Rubber production in guayule: determination of rubber producing potential

Optimum conditions for the rapid, efficient, nondestructive determination of rubber producing potential in guayule (Parthenium argentatum) were established. The rubber producing potential may be defined as the ability of the plant material to synthesize rubber from a precursor under specified conditions. To achieve this, stem slices taken from the first 5 centimeters of branches were incubated with [¹⁴C]acetate as precursor in 0.1 molar phosphate buffer (pH 6.5) at 26°C for 16 hours in the light. The ¹⁴C from labeled acetate and acetyl coenzymeA were efficiently incorporated into rubber whereas the ¹⁴C from both mevalonic acid (MVA) and isopentenylpyrophosphate (IPP) were poorly incorporated. Incorporation of 68.6% of the ¹⁴C from labeled IPP into the acetone extractable material suggests that most of the IPP was channeled down the lower terpenoid branch of the polyisoprene biosynthetic pathway. The incorporation of ¹⁴C from labeled acetate into rubber was most efficient at temperatures between 20 and 25°C. The rubber producing potential was also found to be dependent on light intensity. The roots which represent about one-third of the plant biomass not only had the highest rubber producing potential but also contained the highest amount of rubber (7.6%), indicating that the root system could be a major source of rubber. The mature stem bark also had a high rubber content and rubber producing potential, whereas the young stem had a low rubber content and a lower potential for producing rubber. The leaves showed little potential to incorporate labeled acetate into rubber and no more than 0.5% rubber was found in guayule leaves.     

Structure and development of sieve areas in leaf veins ofWelwitschia

Summary The sieve areas inWelwitschia are essentially similar to those of coniferous sieve cells, consisting of groups of plasmalemma-lined pores, which are joined in the middle of the wall by a median cavity. The median cavity contains membranes which apparently are connected with aggregates of endoplasmic reticulum bordering the sieve areas. The median cavity is formed through union of smaller median enlargements, the median nodules, each initially associated with a plasmodesma, during perforation of the young sieve area. Callose platelets are not associated with developing pores. All fully-developed pores were lined with callose. The sieve cells are connected with only one other cell type, the albuminous cell. On the sieve-cell side of the wall these connections are similar to sievearea pores, on the albuminous-cell side to plasmodesmata. These connections are also characterized by median enlargements.This work was supported in part by a grant from the South African Council for Scientific and Industrial Research and in part by the U.S. National Science Foundation (GB 31417).     

Can enzymes adopt a self-inhibited form? Results of x-ray crystallographic studies of chymosin.

Chymosin molecules in the crystal lattice have Tyr77 occluding the S1/S3 substrate binding pockets suggesting that the enzyme is self-inhibited. An analysis of this structure in conjunction with its comparison with pepsin has shown that this is most probably an intrinsic property of the enzyme. It also indicates that chymosin's substrate specificity may be dependent upon the ability of the substrate to displace the tyrosine ring from the binding pockets. This analysis also implies that active and self-inhibited forms of other aspartic proteinases can exist in solution helping to explain the results of kinetic studies of these enzymes.     

CD4 and CD8 are positive regulators of T cell receptor signal transduction in early T cell differentiation.

Although cortical (CD4+CD8+) thymocytes mobilize intracellular calcium poorly when CD3/TCR is ligated, we have found that murine cortical thymocytes can transduce strong biochemical signals in response to ligation of the CD3/Ti TCR complex (CD3/TCR) and that the signals are regulated by CD4 and CD8 interactions with CD3/TCR. Striking increases in intracellular calcium were observed in cortical thymocytes from transgenic mice containing productively rearranged alpha and beta TCR genes, when CD3 or TCR was cross-linked with CD4 or CD8 using heteroconjugated mAb. However, in mature T cells derived from lymph nodes of these mice, identical stimuli elicited calcium responses that were significantly smaller in magnitude. A thymocyte cell line that expresses a low level of the transgenic TCR and has a phenotype characteristic of cortical thymocytes (CD4+CD8+J11d+Thy-1+) was established from a female alpha beta TCR transgenic mouse. Cross-linking of CD4 or CD8 molecules to CD3/TCR induced strong calcium responses in these cells. Responses were weak or absent when CD3 or TCR were aggregated alone. Heteroconjugates of Thy-1xCD3 did not increase the intracellular calcium concentration in transgenic thymocytes or in the thymocyte cell line, although Thy-1 is highly expressed on immature cells. Enhanced tyrosine phosphorylation was observed when CD3 or TCR was cross-linked with CD4 or CD8 on transgenic thymocytes or on the thymocyte cell line, in comparison with aggregation of CD3/TCR alone. Taken together, these data show that CD4 and CD8 molecules allow the weakly expressed CD3/TCR of cortical thymocytes to transduce strong intracellular signals upon receptor ligation. These signals may be involved in selection processes at the CD4+CD8+ stage of differentiation.     

A metal-mediated hydride shift mechanism for xylose isomerase based on the 1.6 A Streptomyces rubiginosus structures with xylitol and D-xylose

The crystal structure of recombinant Streptomyces rubiginosus D-xylose isomerase (D-xylose keto-isomerase, EC 5.3.1.5) solved by the multiple isomorphous replacement technique has been refined to R = 0.16 at 1.64 A resolution. As observed in an earlier study at 4.0 A (Carrell et al., J. Biol. Chem. 259: 3230-3236, 1984), xylose isomerase is a tetramer composed of four identical subunits. The monomer consists of an eight-stranded parallel beta-barrel surrounded by eight helices with an extended C-terminal tail that provides extensive contacts with a neighboring monomer. The active site pocket is defined by an opening in the barrel whose entrance is lined with hydrophobic residues while the bottom of the pocket consists mainly of glutamate, aspartate, and histidine residues coordinated to two manganese ions. The structures of the enzyme in the presence of MnCl2, the inhibitor xylitol, and the substrate D-xylose in the presence and absence of MnCl2 have also been refined to R = 0.14 at 1.60 A, R = 0.15 at 1.71 A, R = 0.15 at 1.60 A, and R = 0.14 at 1.60 A, respectively. Both the ring oxygen of the cyclic alpha-D-xylose and its C1 hydroxyl are within hydrogen bonding distance of NE2 of His-54 in the structure crystallized in the presence of D-xylose. Both the inhibitor, xylitol, and the extended form of the substrate, D-xylose, bind such that the C2 and C4 OH groups interact with one of the two divalent cations found in the active site and the C1 OH with the other cation. The remainder of the OH groups hydrogen bond with neighboring amino acid side chains. A detailed mechanism for D-xylose isomerase is proposed. Upon binding of cyclic alpha-D-xylose to xylose isomerase, His-54 acts as the catalytic base in a ring opening reaction. The ring opening step is followed by binding of D-xylose, involving two divalent cations, in an extended conformation. The isomerization of D-xylose to D-xylulose involves a metal-mediated 1,2-hydride shift. The final step in the mechanism is a ring closure to produce alpha-D-xylulose. The ring closing is the reverse of the ring opening step. This mechanism accounts for the majority of xylose isomerase's biochemical properties, including (1) the lack of solvent exchange between the 2-position of D-xylose and the 1-pro-R position of D-xylulose, (2) the chemical modification of histidine and lysine, (3) the pH vs. activity profile, and (4) the requirement for two divalent cations in the mechanism.     

Preliminary crystal structure of Acinetobacter glutaminasificans glutaminase-asparaginase

The preliminary structure of a glutaminase-asparaginase from Acinetobacter glutaminasificans is reported. The structure was determined at 3.0-A resolution with a combination of phase information from multiple isomorphous replacement at 4-5-A resolution and phase improvement and extension by two density modification techniques. The electron density map was fitted by a polypeptide chain that was initially polyalanine. This was subsequently replaced by a polypeptide with an amino acid sequence in agreement with the sizes and shapes of the side chain electron densities. The crystallographic R factor is 0.300 following restrained least squares refinement with data to 2.9-A resolution. The A. glutaminasificans glutaminase-asparaginase subunit folds into two domains: the aminoterminal domain contains a five-stranded beta sheet surrounded by five alpha helices, while the carboxyl-terminal domain contains three alpha helices and less regular structure. The connectivity is not fully determined at present, due in part to the lack of a complete amino acid sequence. The A. glutaminasificans glutaminase-asparaginase structure has been used successfully to determine the relative orientations of the molecules in crystals of Pseudomonas 7A glutaminase-asparaginase, in crystals of Vibrio succinogenes asparaginase, and in a new crystal form of Escherichia coli asparaginase (space group 1222, one subunit per asymmetric unit).     

Brain gamma-glutamyltransferase levels in alcohol-fed rabbits

Brain gamma-glutamyltransferase levels were determined in the cortex, cerebellum, medulla and mid-brain of 14 ethanol-fed and 10 control rabbits. In the ethanol-fed animals, brain gamma-glutamyltransferase activity was reduced. This reduction was more pronounced with 3 than with 6 months ethanol feeding, and significantly reduced gamma-glutamyltransferase activity was observed at each brain site when rabbits fed ethanol for 3 months were compared with non-ethanol-fed controls with similar calorie intake. Possible neuropharmacological implications are considered.