets of self-antigens into the antigenic repertoire of an infected cell. These would act as "intracellular antibodies" by virtue of their weak, but specific, aggregation with pathogen proteins. Peptides from the self-antigens, as well as (or instead of) those from the antigens of the pathogen, would then serve as targets for attack by cytotoxic T cells. Thus, polymorphism of intracellular proteins should be of adaptive value, serving to amplify and individualize the immune response to intracellular pathogens.     

The intracellular cyanobacteria of Paulinella chromatophora: endosymbionts or organelles?

Endosymbiotic relationships are common across the tree of life and have had profound impacts on cellular evolution and diversity. Recent molecular investigations of the amoeba Paulinella chromatophora have raised a timely and important question: should obligatory intracellular cyanobacteria in Paulinella be considered new organelles, or do plastids and mitochondria hold a unique stature in the history of endosymbiotic events? We argue that drawing a sharp distinction between these two organelles and all other endosymbionts is not supported by accumulating data, neither is it a productive framework for investigating organelle evolution.     

Regulation of biosynthesis and intracellular localization of rice and tobacco homologues of nucleosome assembly protein 1

The nucleosome assembly protein 1 (NAP1) is considered to be a conserved histone chaperone, facilitating the assembly of nucleosomes in all eukaryotes. However, studies in yeast and animal cells also indicated that NAP1 proteins have diverse functions likely independent of nucleosome-assembly activity. Here, we describe the isolation and characterization of cDNAs encoding NAP1-like proteins from the monocotyledon rice ( Oryza sativa L.) and the dicotyledon tobacco ( Nicotiana tabacum L.). Northern-blot analysis demonstrated that the two rice NAP1-like genes are predominantly expressed in stem tissues such as root and shoot apical meristems as well as in young flowers. During the cell cycle, all four tobacco NAP1-like genes are highly expressed, with one of them showing a slightly increased expression at the G1/S transition. These results are consistent with a role for plant NAP1-like proteins in cell division. In vitro binding assays revealed that different NAP1-like proteins bind, with distinct relative binding strengths, to different classes of histone. Intracellular localization analyses showed that some NAP1-like proteins could be targeted into the nucleus whereas others are exclusively cytoplasm-localized. It is thus likely that different plant NAP1-like proteins have distinct functions in vivo. Plant NAP1-like proteins were observed to concentrate around the metaphase plate and in the phragmoplast, suggesting a role in mitotic events and cytokinesis.     

Kinetics of improved production and thermostability of an intracellular β-glucosidase from a mutant-derivative of Cellulomonas biazotea

The highest productivity (20 IU l(-1) h(-1)) of beta-glucosidase by a mutant of Cellulomonas biazotea was 2.5-fold more than that of the parent organism. The enzyme had a lower activation energy (57 kJ mol(-1)) than the native enzyme (68 kJ mol(-1)). The enzyme from the mutant had enthalpy and entropy values for irreversible intactivation of 95.6 kJ mol(-1) and 60 J.mol(-1) K(-1) compared with 108 kJ mol(-1) and 86 J mol(-1) K(-1) for the native enzyme suggesting that the mutation had stabilized the enzyme.     

Is the intrasomal phase of fast axonal transport driven by oscillations of intracellular calcium?

An hypothesis is presented suggesting that the delivery of vesicle-packaged protein from the neuronal soma to the axonal transport system is physiologically coupled to spontaneous fluctuations of intracellular calcium (Ca_i). Evidence is reviewed that oscillations of Ca_i, commonly detected as agonist-or voltage-triggered waves and spikes propagating through the cytosol, also occur as spontaneous events. Endogenously-generated oscillations are examined since intrasomal transport persists in the absence of extracellular signals or nerve impulse activity. Vesicle budding from the endoplasmic reticulum (ER) may be a key step at which anterograde transport is regulated by events related to the release and reuptake of ER stores of Ca²⁺.Special-issue dedicated to Dr. Sidney Ochs.