Biomedical subjects

A Zarka

Publications and source records attributed to A Zarka.

7 recordsLinked to original sources

PubMed2001

Suitability of Anabaena PCC7120 expressing mosquitocidal toxin genes from Bacillus thuringiensis subsp. israelensis for biotechnological application.

We present evidence that Anabaena PCC7120 (A.7120) strains expressing mosquitocidal toxin genes from Bacillus thuringiensis subsp. israelensis (Bti) have a strong potential for biotechnological application. Characterization of two 4-year-old recombinant A.7120 clones constructed previously in our laboratory [clone 7 and clone 11, each carrying three Bti genes (cry4Aa, cry11Aa, and p20)] revealed three facts. First, the Bti genes were stable in A.7120 even in the absence of antibiotic selection when the genes were integrated in the chromosome (in clone 11); and the genes were also stable as plasmid-borne constructs (in clone 7), provided the cultures were maintained under continued selection. Second, clone 7 (kept under selection) and clone 11 (either kept or not kept under selection) continued to be mosquitocidal through 4 years of culture. Third, growth of the recombinant clones was comparable to the wild type under optimal growth conditions, indicating that growth was not compromised by the expression of toxin genes. These results clear the way for the development of mass production techniques for A.7120 strains expressing Bti toxin genes.

Aedes↗

PubMed2001

Presence of a nonhydrolyzable biopolymer in the cell wall of vegetative cells and astaxanthin-rich cysts of Haematococcus pluvialis (Chlorophyceae).

The green alga Haematococcus pluvialis accumulates massive amounts of the red pigment astaxanthin in response to stimuli inducing it to form cysts. During the encystment process the cell wall undergoes a clear hardening and thickening. In this work, a cell wall fraction withstanding successive acid and basic hydrolysis was isolated and proves to be algaenan by Fourier transform infrared spectroscopy. This compound is equally abundant in nonmotile vegetative cells and astaxanthin-rich cysts. This finding indicates that the synthesis of algaenan does not require the activation of the machinery for the massive production of secondary carotenoids. We conclude that algaenan cannot cause the changes occurring in the cell wall during the encystment process without the involvement of other cell wall components.

Journal Article↗

PubMed1993

Characterization and photoaffinity labeling of the ATP binding site of the ryanodine receptor from skeletal muscle.

The photoaffinity analog of ATP, 3'-O-(4-benzoyl)benzoyl-adenosine 5'-triphosphate (Bz2ATP) was used to covalently label and to identify the ATP binding site of the skeletal muscle ryanodine receptor. Like ATP, Bz2ATP stimulates up to fivefold the binding of ryanodine to its receptor. Photoactivation by ultraviolet light of the benzophenone group in the [alpha-32P]Bz2ATP results in covalent binding of [alpha-32P]Bz2ATP to the 450-kDa polypeptide, the ryanodine receptor's subunit. An apparent molar stiochiometry of Bz2ATP to the tetrameric ryanodine receptor complex of 1.146 +/- 0.087 (n = 2) was estimated. The covalent binding of [alpha-32P]Bz2ATP was inhibited by ATP and analogous compounds in the order: ATP = AdoPP[CH2]P = ADP = Ado = cAMP > AMP > ITP = GTP. Similar specificity was obtained for the stimulation of ryanodine binding by these nucleotides. ATP increased the ryanodine binding affinity by about sixfold. The polycationic dye ruthenium red, known as an inhibitor of Ca2+ release and ryanodine binding, inhibited the labeling of the ryanodine receptor by [alpha-32P]Bz2ATP. Tryptic digestion of the ryanodine receptor revealed a [alpha-32P]Bz2ATP-labeled 76-kDa tryptic fragment. Digestion of either the [alpha-32P]Bz2ATP-labeled 450-kDa or the 76-kDa polypeptides with S. aureus resulted in the appearance of four labeled fragments of 39, 33, 27 and 13 kDa, where the 39-kDa fragment is the precursor of the 27-kDa and 13-kDa fragments. The results suggest that the regulation of Ca2+ release by ATP involves an ATP binding site(s) located on the 27-kDa and 13-kDa fragments of the ryanodine receptor protein.

Adenosine Triphosphate↗

PubMed1992

The interaction of spermine with the ryanodine receptor from skeletal muscle.

The effect of polyamines on ryanodine binding activity of junctional sarcoplasmic reticulum membranes is described. Spermine stimulated the binding of ryanodine to its receptor up to 5-fold, with half-maximal stimulation obtained with 3.5 mM. Spermidine and putrescine also stimulated ryanodine binding, but they were about 12-fold less potent. The degree of stimulation is dependent on the NaCl concentration present in the assay medium. Spermine has no effect on the Ca(2+)-dependency of ryanodine binding but it increases the ryanodine binding affinity (Kd) by about 5.6-fold. Both the rate of ryanodine association with, and dissociation from, its binding site were affected by spermine. Spermine also stimulates the photoaffinity labelling by 3-O-(4-benzoyl)benzoyl[alpha-32P]ATP ([alpha-32P]BzATP) of the ryanodine receptor, increasing the BzATP binding affinity. We suggest that the stimulatory effect of spermine on ryanodine binding is due to its specific interaction with the ryanodine receptor. This spermine interaction enabled us to develop a new, one-step, fast and with high yield method for the purification of ryanodine receptor (Shoshan-Barmatz, V. and Zarka, A. (1992) Biochem. J. 284, in press).

Animals↗

PubMed1992

A simple, fast, one-step method for the purification of the skeletal-muscle ryanodine receptor.

In this paper we describe a simple, fast, one-step method for the purification of the skeletal-muscle ryanodine receptor. The ryanodine receptor from CHAPS-solubilized junctional sarcoplasmic-reticulum membranes was adsorbed to a spermine-agarose column and eluted by 2 mM-spermine. The purified receptor, consisting predominantly of a 450 kDa polypeptide on SDS/PAGE, binds [3H]ryanodine with a specific activity of approximately 300 pmol/mg of protein and with a high affinity (KD = 4.7 +/- 2 nM). The purified receptor appears to retain the pharmacological properties of the receptor in the original membranes. The purification resulted in over 80% recovery of the initial ryanodine-binding sites and about 30-96-fold purification. This simple and fast method is highly reproducible and suitable for purification of small as well as large quantities of ryanodine receptor.

Animals↗

PubMed1988

Trypsin destruction of the high affinity ryanodine binding sites of the junctional sarcoplasmic reticulum.

Tryptic digestion of the junctional sarcoplasmic reticulum membranes in sucrose but not NaCl buffer leads to complete loss of ryanodine binding capacity. The presence of MgCl2 in the sucrose buffer prevents the loss of ryanodine binding by the trypsin treatment. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the treated membranes reveal that the 400-kDa protein band disappeared under all the different digestion conditions. However, the presence of 135-kDa tryptic fragment is observed only when ryanodine binding is retained. Quantitative analysis of the gels shows that the loss of ryanodine binding is well correlated with the cleavage of the 135-kDa tryptic fragment. This correlation is obtained when the cleavage was controlled either by the digestion time or by NaCl or MgCl2 concentrations. The same concentrations of MgCl2 and NaCl affect the ryanodine binding activity, the cleavage of the 135-kDa tryptic fragment, and the solubility and stability of the [3H]ryanodine-receptor complex in a detergent-containing medium. Tryptic digestion of the ryanodine receptor/junctional Ca2+ release channel, which leads to complete loss of ryanodine binding capacity, has no effect or slightly stimulates the Ca2+ accumulation activity of these membranes.

Adenosine Triphosphate↗

PubMedDate not supplied

Does astaxanthin protect Haematococcus against light damage?

The photoprotective function of the ketocarotenoid astaxanthin in Haematococcus was questioned. When exposed to high irradiance and/or nutritional stress, green Haematococcus cells turned red due to accumulation of an immense quantity of the red pigment astaxanthin. Our results demonstrate that: 1) The addition of diphenylamine, an inhibitor of astaxanthin biosynthesis, causes cell death under high light intensity; 2) Red cells are susceptible to high light stress to the same extent or even higher then green ones upon exposure to a very high light intensity (4000 mumol photon m(-2)s(-1)); 3) Addition of 1O2 generators (methylene blue, rose bengal) under noninductive conditions (low light of 100 mumol photon m(-2)s(-1) induced astaxanthin accumulation. This can be reversed by an exogenous 1O2 quencher (histidine); 4) Histidine can prevent the accumulation of astaxanthin induced by phosphate starvation. We suggest that: 1) Astaxanthin is the result of the photoprotection process rather than the protective; 2) 1O2 is involved indirectly in astaxanthin accumulation process.

Chlorophyta↗