On the nature of the fluorescence decrease due to phosphorylation of chloroplast membrane proteins

1. Phosphorylation of chloroplast membranes by illumination in the presence of ATP results in a 15–20% increase in the rate of Photosystem I electron transfer at low light intensity. 2. Phosphorylated membranes when depleted of Mg²⁺ and resuspended in a low salt medium still show a 17% lower yield of Photosystem II fluorescence than do unphosphorylated membranes. A 31% difference is seen after restoration of the maximal yield by addition of Mg²⁺. 3. The concentration of Mg²⁺ required to induce a half-maximal increase in fluorescence is 0.9 mM for control and 1.8 mM for phosphorylated chloroplasts. Phosphorylation at 1 mM Mg²⁺ can therefore cause more than double the amount of decrease in fluorescence yield from Photosystem II compared to phosphorylation at 5 mM. 4. The above results are discussed in terms of the mechanism of the ATP-induced fluorescence changes and a suggestion is made that the apparent interaction between phosphorylation and Mg²⁺ concentration may be a physiologically important phenomenon.     

Autophagic dysfunction in a lysosomal storage disorder due to impaired proteolysis

Alterations in macroautophagy (hereafter referred to as “autophagy”) are a common feature of lysosomal storage disorders, and have been hypothesized to play a major role in the pathogenesis of these diseases. We have recently reported multiple defects in autophagy contributing to the lysosomal storage disorder Niemann-Pick type C (NPC). These include increased formation of autophagosomes, slowed turnover of autophagosomes secondary to impaired lysosomal proteolysis, and delivery of stored lipids to the lysosome via autophagy. The study summarized here describes novel methods for the interrogation of individual stages of the autophagic pathway, and suggests mechanisms by which lipid storage may result in broader lysosomal dysfunction.     

Light-induced decrease in DCF fluorescence of wheat leaves in the presence of salicyl hydroxamate

5-(and-6)-Carboxy-2',7'-dichlorodihydrofluorescein diacetate (DCF-DA), a permeative indicator of oxidative stress, was loaded into dissected leaves of wheat in order to monitor the temporal development of reactive oxygen species. DCF fluorescence was found to be constant under dark conditions. Upon loading the leaves with salicyl hydroxamate, a blocker of the alternative oxidase, DCF fluorescence linearly increased in the dark. This indicates a function of alternative oxidase in preventing reactive oxygen radicals in the mitochondria. Upon illumination, the DCF signal decreased within 5 min. As illuminated chloroplasts would increase the load of reactive oxygen species, the observed decrease cannot be assigned to the production of reactive oxygen species in the chloroplasts. Three different putative mechanisms are considered which all assign an important role to light-induced delivery of NAD(P)H: (1) direct quenching of DCF fluorescence by light-generated NAD(P)H, (2) light-stimulated activation of scavenging enzymes, or (3) redirection of mitochondrial electron fluxes as caused by the delivery of excess redox equivalents (NADH) from the chloroplasts.     

Facilitation of the structural and functional disorders of liver lysosomes in toxic hepatitis due to the suppression of intralysosomal proteolysis

Suramin that accumulates in rat liver Kupffer cell lysosomes and inhibits the intralysosomal proteolysis was used to suppress the functional activity of these particles during liver damage (acute CCl4 hepatitis). Polyvinylpyrrolidone that does not disturb protein catabolism in liver lysosomes was employed for reference. According to the characteristic changes in lysosomes induced by suramin (inhibition of acid phosphatase, decrease of the rate of the intralysosomal proteolysis in the liver) and PVP the damaged liver was able to accumulate the lysosomotropic substances under study. Suramin aggravated liver damage and increased the lysosomal labilization, whereas PVP exhibited the protective action. The unfavourable effect of suramin may be linked with the suppression of catabolism of Kupffer cell lysosomes. The data obtained suggest the lack of safety of using the inhibitors of intralysosomal proteolysis in patients with acute hepatitis.     

Hyperglycemia impairs osteoblast cell migration and chemotaxis due to a decrease in mitochondrial biogenesis

Molecular and Cellular Biochemistry - Diabetes is associated with an increase in skeletal fragility and risk of fracture. However, the underlying mechanism for the same is not well understood....     

Causes of fluorescence in bidistilled water after electrochemical treatment

Fluorescence spectroscopy was used to study the shortwave fluorescence of bidistilled water treated in the cathode and anode chambers of two types of electrolyzers made of different materials. Electrochemical treatment in the quartz glass electrolyzer did not induce intrinsic fluorescence of the anolyte or catholyte. An increase in the shortwave fluorescence of the anolyte and catholyte was observed in the electrolyzer made of Plexiglass, which was probably due to the release of microcontaminants from components of the electrolyzer.     

Human male infertility may be due to a decrease of the protamine P2 content in sperm chromatin

Basic chromosomal proteins were extracted from the sperm of fertile and infertile human males. The relative proportions of protamine 1, 2, and 3 were determined by scanning microdensitometry following electrophoresis of total protamine in polyacrilamide gels. The findings were as follows: (1) The proportion of protamine P(2 + 3) in sperm obtained from infertile males was lower than that in fertile males. (2) Protamine P(2 + 3) in infertile human males showed reduced affinity to DNA. The possibility that some cases of human male infertility may be due to mutation within the protamine P2 gene is discussed. © 1993 Wiley-Liss, Inc.     

The decrease of Xenopus egg membrane capacity during activation might be due to endocytosis

An endocytotic process in Xenopus eggs was studied by direct visualization of the fluorescent tracer lucifer yellow CH in 1-μm-thick sections. Eggs fixed 3 min after activation show small fluorescent vesicles in the cortex; those fixed after 30 min show mainly large vesicles. This process is related to membrane recycling after cortical granule exocytosis and can account for the observed decrease of the effective membrane capacity.     

Wall turnover deficiency of Bacillus subtilis Ni15 is due to a decrease in teichoic acid

Bacillus subtilis Ni15 is deficient in cell wall turnover. The deficiency is removed if the medium contains 0.2 M NaCl, which does not affect growth. The levels of amidase and glucosaminidase, the most likely enzymes involved in turnover, were, in stationary phase Ni15 cells, similar to those in late-exponential phase cells of a standard strain. The Ni15 enzymes were not salt sensitive. However, the Ni15 walls contained 4.7-fold less phosphorus than the walls of the standard strain. Since the phosphorus content of B. subtilis walls reflects the level of teichoic acid, it is proposed that the turnover deficiency of this strain is due to a decrease in wall teichoic acid.     

Substates of cardiac sodium channels are due to both decrease in conductance and changes in selectivity.

Currents through DPI 201-106 modified single cardiac sodium channels in guinea pig ventricular cells were measured using the patch clamp technique in the cell-free configuration to control the sodium concentrations on both sides of the patch membrane. Current-voltage relationships of the single channels were obtained by application of linear voltage ramps from -140 to 100 mV. With 10 mmol/l Na+ at the inner surface of the patch, openings of sodium channels with conductances of 17 pS (selectivity ratios PK/PNa = 0.083 and PK/PNa = 0.58) and 12 pS (selectivity ratios PK/PNa = 0.084 and PK/PNa = 1.832) were obtained. With 30 mmol/l internal sodium, conductances of 20, 10, and 7 pS and selectivity ratios of 0.084, 0.386, and 0.543, respectively, could be measured. It is concluded that substates of sodium channel currents are due to changes in single channel conductance as well as in selectivity, or to changes of both independently of each other which accounts for the variability of conductance levels of cardiac Na channels.     

Decreased expression of MUC2 due to a decrease in the expression of lectins and apoptotic defects in colitis patients

IntroductionThe involvement of mucin, lectin, and apoptosis in colitis is still unclear. This study aimed to investigate changes in MUC2 expression, inflammation, and changes in lectin expression in colitis patients.MethodsA total of 17 patients were divided into two groups including 11 hemorrhoid patients as a control group and 6 colitis patients. MUC2 mutation analysis was carried out using immunofluorescent and FISH techniques. Assessment of caspase-3, Ki-67, NF-kB, and lectin expressions was also carried out by immunofluorescent technique then analyzed by confocal laser scanning microscope.ResultsThe MUC2, caspase-3, and lectin expressions were significantly lower in the colitis group than in the control group (p < 0.05).ConclusionsIt was concluded that in colitis there was a change in MUC2 expression due to changes in lectins accompanied by apoptotic defects.conclusion     

Bacterial Cooperation Causes Systematic Errors in Pathogen Risk Assessment due to the Failure of the Independent Action Hypothesis

The Independent Action Hypothesis (IAH) states that pathogenic individuals (cells, spores, virus particles etc.) behave independently of each other, so that each has an independent probability of causing systemic infection or death. The IAH is not just of basic scientific interest; it forms the basis of our current estimates of infectious disease risk in humans. Despite the important role of the IAH in managing disease interventions for food and water-borne pathogens, experimental support for the IAH in bacterial pathogens is indirect at best. Moreover since the IAH was first proposed, cooperative behaviors have been discovered in a wide range of microorganisms, including many pathogens. A fundamental principle of cooperation is that the fitness of individuals is affected by the presence and behaviors of others, which is contrary to the assumption of independent action. In this paper, we test the IAH in Bacillus thuringiensis (B.t), a widely occurring insect pathogen that releases toxins that benefit others in the inoculum, infecting the diamondback moth, Plutella xylostella. By experimentally separating B.t. spores from their toxins, we demonstrate that the IAH fails because there is an interaction between toxin and spore effects on mortality, where the toxin effect is synergistic and cannot be accommodated by independence assumptions. Finally, we show that applying recommended IAH dose-response models to high dose data leads to systematic overestimation of mortality risks at low doses, due to the presence of synergistic pathogen interactions. Our results show that cooperative secretions can easily invalidate the IAH, and that such mechanistic details should be incorporated into pathogen risk analysis.     

Origin of a fluorescence increase accompanying the limited proteolysis of fluorescein-labeled human prothrombin by Factor Xa

In a search for a probe which would report its proteolysis to thrombin, the human blood coagulation zymogen prothrombin was covalently labeled with fluorescein. Fluorescein isothiocyanate (FITC) and dichlorotriazinylaminofluorescein (DCTAF) both introduced approximately 1 molecule of dye, but labeling occurred at different locations, as FITC had no effect on clotting activity whereas DCTAF caused 95% inactivation. At pH 9.0 DCTAF, but not FITC, could induce labeling up to 4 mol/mol. All derivatives were activated normally by prothrombinase (the activating complex of Factor Xa, Factor V(a), Ca2+ and phospholipids), as indicated by the pattern of bands on SDS gel electrophoresis and an unaltered yield of activity toward a chromogenic substrate for thrombin. Upon undergoing this limited proteolysis, the most heavily labeled derivative showed a 40% increase in fluorescence of the fluorescein at 520 nm (lambda ex 480 nm). In contrast, the fluorescence of lightly labeled forms was more intense but increased by only 0-5% upon activation. The data suggest that the lower fluorescence of the most labeled form is due to an intramolecular quenching effect between the dye molecules on individual polypeptide chains that is partly relieved when activation occurs.     

Iron deficiency causes changes in chlorophyll fluorescence due to the reduction in the dark of the Photosystem II acceptor side

Iron deficiency was found to affect the redox state of the Photosystem II acceptor side in dark-adapted, attached leaves of sugar beet (Beta vulgaris L.). Dark-adapted iron-deficient leaves exhibited relatively high F_o and F_pl levels in the Kautsky chlorophyll fluorescence induction curve when compared to the iron-sufficient controls. However, far-red illumination led to marked decreases in the apparent F_o and F_pl levels. Modulated fluorescence showed that far-red light decreased the fluorescence yield to the true F_o levels by increasing photochemical quenching, without inducing changes in the level of non-photochemical quenching. In dark-adapted, iron-deficient leaves, far-red illumination induced a faster fluorescence decay in the µs-ms time domain, indicating an improvement in the electron transport after the primary quinone acceptor in the reducing side of Photosystem II. All these data indicate that in iron-deficient leaves the plastoquinone pool was reduced in the dark. The extent of the plastoquinone reduction in sugar beet depended on the chlorophyll concentration of the leaf, on the time of preillumination and on the duration of dark adaptation. The dark reduction of plastoquinone was observed not only in sugar beet but also in other plant species affected by iron deficiency both in controlled conditions and in the field.     

A specific decrease of the fluorescence depolarization of perylene in muscle membranes from mice with muscular dystrophy

The microviscosity of erythrocyte membranes and muscle microsomes from age matched 6-week old control mice REJ 129 Dy/Dy, and mice with muscular dystrophy REJ 129 dy/dy has been estimated by measuring the fluorescence depolarization of perylene. There was no difference between the erythrocyte membranes. The muscle microsomes from dystrophic animals had about 20% lower values than the controls. The temperature dependence indicated that a transition occurs in both sets of muscle microsomes, but the transition temperature was lower in the dystrophic microsomes. Cholesterol, phospholipid and triglyceride analyses of the membranes showed no difference between the erythrocyte membranes. The largest difference in the muscle microsomes was a two-fold increase in cholesterol level found in the dystrophic microsomes. No simple correlation could be made between the lipid analysis and the microviscosity measurements. Since the change in microviscosity is found in membranes isolated from the tissue primarily affected by the dy gene, we suggest that the change in microviscosity may be important in the development of the disease.     

Recombinant expression analysis of natural and synthetic bovine alpha-casein in Escherichia coli

As a prelude to developing a yeast-based fermentation process for the production of phenylalanine-free alpha-casein as a foodstuff for patients suffering from phenylketonuria, we cloned the gene encoding bovine alpha-casein. We synthesised a modified gene sequence encoding the same, but devoid of phenylalanine codons and with a codon bias similar to that of naturally occurring highly expressed genes in Saccharomyces cerevisiae. The results show that both gene sequences are readily expressed in Escherichia coli when cloned in an E. coli bacteriophage T7 promoter-driven plasmid vector. In this host, the natural and synthetic casein proteins were produced at levels equating to 18.0% and 7.6% of the cell's soluble protein, respectively. Received: 18 January 2000 / Received revision: 22 May 2000 / Accepted: 26 May 2000