A high-resolution add-on in-lens attachment for scanning electron microscopes.

A compact add-on objective lens for the scanning electron microscope (SEM) has been designed and tested. The lens is < 35 mm high and can be fitted on to the specimen stage as an easy-to-use attachment. Initial results show that it typically improves the spatial resolution of the SEM by a factor of three. The add-on unit is based upon a permanent magnet immersion lens design. Apart from the extra attachment to the specimen stage, the SEM with the add-on lens functions in the normal way. The in-lens unit can comfortably accommodate specimen heights up to 10 mm. The new add-on lens unit opens up the possibility of operating existing SEMs in the high-resolution in-lens mode. By using a deflector at the top of the add-on lens unit, it can also operate as a quantitative multichannel voltage contrast spectrometer, capable of recording the energy spectrum of the emitted secondary electrons. Initial experiments confirm that a significant amount of voltage contrast can be obtained.

Cyclodextrins as protein folding aids.

Aggregation of proteins is a frequent occurrence during their transition from random coil to native structure. The influence of cyclodextrins in the refolding of carbonic anhydrase under aggregating conditions was studied. Cyclodextrin prevented formation of protein aggregates during renaturation of carbonic anhydrase. In addition, over 90% of active enzyme was recovered even at protein concentrations as high as 67 microM. The enhanced protein reactivation by cyclodextrins may be due to their ability to bind to hydrophobic sites in protein folding intermediate(s) followed by their subsequent removal as the protein refolds.

Kinetic induction of oat shoot pulvinus invertase mRNA by gravistimulation and partial cDNA cloning by the polymerase chain reaction.

An asymmetric (top vs. bottom halves of pulvini) induction of invertase mRNA by gravistimulation was analyzed in oat shoot pulvini. Total RNA and poly(A)+ RNA, isolated from oat pulvini, and two oligonucleotide primers, corresponding to two conserved amino acid sequences (NDPNG and WECPD) found in invertase from other species, were used for the polymerase chain reaction (PCR). A partial length cDNA (550 bp) was obtained and characterized. A 62% nucleotide sequence homology and 58% deduced amino acid sequence homology, as compared to beta-fructosidase of carrot cell wall, was found. Northern blot analysis showed that there was an obviously transient induction of invertase mRNA by gravistimulation in the oat pulvinus system. The mRNA was rapidly induced to a maximum level at 1 h after gravistimulation treatment and gradually decreased afterwards. The mRNA level in the bottom half of the oat pulvinus was significantly higher than that in the top half of the pulvinus tissue. The kinetic induction of invertase mRNA was consistent with the transient accumulation of invertase activity during the graviresponse of the pulvinus. This indicates that the expression of the invertase gene(s) could be regulated by gravistimulation at the transcriptional level. Southern blot analysis showed that there were two to three genomic DNA fragments which hybridized with the partial-length invertase cDNA.

Cell wall and enzyme changes during the graviresponse of the leaf-sheath pulvinus of oat (Avena sativa).

The graviresponse of the leaf-sheath pulvinus of oat (Avena sativa) involves an asymmetric growth response accompanied by several asymmetric processes, including degradation of starch and cell wall synthesis. To understand further the cellular and biochemical events associated with the graviresponse, changes in cell walls and their constituents and the activities of related enzymes were investigated in excised pulvini. Asymmetric increases in dry weight with relatively symmetric increases in wall weight accompanied the graviresponse. Starch degradation could not account for increases in wall weight. However, a strong asymmetry in invertase activity indicated that hydrolysis of exogenous sucrose could contribute significantly to the increases in wall and dry weights. Most cell wall components increased proportionately during the graviresponse. However, beta-D-glucan did not increase symmetrically, but rather increased in proportion in lower halves of gravistimulated pulvini. This change resulted from an increase in glucan synthase activity in lower halves. The asymmetry of beta-D-glucan content arose too slowly to account for initiation of the graviresponse. A similar pattern in change in wall extensibility was also observed. Since beta-D-glucan was the only wall component to change, it is hypothesized that this change is the basis for the change in wall extensibility. Since wall extensibility changed too slowly to account for growth initiation, it is postulated that asymmetric changes in osmotic solutes act as the driving factor for growth promotion in the graviresponse, while wall extensibility acts as a limiting factor during growth.

A comparative kinetic study of bovine calmodulin-dependent cyclic nucleotide phosphodiesterase isozymes utilizing cAMP, cGMP and their 2'-O-anthraniloyl-,2'-O-(N-methylanthraniloyl)-derivatives as substrates.

Calmodulin-dependent 3',5'-cyclic nucleotide phosphodiesterase (EC 3.1.4.17) Mr 63,000 and Mr 60,000 from the brain as well as Mr approximately 59,000 species from the heart, have been compared with respect to their steady-state kinetic parameters for the hydrolysis of cAMP, cGMP and their 2'-O-anthraniloyl- and 2'-O-(N-methylanthraniloyl)-derivatives. Kinetic studies with the native substrates indicate high Mr brain enzyme to be cGMP specific whereas low Mr brain and heart enzymes to be nonspecific. In addition, the isozymes studied here appear to be kinetically distinct from those previously isolated form bovine brain tissues. Substitution at 2'-O-position of the cyclic nucleotides gave rise to Vmax values ranging 1-11% of those observed with the native substrates, with minimal effect on Km. The isozymes with exception of heart isoform gave higher Km and Vmax with the anthraniloyl derivatives. This effect is thought to be related to the formation of an intramolecular hydrogen bond which leads to decreased electrostatic interactions between the active-site side chains and the pseudo-substrates.

Purification and characterization of soluble (cytosolic) and bound (cell wall) isoforms of invertases in barley (Hordeum vulgare) elongating stem tissue.

Three different isoforms of invertases have been detected in the developing internodes of barley (Hordeum vulgare). Based on substrate specificities, the isoforms have been identified to be invertases (beta-fructosidases EC 3.2.1.26). The soluble (cytosolic) invertase isoform can be purified to apparent homogeneity by diethylaminoethyl cellulose, Concanavalin-A Sepharose, organo-mercurial Sepharose, and Sephacryl S-300 chromatography. A bound (cell wall) invertase isoform can be released by 1 molar salt and purified further by the same procedures as above except omitting the organo-mercurial Sepharose affinity chromatography step. A third isoform of invertase, which is apparently tightly associated with the cell wall, cannot be isolated yet. The soluble and bound invertase isoforms were purified by factors of 60- and 7-fold, respectively. The native enzymes have an apparent molecular weight of 120 kilodaltons as estimated by gel filtration. They have been identified to be dimers under denaturing and nondenaturing conditions. The soluble enzyme has a pH optimum of 5.5, Km of 12 millimolar, and a Vmax of 80 micromole per minute per milligram of protein compared with cell wall isozyme which has a pH optimum of 4.5, Km of millimolar, and a Vmax of 9 micromole per minute per milligram of protein.

Selective adsorption of 2'-O-anthraniloyl-AMP on DEAE-Sephadex: the basis of a direct, fluorescent assay for cyclic nucleotide phosphodiesterase.

The fluorescent, 2'-O-anthraniloyl derivative of AMP was selectively absorbed onto DEAE-Sephadex in the presence of zirconyl chloride in citrate buffer. Under these conditions 2'-O-anthraniloyl-cAMP was eluted from the column. The selective adsorption of the AMP derivative onto DEAE-Sephadex, in the presence of zirconyl chloride, was adapted to the direct discontinuous assay of cyclic nucleotide phosphodiesterase. In this assay the enzyme is incubated for 4 min with 2'-O-anthraniloyl-cAMP; after quenching of the reaction by boiling, zirconyl chloride is added and the product (2'-O-anthraniloyl-AMP) is separated from the substrate on a column (0.6 ml) of DEAE-Sephadex. 2'-O-Anthraniloyl-AMP is then eluted with NaCl (2 M) and quantitated spectrofluorometrically. Under the conditions employed, 2'-O-anthraniloyl-AMP concentrations as low as 0.1 nmol can be detected. In the present study, this assay has been used to estimate Km and Vmax values for 2'-O-anthraniloyl-cAMP hydrolysis catalyzed by highly purified, as well as crude, preparations of cyclic nucleotide phosphodiesterase from bovine brain.

The differential stimulation of brain and heart cyclic-AMP phosphodiesterase by oncomodulin.

Ca2+/calmodulin dependent cyclic nucleotide phosphodiesterase, from the bovine heart and brain, purified by monoclonal antibody chromatography were tested with respect to activation by oncomodulin. The heart and brain enzymes which have previously been shown to have slightly different electrophoretic mobilities (1), were found to also differ in the oncomodulin dose-dependent activation of cAMP hydrolysis. Oncomodulin was shown to activate the heart enzyme to the same extent as calmodulin. However, this study indicates that the heart phosphodiesterase has approximately 25-fold higher affinity for oncomodulin than the brain enzyme. The oncomodulin concentration required for the half-maximal activation of the heart phosphodiesterase was estimated to be 2 X 10(-7)M. In addition, the possibility of the observed activation by oncomodulin being due to calmodulin contamination can be ruled out as the oncomodulin activation profiles were unaltered subsequent to chromatography on organomercurial agarose and the activation by oncomodulin could not be reversed by anti-calmodulin IgG.