Synthesis of two carboxylic haptens for raising antibodies to 25-hydroxyvitamin D3 and 1alpha,25-dihydroxyvitamin D3

Hapten derivatives of 25-hydroxyvitamin D(3) and 1alpha,25-dihydroxyvitamin D(3) were synthesized using the Wittig-Horner approach. Both haptens bearing a carboxylic group at the side chain that can be linked to a protein for raising antibodies of potential utility for the determination of 25-hydroxyvitamin D(3), 1alpha,25-dihydroxyvitamin D(3) and 1alpha-hydroxylated vitamin D(3) analogues.     

Water stress effects on the content and organization of chlorophyll in mesophyll and bundle sheath chloroplasts of maize

The consequences of drought stress on the organization of chlorophyll into photosynthetic units and on the chlorophyll-protein composition of mesophyll and bundle sheath chloroplasts of Zea mays L. were studied. It was found that the majority of chlorophyll lost in response to water stress occurs in the mesophyll cells with a lesser amount being lost from the bundle sheath cells. All of the chlorophyll loss could be accounted for by reduction in the lamellar content of the light-harvesting chlorophyll a/b-protein, a rather specific target for water stress. The decreased content of this chlorophyll-protein accounts for the elevated chlorophyll a/b ratios and the reduced photosynthetic unit sizes of the two cell types in stressed plants. The implications of the selective catabolism of this major membrane component are discussed.     

El carcinoma diferenciado incidental de tiroides es menos prevalente en la enfermedad de Graves que en el bocio multinodular

ObjectiveRisk factors for differentiated thyroid carcinoma (DTC) are poorly understood, but serum TSH levels, thyroid nodularity, and presence of autoimmunity are well-recognized factors that modulate DTC prevalence. TSH stimulates proliferation of both normal and neoplastic follicular cells. Consequently, thyroid-stimulating immunoglobulins (TSI), because of its TSH-like action, should induce DTC progression in patients with Graves’ disease (GD). The study objective was to compare the prevalence of incidental DTC in patients undergoing thyroidectomy for benign thyroid disease.MethodsThe pathology reports of 372 patients with preoperative diagnosis of euthyroid multinodular goiter (EMG) or hyperthyroidism were reviewed. Scintigraphy results and serum TSI levels were used to diagnosed either GD or hyperactive MG (HMG) to hyperthyroid subjects. Prevalence of DTC in each category was calculated using a Chi-square test.ResultsEMG, GD, and HMG were diagnosed in 221, 125, and 26 patients. There were 58 DTCs, distributed as follows [n (%)]: EMG, 49 (22.2%); GD, 8 (6.4%), and HMG, 1 (3.8%). Difference in prevalence of incidental DTC between the groups was statistically significant (p < 0.001). After adjustment for age, patients with EMG had a greater DTC prevalence than GD patients, with an OR of 4.17 (p < 0.001). Tumor size (mm, mean ± SD) was 6.92 ± 11.26, 1.97 ± 1.85, and 9.0 for EMG, GD and HMG respectively (p = 0.017).ConclusionsIncidental DTC was less prevalent in GD as compared to EMG irrespective of age. This finding may suggest a predisposition to develop DTC in patients with thyroid nodular disease and/or a potential effect of autoimmunity to protect against development of neoplastic disease.     

PHOTOSYNTHETIC LIGHT-HARVESTING FUNCTION OF VIOLAXANTHIN IN NANNOCHLOROPSIS SPP. (EUSTIGMATOPHYCEAE)1

Whole cell absorption spectra of the Eustigmatophycean algae Nannochloropsis salina Bourrelly and Nannochloropsis sp. reveal the presence of a distinct absorption peak at 490 nm. The lack of chlorophylls b and c in these species indicates that this peak must be attributed to carotenoid absorption. In vivo fluorescence excitation spectra for chlorophyll a emission show a corresponding maximum at 490 nm. This peak is more clearly resolved than carotenoid maxima in other algal classes due to the absence of accessory chlorophylls. The carotenoid composition of the two Nannochloropsis species shows that violaxanthin and vaucheriaxanthin are the main contributors to 490 nm absorption. Violaxanthin accounts for approximately 60% of the total carotenoid in both clones. We conclude that light absorption by violaxanthin, and possibly by vaucheriaxanthin, is coupled in energy transfer to chlorophyll a and that violaxanthin is the major light-harvesting pigment in the Eustigmatophyceae. This is the first report of the photosynthetic light-harvesting function of this carotenoid.     

PHYLOGENETIC DISTRIBUTION OF THE MAJOR DIATOM LIGHT-HARVESTING PIGMENT-PROTEIN DETERMINED BY IMMUNOLOGICAL METHODS 1

Monospecific, polyclonal antibodies raised against the apoprotein of the major light-harvesting pigment-protein of Phaeodactylum tricornutum Bohlin UTEX 646 were used to determine (1) whether this complex was common to the class Bacillariophyceae, whose members contain chlorophylls a and c and fucoxanlhin; (2) whether antigenically-related apoproteins were present in other chlorophyll c-containing groups, and (3) whether there was immunological homology with the light-hanvsting chlorophyll a/b protein of similar photosynthetic function in the Chlorophyta and vascular plants. We have used protein blotting techniques to show that antibodies against the two P. tricornutum light-harvesting complex polypeptides cross-reacted with one or two polypeptides of similar molecular weight (17–21 kD) in all ten diatom species examined, representing two orders and six families. No cross-reactivity was obtained with total membrane polypeptides from isolated representatives of three chromophyte algal divisions (Chrysophyta, Cryptophyta, Pyrrophyta), all of which contained chlorophyll c. No cross-reactivity was observed with membrane Polypeptides isolated from members of two classes of Chlorophte algae. These data suggest that the Bacillariophyceae may be monophyletic, and that the primary structure of the diatom light-harvesting complex is not closely related to pigment-protein complexes with similar function in other chlorophyll c-containing unicellular algal groups. Lastly, it may be possible to use the antibodies to the diatom light-harvesting polypeptides as specific markers for diatoms in natural phytoplankton assemblages.     

SEASONAL PATTERNS OF PHOTOSYNTHESIS AND LIGHT-INDEPENDENT CARBON FIXATION IN MARINE MACROPHYTES1

The contribution of light-independent carbon fixation (LICF) to the overall carbon gain and the seasonal patterns of maximum photosynthesis (P_max and LICF were characterized in a broad taxonomic range of macrophytes from Monterey Bay, California. P_max and LICF rates (nmol C.g filtered seawater^?1.min^?1) varied among species and taxonomic groups examined, and as a function of tissue type in the phaeophyte Laminaria setchellii Silva (Phaeophyceae). On average, P_max values were higher in the Rhodophyta, whereas LICF rates were greater in the Phaeophyceae. LICF rates were generally less than 5% of P_max in the marine macrophytes studied and, as a consequence, cannot fully compensate for respiratory carbon losses, which usually are greater than 10% of P_max. All species studied possessed the highest P_max and LICF rates when irradiance levels were highest and decreased during periods of low incident irradiance. Seasonal patterns of P_max and LICF in most of the macrophytes from the stenothermal environment of Monterey Bay were strongly correlated with photosynthetic photon flux rather than seawater temperature. The concomitant decrease of LICF and P_max rates in all species examined argues against LICF playing a major role in carbon acquisition under light-limiting conditions as suggested previously. Rather, the strong positive correlation of P_max and LICF indicates the direct coupling of photosynthate (e.g. 3-phosphoglyceric acid) generation with production of substrates for LICF reactions. Our results also suggest that LICF might be a useful indicator of photosynthetic metabolism in marine macrophytes.     

Isolation and characterization of glutamine synthetase from the marine diatom Skeletonema costatum.

Two peaks of glutamine synthetase (GS) activity were resolved by anion-exchange chromatography from the marine diatom Skeletonema costatum Grev. The second peak of activity accounted for greater than 93% of total enzyme activity, and this isoform was purified over 200-fold. Results from denaturing gel electrophoresis and gel-filtration chromatography suggest that six 70-kD subunits constitute the 400-kD native enzyme. The structure of the diatom GS, therefore, appears more similar to that of a type found in bacteria than to the type common among other eukaryotes. Apparent Michaelis constant values were 0.7 mM for NH4(+), 5.7 mM for glutamic acid, and 0.5 mM for ATP. Enzyme activity was inhibited by serine, alanine, glycine, phosphinothricin, and methionine sulfoximine. Polyclonal antiserum raised against the purified enzyme localized a single polypeptide on western blots of S. costatum cell lysates and recognized the denatured, native enzyme. Western analysis of the two peak fractions derived from anion-exchange chromatography demonstrated that the 70-kD protein was present only in the later eluting peak of enzyme activity. This form of GS does not appear to be unique to S. costatum, since the antiserum recognized a similar-sized protein in cell lysates of other chromophytic algae.     

Regulation of synthesis of the photosystem I reaction center

The in vivo biosynthesis of the P700 chlorophyll a-apoprotein was examined to determine whether this process is light regulated and to determine its relationship to chlorophyll accumulation during light- induced chloroplast development in barley (Hordeum vulgare L.). Rabbit antibodies to the 58,000-62,000-mol-wt apoprotein were used to measure relative synthesis rates by immunoprecipitation of in vivo labeled leaf proteins and to detect apoprotein accumulation on nitrocellulose protein blots. 5-d-old, dark-grown barley seedlings did not contain, or show net synthesis of, the 58,000-62,000-mol-wt polypeptide. When dark- grown barley seedlings were illuminated, net synthesis of the apoprotein was observed within the first 15 min of illumination and accumulated apoprotein was measurable after 1 h. After 4 h, P700 chlorophyll a-apoprotein biosynthesis accounted for up to 10% of the total cellular membrane protein synthesis. Changes in the rate of synthesis during chloroplast development suggest coordination between production of the 58,000-62,000-mol-wt polypeptide and the accumulation of chlorophyll. However, when plants were returned to darkness after a period of illumination (4 h) P700 chlorophyll a-apoprotein synthesis continued for a period of hours though at a reduced rate. Thus we found that neither illumination nor the rate of chlorophyll synthesis directly control the rate of apoprotein synthesis. The rapidity of the light-induced change in net synthesis of the apoprotein indicates that this response is tightly coupled to the primary events of light-induced chloroplast development. The data also demonstrate that de novo synthesis of the apoprotein is required for the onset of photosystem I activity in greening seedlings.     

The role of cytokinins in chloroplast lamellar development

The accumulation of chlorophyll, production of two specific lamellar chlorophyll-protein complexes, onset of O(2) evolution, and detection of P700 were examined in intact Jack bean (Canavalia ensiformis [L.] D.C.) leaves treated with 10(-5)m kinetin or benzyladenine and allowed to green under low (30-35%) and high (80-85%) relative humidity. In contrast to reports of the promotion of chlorophyll accumulation by cytokinin treatment in excised tissue or cotyledons, intact greening leaves showed neither promotion of chlorophyll accumulation nor alteration in formation of the lamellar chlorophyll-protein complexes or development of photosynthetic function. Furthermore, cytokinin was ineffective in relieving the consequences of low relative humidity water stress on chlorophyll accumulation and on the formation of at least one lamellar chlorophyll-protein.     

P(700) Chlorophyll a-Protein : Purification, Characterization, and Antibody Preparation

The P₇₀₀ chlorophyll α-protein was purified by preparative sodium dodecyl sulfate (SDS) gel electrophoresis from SDS-solubilized barley (Hordeum vulgare L., cv Himalaya) chloroplast membranes. After elution from the gel in the presence of 0.05 to 0.1% Triton X-100, the recovered protein had a chlorophyll/P₇₀₀ ratio of 50 to 60/1 and contained no chlorophyll b or cytochromes. Analysis of the polypeptide composition of the chlorophyll-protein revealed a 58 to 62 kilodalton (kD) polypeptide component but no lower molecular weight polypeptides. The 58 to 62 kD component was further resolved into two distinct polypeptide bands which were subsequently mapped by partial cyanogen bromide digestion and Staphylococcus aureus proteolysis. Based on results from the mapping experiments and other data, we suggest that the two components are conformational variants of a single polypeptide. Measurement of the chlorophyll to protein ratio by quantitative amino acid analysis and consideration of the yield of P₇₀₀ in the protein isolate suggest that, contrary to previous models (Bengis and Nelson, 1975, 1977), P₇₀₀in vivo is associated with a minimum of four subunits of approximately 60 kD.

Antibodies raised against the photochemically active chlorophyll-protein complex from barley reacted specifically with the 58 to 62 kD apoprotein. The same preparative electrophoresis procedure was used to isolate photochemically active P₇₀₀ chlorophyll a-protein from soybean (Glycine max L.), tobacco (Nicotiana tobacum L.), petunia (Petunia × hybrida), tomato (Lycopersicum esculentum), and Chlamydomonas reinhardti. The isolated complex from all species exhibited identical polypeptide compositions and chlorophyll/P₇₀₀ ratios. Antibodies to the barley protein cross reacted with all species tested demonstrating the highly conserved structure of the apoprotein.