Purification and characterization of a membrane-bound protein kinase from spinach thylakoids

A protein kinase was isolated from spinach thylakoid membranes by solubilization with octyl glucoside and cholate. The enzyme was purified to apparent homogeneity by ammonium sulfate precipitation, gel filtration, and sucrose density centrifugation, followed by affinity chromatography on either Affi-Gel blue (yielding denatured enzyme) or on histone cross-linked to Sepharose (yielding active enzyme). Electrophoresis on denaturing polyacrylamide gels, followed by staining with silver, revealed the kinase as a single band corresponding to an apparent molecular mass of 64 kDa. The active enzyme underwent autophosphorylation and could be detected by autoradiography following incubation with [gamma-32P]ATP and Mg2+ ion. The specific phosphotransferase activity of purified kinase was approximately 30 nmol of phosphate min-1 (mg protein)-1 with lysine-rich histone (III-S or V-S) as substrate; casein was phosphorylated at approximately 30% of this rate. The physiological substrate for the kinase is presumed to be light-harvesting chlorophyll a/b protein complex. In solubilized form, this was phosphorylated at approximately 10% of the rate observed with histone III-S as substrate, or 10-100 times slower than the estimated rate of phosphorylation of the light-harvesting complex in situ. Possible reasons for this shortfall are considered. The kinase is proposed as the principal effector of thylakoid protein phosphorylation and associated State transition phenomena.     

Evolution of the immunodominant domain of the circumsporozoite protein gene from Plasmodium vivax. Implications for vaccines

Recent work directed toward the development of a malarial vaccine has focused on the identification and production of the immunodominant repeating peptide of the circumsporozoite protein of the human malaria parasites as an antigen. An important factor which relates to the usefulness of this antigen in a vaccine is the rate at which the molecule changes in sequence. We have determined the sequence and arrangement of the repeating epitope of the circumsporozoite protein gene from a Plasmodium vivax isolate from La Paz, El Salvador (Sal-I). This is compared with a portion of the previously published sequence of the circumsporozoite protein gene from a P. vivax isolate from Belém, Brazil. The genes appear to be very similar in the repeat region. There are 20 similar repeating units in the El Salvador strain and only 19 units are conserved in the Brazilian strain. Following this there are degenerate repeats in both strains. Even the pattern of silent mutations in the repeat area are similar; however, they are not necessarily in the identical location and appear to have shifted. The data suggest that the repeat region of these genes may be evolving by an accelerated mechanism(s). Such a phenomenon could severely decrease the long-term efficacy of a repeat-based anti-sporozoite vaccine.     

Metabolic Syndrome Is Associated with Increased Oxo-Nitrative Stress and Asthma-Like Changes in Lungs

Epidemiological studies have shown an increased obesity-related risk of asthma. In support, obese mice develop airway hyperresponsiveness (AHR). However, it remains unclear whether the increased risk is a consequence of obesity, adipogenic diet, or the metabolic syndrome (MetS). Altered L-arginine and nitric oxide (NO) metabolism is a common feature between asthma and metabolic syndrome that appears independent of body mass. Increased asthma risk resulting from such metabolic changes would have important consequences in global health. Since high-sugar diets can induce MetS, without necessarily causing obesity, studies of their effect on arginine/NO metabolism and airway function could clarify this aspect. We investigated whether normal-weight mice with MetS, due to high-fructose diet, had dysfunctional arginine/NO metabolism and features of asthma. Mice were fed chow-diet, high-fat-diet, or high-fructose-diet for 18 weeks. Only the high-fat-diet group developed obesity or adiposity. Hyperinsulinemia, hyperglycaemia, and hyperlipidaemia were common to both high-fat-diet and high-fructose-diet groups and the high-fructose-diet group additionally developed hypertension. At 18 weeks, airway hyperresponsiveness (AHR) could be seen in obese high-fat-diet mice as well as non-obese high-fructose-diet mice, when compared to standard chow-diet mice. No inflammatory cell infiltrate or goblet cell metaplasia was seen in either high-fat-diet or high-fructose-diet mice. Exhaled NO was reduced in both these groups. This reduction in exhaled NO correlated with reduced arginine bioavailability in lungs. In summary, mice with normal weight but metabolic obesity show reduced arginine bioavailability, reduced NO production, and asthma-like features. Reduced NO related bronchodilation and increased oxo-nitrosative stress may contribute to the pathogenesis.     

Characterization of immunodominant epitopes of gag and pol gene-encoded proteins of human T-cell lymphotropic virus type I.

A series of synthetic peptides derived from the corresponding regions of the gag, pol, and env proteins of human T-cell lymphotropic virus types I (HTLV-I) and II (HTLV-II) were used in an enzyme immunoassay to map the immunodominant epitopes of HTLV. Serum specimens from 79 of 87 (91%) HTLV-I-infected patients reacted with the synthetic peptide Gag-1a (amino acids [a.a.] 102 to 117) derived from the C terminus of the p19gag protein of HTLV-I. Minimal cross-reactivity (11%) was observed with serum specimens from HTLV-II-infected patients. Peptide Pol-3, encoded by the pol region of HTLV-I (a.a. 487 to 502), reacted with serum specimens from both HTLV-I- and HTLV-II-infected patients (94 and 86%, respectively). The antibody levels to Pol-3 were significantly higher (P less than 0.01) in patients with HTLV-I-associated myelopathy/tropical spastic paraparesis than in either adult T-cell leukemia patients or HTLV-I-positive asymptomatic carriers. None of the other peptides studied demonstrated significant binding to serum specimens obtained from HTLV-I- or HTLV-II-infected individuals. While Gag-1a did not react with serum specimens from normal controls, Pol-3 demonstrated some reaction with specimens from seronegative individuals (11.4%). The antibodies to Gag-1a and Pol-3 in serum specimens from HTLV-I-infected patients could be specifically inhibited by the corresponding synthetic peptides and by a crude HTLV-I antigen preparation, indicating that these peptides mimic native epitopes present in HTLV-I proteins that are recognized by serum antibodies from HTLV-I- and -II-infected individuals.     

Construction of a hybrid plasmid capable of replication in Amycolatopsis mediterranei

A new plasmid, pA387, has been isolated from "Amycolatopsis sp." (DSM 43387). This plasmid could be isolated from liquid culture as well as mycelium from agar plates by a modified procedure. Plasmid pA387 is about 29.6 kb and can be cured at low frequency by protoplasting and ethidium bromide and heat treatment. Hybridization experiments showed that this plasmid is present in free form and does not integrate into the chromosome. A hybrid plasmid was constructed by cloning a 5.1-kb fragment of pA387 into the Escherichia coli vector pDM10. This hybrid plasmid, termed pRL1, could be transformed into Amycolatopsis mediterranei and A. orientalis by electroporation. A transformation frequency of 2.2 x 10(3) transformants per micrograms of DNA at 12.5 kV/cm and a pulse duration of 10.8 ms was obtained in A. mediterranei, whereas 1.1 x 10(5) transformants per microgram of DNA were obtained at a field strength of 7.5 kV/cm and a pulse duration of 7.6 ms in A. orientalis. Plasmid pRL1 is the first hybrid plasmid which could be used successfully for the transformation of A. mediterranei. The plasmid has a rather high copy number, is genetically stable, and can be easily reisolated from A. mediterranei. Plasmid pRL1 will be useful for further construction of a shuttle vector for E. coli and A. mediterranei and becomes the basis for the development of gene cloning techniques in Amycolatopsis spp.     

Endogenous phosphorylation of retinal photoreceptor outer segment proteins by calcium phospholipid-dependent protein kinase

A calcium phospholipid-dependent protein kinase (C-kinase) activity was detected in the soluble fraction of rod outer segments (ROS) of the bovine retina. The enzyme required calcium, phosphatidylserine (PS) and diacylglycerol for maximal activity. In the presence of calcium and PS, C-kinase endogenously phosphorylated proteins with molecular weights of 95,000, 91,000, 31,000, 21,000, 19,000, 18,000, 16,000, 14,000 and 11,000. Addition of diolein in the reaction mixture further enhanced the endogenous phosphorylation of these proteins. Retinal was found to inhibit the phosphorylation of endogenous proteins by C-kinase in a concentration dependent manner. Half-maximal inhibition of enzyme activity was obtained at a retinal concentration of about 12μM. These results suggest that calcium, phospholipids and the C-kinase enzyme may play an important role in the functional regulation of rod photoreceptors and, with retinal, perhaps in the visual process as well.     

Kinetic evidence for involvement of two cytochrome b-563 hemes in photosynthetic electron transport

The effect of 2-(n-heptyl)-4-hydroxyquinoline N-oxide (HQNO) on the kinetics of cytochrome b-563 and cytochrome c² turnovers following single-turnover flashes was measured in isolated heterocysts. Low concentrations of HQNO (below 3 μM) blocked reoxidation of cytochrome b-563, whereas higher concentrations (above 5 μM) resulted in additional inhibition of cytochrome b-563 oxidation and also inhibited reduction of cytochrome b-563 and cytochrome c. Similar effects on cytochrome b-563 reduction and reoxidation were obtained with a combination of 5 μM HQNO and 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (1–7 μM). In HQNO-inhibited heterocysts, cytochrome c reduction following a flash occurred in three phases with half-times of 0.5, 2.8 and 45 ms. The second phase nearly equalled the cytochrome b-563 reduction in half-time and magnitude. In the presence of HQNO, the reoxidation of cytochrome b-563 following two closely spaced actinic flashes displayed biphasic kinetics. The two phases correspond to reoxidation of cytochrome b-563 in which one or both of the cytochrome b-563 hemes in the cytochrome b–f complex are reduced. These results are interpreted in terms of a Q-loop in which HQNO, at low concentrations, blocks the site of rapid cytochrome b-563 reoxidation and at higher concentrations, also inhibits the site of electron donation by plastoquinol to the cytochrome b-f complex.