The calmodulin and F-actin binding sites of smooth muscle caldesmon lie in the carboxyl-terminal domain whereas the molecular weight heterogeneity lies in the middle of the molecule

Caldesmons are major Ca2+-calmodulin regulated F-actin binding proteins of smooth and non-muscle cells that have been implicated as components of a thin filament regulatory system. Chicken gizzard caldesmons are monomeric proteins of Mr 140,000 and 135,000. We have employed enzymatic and chemical cleavage methods in order to dissect the protein to locate the Ca2+-calmodulin and F-actin binding domain and the site of molecular weight heterogeneity. Using a novel mapping procedure that employs partial chemical cleavage at cysteine residues, we show that both caldesmon polypeptides contain 2 cysteine residues located approximately 28,000 from the protein's amino terminus and the second approximately 25,000 from the carboxyl terminus. Identification of the composition of partial cleavage products with region-specific antibodies is consistent with this derived map. The apparent molecular weight heterogeneity was found to lie in the approximately 80,000 region between the 2 cysteine residues and therefore is not due to proteolytic processing. Digestion with alpha-chymotrypsin yields a relatively stable basic Mr 40,000 Ca2+-calmodulin and F-actin binding fragment that we have purified and characterized. The chymotryptic 40,000 fragment contains the 25,000 carboxyl-terminal fragment and therefore is derived from the carboxyl-terminal region of caldesmon. The 25,000 fragment obtained after chemical cleavage at cysteine under native conditions has also been purified and shown to bind F-actin and Ca2+-calmodulin. Surprisingly, the purified carboxyl 25,000 fragment, unlike the reduced intact monomer, cross-links F-actin into tightly ordered bundles in which the filaments are in register.     

In vitro propagation of seven <Emphasis Type="Italic">Daphne</Emphasis> L. species

Cultures of seven Daphne species: Daphne caucasica, D. cneorum, D. giraldii, D. retusa, D. jasminea, D. laureola and D. tangutica were established in vitro on MS/WPM based media. Five of the species responded best on MS-based media (D. tangutica, D. laureola, D. caucasica, D. retusa and D. giraldii), while the remaining two species performed best on WPM-based media (D. cneorum, and D. jasminea). Shoot proliferation was achieved from both apical and nodal explants. Shoots were sub-cultured from stock cultures, cut into nodal explants 3–5 cm long and place vertically on basal media supplemented with different concentrations and combinations of cytokinins and auxins. Individual species displayed different responses to the various cytokinins and auxins. Among species, D. jasminea produced the greatest proliferation rate with an average of 7.84 + 0.6 shoots per explant on WPM supplemented with 2.32 μM BA + 0.0045 μM TDZ + 0.054 μM NAA, while the best multiplication rate for the same species grown on the same media supplemented with a single cytokinin (BA) and no auxin was 2.60 + 1.3 shoots per explant. Following multiplication, new shoots transferred to the elongation trails and then 50–100 mm Shoots used for rooting experiments. Increased rooting efficiencies were observed on in vitro-generated shoots with the two-layer medium or dipping methods over when PGRs were uniformly incorporated into the medium. Maximum rooting frequencies (average) ranged from 59% in D. tangutica to 85% in D. jasminea. Following in vitro rooting, rooted shoots immersed in 0.01% solution of humates and planted into a standard horticultural substrate composed and watered weekly with a solution containing half-strength MS salts.     

Organogenesis plant regeneration from leaf explants of Exacum Styer Group

Exacum Styer Group plantlets were regenerated through direct organogenesis from leaf explants. Four genotypes were evaluated on MS media supplemented with combinations of BA (0, 0.44, 2.22, 4.44, or 8.88 μM) and NAA (0, 0.05, 0.54, or 2.69 μM) for direct shoot organogenesis without an intervening callus phase. Regression analyses were used to analyze and interpret the data. There were significant genotype, media, and genotype × media interactions for several variables. Genotypes 01-09-01 and 01-37-61 had the highest number of shoots per explant across media (10.2 and 6.6, respectively) while the 4.44 μM BA plus 0.54 μM NAA treatment induced the greatest number of shoots among the genotypes evaluated.     

Effects of iron concentration on pigment composition in Phaeocystis antarctica grown at low irradiance

Interpretation of photosynthetic pigment data using iterative programs such as CHEMTAX are widely used to examine algal community structure in the surface ocean. The accuracy of such programs relies on understanding the effects of environmental parameters on the pigment composition of taxonomically diverse algal groups. Phaeocystis antarctica is an important contributor to total autotrophic production and the biogeochemical cycling of carbon and sulfur in the Southern Ocean. Here we report the results of a laboratory culture experiment in which we examined the effects of ambient dissolved iron concentration on the pigment composition of colonial P. antarctica, using a new P. antarctica strain isolated from the southern Ross Sea in December 2003. Low-iron (<0.2 nM dissolved Fe) filtered Ross Sea seawater was used to prepare the growth media, thus allowing sub-nanomolar iron additions without the use of EDTA to control dissolved iron concentrations. The experiment was conducted at relatively low irradiance (∼20 μE m⁻² s⁻¹), with P. antarctica primarily present in the colonial form—conditions that are typical of the southern Ross Sea during austral spring. Relative to the iron-limited control treatments (0.22 nM dissolved Fe), iron addition mediated a decrease in the ratio of 19′-hexanoyloxyfucoxanthin to chlorophyll a, and an increase in the ratio of fucoxanthin to chlorophyll a. Our results also suggest that the ratio of 19′-hexanoyloxyfucoxanthin to chlorophyll c₃ (Hex:Chl c₃ ratio) may be a characteristic physiological indicator for the iron-nutritional status of colonial P. antarctica, with higher Hex:Chl c₃ ratios (>3) indicative of Fe stress. We also observed that the ratio of fucoxanthin to 19′-hexanoyloxyfucoxanthin (Fuco:Hex ratio) was highly correlated (r ² = 0.82) with initial dissolved Fe concentration, with Fuco:Hex ratios <0.05 measured under iron-limited conditions (dissolved Fe <0.45 nM). Our results corroborate and extend the results of previous experimental studies, and, combined with pigment measurements from the southern Ross Sea, are consistent with the hypothesis that the interconversion of fucoxanthin and 19′-hexanoyloxyfucoxanthin by colonial P. antarctica is used as a photo-protective or light-harvesting mechanism, according to the availability of dissolved iron.     

Insecticidal activity of selected monoterpenoids and rosemary oil to Agriotes obscurus (Coleoptera: Elateridae)

Acute toxicities of three naturally occurring monoterpenoid essential oil constituents and the essential oil of rosemary were tested against late instars of Agriotes obscurus (L.) (Coleoptera: Elateridae). Both contact and volatile toxicities of thymol, citronellal, eugenol, and rosemary oil were determined. Also, phytotoxicity of these compounds was evaluated on corn germination and seedling development. Thymol had the greatest contact toxicity (LD50 = 196.0 microg/larva), whereas citronellal and eugenol were less toxic (LD50 = 404.9 and 516.5 microg/larva, respectively). Rosemary oil did not show any significant contact toxicity, even at 1,600 microg/larva. In terms of volatile toxicity, citronellal was the most toxic to wireworm larvae (LC50 = 6.3 microg/cm3) followed by rosemary oil (LC50 = 15.9 microg/cm3), thymol (LC50 = 17.1 microg/cm3), and eugenol (LC50 = 20.9 microg/cm3). Thymol, eugenol, and citronellal significantly inhibited corn seed germination and development, whereas rosemary oil had only minimal phytotoxic effects.     

Smooth muscle caldesmon is an extended flexible monomeric protein in solution that can readily undergo reversible intra- and intermolecular sulfhydryl cross-linking. A mechanism for caldesmon's F-actin bundling activity

Caldesmon is a major F-actin binding protein of smooth muscle that has been implicated as a component of a thin filament regulatory system. Chicken gizzard caldesmon consists of polypeptides of Mr-135,000 and 140,000 which are closely related as determined by analysis of cyanogen bromide cleavage fragments. It is a highly extended flexible protein having a contour length of about 146 nm and a secondary structure composed primarily of random coil. Physical and chemical cross-linking data suggest that caldesmon exists as a monomer in solution. The cysteine content of caldesmon was determined to be 2 residues/polypeptide. Remarkably, in solution it readily undergoes sulfhydryl oxidation to form either an internal disulfide bridge in the protein or cross-links between individual polypeptides to form dimers, trimers, tetramers, etc. The internally cross-linked species have a smaller Stokes radius than the reduced molecules, indicating that the cross-link "trapped" the molecule in a compact conformation. Oxidized protein containing caldesmon oligomers is a potent F-actin bundling protein. Complete reduction of caldesmon abolishes the F-actin bundling activity. Since a vast excess of reducing agent is required to convert caldesmon from an oxidized to reduced state, it may exist in either state in vivo. Thus, the ability of caldesmon to undergo reversible sulfhydryl cross-linking, and thereby reversible F-actin cross-linking, may be of physiological significance.     

Evidence for high iron requirements of colonial Phaeocystis antarctica at low irradiance

We have carried out field and laboratory experiments to examine the iron requirements of colonial Phaeocystis antarctica in the Ross Sea. In December 2003, we performed an iron/light-manipulation bioassay experiment in the Ross Sea polynya, using an algal assemblage dominated by colonial Phaeocystis antarctica, collected from surface waters with an ambient dissolved Fe concentration of ∼0.4 nM. Results from this experiment suggest that P. antarctica growth rates were enhanced at high irradiance (∼50% of incident surface irradiance) but were unaffected by iron addition, and that elevated irradiance mediated a significant decrease in cellular chlorophyll a content. We also conducted a laboratory iron dose–response bioassay experiment using a unialgal, non-axenic strain of colonial P. antarctica and low-iron (<0.2 nM) filtered seawater, both collected from the Ross Sea polynya in December 2003. By using rigorous trace-metal clean techniques, we performed this dose–response iron-addition experiment at ∼0°C without using organic chelating reagents to control dissolved iron levels. At the relatively low irradiance of this experiment (∼20 μE m⁻² s⁻¹), estimated nitrate-specific growth rate as a function of dissolved iron concentration can be described by a Monod relationship, yielding a half-saturation constant with respect to growth of 0.45 nM dissolved iron. This value is relatively high compared to reported estimates for other Antarctic phytoplankton. Our results suggest that seasonal changes in the availability of both iron and light play critical roles in limiting the growth and biomass of colonial Phaeocystis antarctica in the Ross Sea polynya.     

Physiological and morphological traits associated with zinc efficiency in Exacum

Interspecific hybrids of Exacum exhibit variation in the expression of zinc efficiency. This research investigated the genetic basis for this variation and evaluated a series of physiological and morphological traits for their association with zinc efficiency. Chi-square analyses of self-pollinated progeny from both zinc-efficient and zinc-inefficient parents indicate a significant genetic component. One hundred percent of the progeny from the inefficient parent were classified as inefficient, while the progeny from the efficient parent segregated 32% inefficient to 68% efficient. Six plants from each phenotypic class (efficient and inefficient) of the efficient parent were utilized in analyses of plant traits. Statistically significant associations were identified between the zinc-efficient phenotype and mol Zn uptake mg^-1 root, root-to-shoot ratio, specific root length, mol Zn uptake cm^-2 root surface area, and Zn uptake cm^-1 root length. No association was identified between zinc-efficient phenotype and root diameter, transpiration rate, or H⁺ production. Zinc uptake cm^-1 root length had the greatest association with the zinc-efficiency phenotype and was able to discriminate the two phenotypic classes. We suggest that Zn uptake cm^-1 root length is the most significant factor explaining the variation between the zinc-efficient and zinc-inefficient phenotypes in Exacum.