The antihypertensive drug carvedilol inhibits the activity of mitochondrial NADH-ubiquinone oxidoreductase

A study is presented on the interaction of carvedilol with mitochondria isolated from several rat organs. It is shown that carvedilol causes a moderate uncoupling effect under non phosphorylating succinate supported respiration of intact mitochondria, as well as a marked inhibition of coupled respiration with NAD-dependent substrates. The inhibitory effect was also found in the bovine heart purified Complex I as well as in experiments with mitochondrial particles, where the individual redox segments of the respiratory chain were analysed. It is also shown that carvedilol, though exhibiting an intrinsic scavenger activity, caused reactive oxygen species to be produced as a consequence of its inhibitory effect on the steady-state respiration. Under these conditions the pro-oxidant activity of carvedilol appears to prevail over its scavenging activity, and a net generation of ROS is promoted.     

Membrane biogenesis in anoxygenic photosynthetic prokaryotes

Following the discovery of photosynthetic bacteria in the nineteenth century, technical developments of the 1950s led to their use in membrane biogenesis studies. These investigations had their origins in the isolation of subcellular particles designated as ‘chromatophores’ by Roger Stanier and colleagues, which were shown to be photosynthetically competent by Albert Frenkel, and to originate from the intracytoplasmic membrane (ICM) continuum observed in electron micrographs. These ultrastrucutral studies by the G. Drews group, Germaine Cohen-Bazire and others also suggested that the ICM originates by invagination of the cytoplasmic membrane, as later established in the biochemical and biophysical work of the R. Niederman and Drews groups. Through a combination of genetic approaches, first introduced in the early 1980s by Barry Marrs, and the atomic resolution structures determined for light-harvesting antennae and reaction centers, a detailed understanding is emerging of mechanisms regulating their levels in the membrane and the roles played by specific protein domains and additional factors in their assembly and supramolecular organization. Prospects for additional progress during the twenty-first century include further elucidation of molecular aspects of the assembly process and the application of newer spectroscopic probes to photosynthetic unit formation. This revised version was published online in June 2006 with corrections to the Cover Date.     

The discovery of Rubisco activase – yet another story of serendipity

A brief history of Rubisco (ribulose bisphosphate carboxylase oxygenase) research and the events leading to the discovery and initial characterization of Rubisco activase are described. Key to the discovery was the chance isolation of a novel Arabidopsis photosynthesis mutant. The characteristics of the mutant suggested that activation of Rubisco was not a spontaneous process in vivo, but involved a heritable factor. The search for the putative factor by 2D electrophoresis identified two polypeptides, genetically linked to Rubisco activation, that were missing in chloroplasts from the mutant. An assay for the activity of these polypeptides, which were given the name Rubisco activase, was developed after realizing the importance of including ribulose bisphosphate (RuBP) in the assay. The requirement for ATP and the subsequent identification of activase as an ATPase came about fortuitously, the result of a RuBP preparation that was contaminated with adenine nucleotides. Finally, the ability of activase to relieve inhibition of the endogenous Rubisco inhibitor, 2-carboxyarabinitol 1-phosphate, provided an early indication of the mechanism by which activase regulates Rubisco. This revised version was published online in June 2006 with corrections to the Cover Date.     

POR structural domains important for the enzyme activity in R. capsulatus complementation system

NADPH:protochlorophyllide oxidoreductase (POR) catalyzes hydrogen transfer from NADPH to protochlorophyllide (PChlide) in the course of chlorophyll biosynthesis in photosynthetic organisms and is involved in the regulation of the development of photosynthetic apparatus in higher plants, algae and cyanobacteria. To approach molecular factors determining the enzyme activity in a living cell, several mutants of POR from pea (Pisum sativum) with site-directed modifications in different parts of the enzyme were generated. The mutant enzymes were expressed in a R. capsulatus mutant deficient in BChl biosynthesis, and their catalytic activity and ability to integrate in bacterial metabolism were analyzed. Our results demonstrate that in heterologous bacterial cell system, higher plant POR is integrated in the porphyrin biosynthesis network and its activity leads to the formation of photosynthetic chlorophyll-proteins (CPs). The study of POR mutants in R. capsulatus reveals several POR domains important for the association of the enzyme with other subcellular components and for its catalytic activity, including identification of putative enzyme reaction center and substrate binding site. The study also demonstrated that an unknown structural factor is important for the formation of the enzyme photoactive complex in etiolated plants. Moreover, our findings suggest that POR might be directly involved in the regulation of the metabolism of other porphyrins. This revised version was published online in June 2006 with corrections to the Cover Date.     

14-3-3 protein regulation of proton pumps and ion channels

In addition to their regulation of cytoplasmic enzymes, the 14-3-3 proteins are important regulators of membrane localised proteins. In particular, many of the cells' ion pumps and channels are either directly or indirectly modulated by 14-3-3 proteins. Binding of 14-3-3 can lead to the activation of pump activity as in the case of the plasma membrane H⁺-ATPase or inhibition as in the case of the F-type ATP synthase complexes. 14-3-3 binding can also lead to surprising results such as the recruitment of `sleepy' outward rectifiying K⁺ channels in tomato cells. Our present knowledge extends to an initial understanding of isoform-specific binding of 14-3-3 to certain membrane proteins and a perception of the protein kinases and phosphatases that maintain the regulatory process in a state of flux.     

Molecular and Biochemical Diagnosis of Esterase-Mediated Pyrethroid Resistance in a Mexican Strain of Boophilus microplus (Acari: Ixodidae)

We examined pyrethroid resistant Mexican strains of Boophilus microplus using biochemical and molecular tests to determine the mechanisms conferring resistance. Permethrin hydrolysis assays and esterase activity gels indicated enhanced esterase-mediated metabolic detoxification in the Cz strain, while one other pyrethroid resistant strain, SF, and two pyrethroid susceptible strains had lower levels of permethrin hydrolysis. Results from assays using a PCR-based test to detect a pyrethroid target site resistance-associated mutation in the tick sodium channel gene found only low levels of mutations in the Cz strain, while the SF strain had a high level of the mutated sodium channel alleles. A specific esterase, designated CzEst9, believed to be responsible for the esterase-mediated pyrethroid resistance in the Cz strain was purified, and the gene encoding CzEst9 cloned. This revised version was published online in July 2006 with corrections to the Cover Date.     

Markovian models for the developmental study of social behavior

Behavioral development involves changes in the probabilities of both social and nonsocial activities and the sequential pattern of activities over time. A number of methods have been offered for the analysis of these patterns of behavioral sequences. However, there continue to be problematic issues, including the analysis of nonstationary data; accommodation of changes in patterns within an observation period, or over repeated observations or age; and identification of differences in pattern changes between individuals or groups, and the factors responsible for these differences. In this work, we analyze data from 15 young monkeys (Macaca nemestrina) using classification and Markovian methods, including a new approach to nonstationary data called the double-chain Markov model (DCCM). These methods allowed us to identify differences in behavior patterns that differentiate between normal subjects and those presenting developmental anomalies.     

Aggregation of recombinant bovine granulocyte colony stimulating factor in solution

Aggregation of recombinant bovine granulocyte colony-stimulating factor (rbG-CSF) was examined by the techniques of size exclusion chromatography (SEC), multiangle laser light scattering (MALS), and SDS-PAGE. Solutions of rbG-CSF in different buffers and pH were exposed to an elevated temperature of 50°C to induce aggregation. The formation of noncovalent soluble aggregates with molecular weight in the millions of Daltons was observed when a solution of rbG-CSF at pH 2.9 was exposed to 50°C. Precipitated protein was the main product of rbG-CSF aggregation in citrate and phosphate buffers at a pH greater than 4. It was demonstrated that precipitant was a mixture of covalent and noncovalent aggregates. The ratio of covalent to noncovalent binding increased with increase in pH of the protein solution. The covalent binding that occurred was primarily due to disulfide linkages via intermolecular disulfide scrambling as demonstrated by SDS-PAGE.     

Protein acylation in the cardiac muscle like cell line,H9c2

Besides serving as oxidisable substrates, fatty acids (FA) are involved in co- and post-translational modification of proteins (protein acylation). Despite the high rate of fatty acid utilisation in the heart, information on protein acylation in cardiac muscle is scarce. To explore this subject in more detail, we used the H9c2 cell line as an experimental model. After incubation with ³H-palmitate or ³H-myristate, cells were lysed and proteins precipitated, followed by extensive delipidation. The delipidated proteins were subjected to SDS-PAGE and transferred to nitro-cellulose prior to autoradiography. In addition, TLC was used to separate the various lipid classes. The first aspect we addressed was the extent of protein acylation as a function of time, relative to fatty acid incorporation into various lipid classes. Cells were incubated for 30 min, 1 h and 2 h with 100 Ci palmitate (PA, 2.3 nmol) or 125 Ci myristate (MA, 2.5 nmol). Palmitoylation increased from 0.48 ± 0.25 to 1.25 ± 0.56 Ci/mg protein between 30 min to 2 h, while myristoylation increased from 0.25 ± 0.12 to 0.77 ± 0.36 Ci/mg protein. Furthermore, delipidated proteins subjected to autoradiography showed that a set of distinct proteins was labelled with ³H-palmitate. Incorporation into phospholipids (PL) increased from 40–60% of the total amount of radio-labelled PA or MA supplied between 30 min and 2 h. Only the FA pool differed between MA and PA, with a higher FA content present after incubations with MA. Second, we investigated palmitoylation and incorporation into cellular lipids as a function of the amount of PA applied. Palmitoylation showed saturation at high PA concentrations. The percentage incorporation of ³H-PA in the various lipids depended on the amount of PA added: a decline in the PL pool with a concomitant increase in the size of the diacylglycerol pool at high PA concentrations. Third, inhibition of palmitoylation by cerulenin and tunicamycin was investigated. While both were able to inhibit palmitoylation, cerulenin also inhibited the incorporation of PA into various lipid classes, indicating differences in inhibitory action.     

Structural basis of the rind disorder oleocellosis in Washington navel orange (Citrus sinensis L. Osbeck)

Oleocellosis, a physiological rind disorder of citrus fruit, is an unattractive surface blemish caused by phytotoxic effects of released rind oils. The development of oleocellosis in Washington navel orange (Citrus sinensis L. Osbeck) was examined by following a time sequence of surface symptoms and microscopic rind changes. The two natural causes of oleocellosis were simulated: mechanical damage to the fruit and transfer of rind oil between fruit. Mechanical fruit injury resulted in rupture of the epidermis above oil glands. Released surface oil appeared to infiltrate the rind via the ruptured epidermis resulting in rapid degeneration of cortical, but not epidermal, cell contents. Oil application to the rind surface produced a more severe blemish than did mechanical damage. The oil appeared to diffuse through the cuticle causing degeneration of the contents of all cell layers, including the epidermis. Loss of membrane integrity was detected within 30 min, followed by cell content degeneration and cell collapse. The resulting blemish, characterized by rind collapse and darkening, developed substantially within 3 d and was attributed to the cellular damage.