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Biomedical subjects

E H Newcomb

Publications and source records attributed to E H Newcomb.

At least 19 recordsLinked to original sources

Results of intraocular lens implantation in paediatric aphakia.

Intraocular lenses were implanted in 16 eyes of 13 patients with congenital cataract, and visual progress was plotted using a preferential-looking technique. Initial surgery was by lens aspiration with preservation of the posterior capsule, and subsequent posterior capsulotomy without anterior vitrectomy. Poly-HEMA posterior chamber lenses were used, usually as a primary procedure but in four cases as a secondary procedure after contact lens failure. No serious complications were encountered. Most eyes achieved a very significant visual improvement, and none were worse than preoperatively. Residual refractive error was highly unpredictable, but did not exceed 6 dioptres. The importance of rigorous occlusion therapy is stressed. With close follow-up, this procedure offers an effective and safe method for the correction of unilateral paediatric aphakia, and, in selected cases only, for bilateral aphakia.

Cataract↗

Localization of xanthine dehydrogenase in cowpea root nodules: implications for the interaction between cellular compartments during ureide biogenesis.

Immunocytochemistry was used to assess the location of xanthine dehydrogenase (EC 1.1.1.204) in the infected region of nodules of cowpea (Vigna unguiculata [L.] Walpers cv. Queen Anne Blackeye). Polyclonal antibodies raised against purified cowpea xanthine dehydrogenase were used to localize this enzyme at the electron microscopic level. Sparse nonspecific labeling was observed after treatment of nodule sections with preimmune serum. Although immune serum cross-reacted with the ground cytoplasm of both infected and uninfected cells, significantly more labeling was observed in the uninfected cells. No labeling above background was observed in peroxisomes, mitochondria, proplastids, endoplasmic reticulum, cytoplasmic or peribacteroid membranes, peribacteroid spaces, or bacteroids. The enzyme is soluble and not present in any organelle or membrane. The greater concentration of xanthine dehydrogenase in the uninfected cells suggests that xanthine or a precursor to xanthine, rather than uric acid, is the intermediate that moves from infected to uninfected cells during ureide biogenesis.

Journal Article↗

Nodule initiation elicited by noninfective mutants of Rhizobium phaseoli.

Rhizobium phaseoli CE106, CE110, and CE115, originally derived by transposon mutagenesis (Noel et al., J. Bacteriol. 158:149-155, 1984), induced the formation of uninfected root nodule-like swellings on bean (Phaseolus vulgaris). Bacteria densely colonized the root surface, and root hair curling and initiation of root cortical-cell divisions occurred normally in mutant-inoculated seedlings, although no infection threads formed. The nodules were ineffective, lacked leghemoglobin, and were anatomically distinct from normal nodules. Ultrastructural specialization for ureide synthesis, characteristic of legumes that form determinate nodules, was absent. Colony morphology of the mutant strains on agar plates was less mucoid than that of the wild type, and under some cultural conditions, the mutants did not react with Cellufluor, a fluorescent stain for beta-linked polysaccharide. These observations suggest that the genetic lesions in these mutants may be related to extracellular polysaccharide synthesis.

Mutation↗

Separation and characterization of inner and outer envelope membranes of pea chloroplasts.

A procedure for purifying the chloroplast envelope subfractionates it into two membrane fractions of comparable quantities. This procedure differs from previous ones in that the chloroplasts are ruptured by freezing and thawing in hypertonic medium rather than by osmotic shock. The two membrane fractions have qualitatively similar polar lipid compositions but differ in their content of individual lipids, specifically monogalactosyldiacylglycerol and phosphatidylcholine. The two fractions also differ in their constituent polypeptides and in their appearance when examined by electron microscopy. The light (density = 1.08 g/ml) and heavy (density = 1.13 g/ml) membrane fractions have been tentatively identified as the outer and inner envelope membranes, respectively.

Journal Article↗

Identification of chlorophyll b in extracts of prokaryotic algae by fluorescence spectroscopy.

Solvent extracts of three different prokaryotic algae from three species of didemnid ascidians contained pigments identified, on the basis of their fluorescence excitation (E)and fluorescence emission (F)spectral maxima (measured in nm) at 77K, as chlorophyll a (E 449, F 678) and chlorophyll b (E 478, F 658). The release of algae on cutting or freezing Diplosoma virens was accompanied by a strong unidentified acid that converted these pigments to pheophytins. This unexpected finding provided further confirmation of the identity of the chlorophylls on the basis of the fluorescence spectra at 77K of pheophytin a (E 415, F 669) and pheophytin b (E 439, F 655). There was no evidence for the presence of the fluorescent bilin pigments found in other prokaryotic blue-green algae. Chlorophyll a/b ratios ranged from 2.6 to 12.0 in algae from different ascidians. The photosynthetic membranes were not organized into appressed thylakoids or grana in the algae from any of the three species of ascidians. The relationship between these observations and those in higher eukaryotic organisms is discussed.

Chlorophyll↗

Structure and development of P-protein in phloem parenchyma and companion cells of legumes.

Phloem parenchyma and companion cells from four species of legumes, Phaseolus vulgaris L., Melilotus alba Desr., Desmodium canadense, L. and Dolichos lablab L., were examined electron microscopically to estabish the presence, structure and development of P-protein. P-protein components consisting of granular, fibrillar, tubular or crystalline structures were found in parenchyma cells of all species and in companion cells of M. alba. The earliest stages of P-protein formation were closely associated with dictyosome cisternae, dictyosome vesicles and/or spiny vesicles. After their formation, the P-protein bodies were frequently transformed into one or more structurally different components. Although these components appeared to be develop-mentally related, their specific associations and transformations differed in each species examined.

Inclusion Bodies↗

The development of microbodies and peroxisomal enzymes in greening bean leaves.

The ontogeny of leaf microbodies (peroxisomes) has been followed by (a) fixing primary bean leaves at various stages of greening and examining them ultrastructurally, and (b) homogenizing leaves at the same stages and assaying them for three peroxisomal enzymes. A study employing light-grown seedlings showed that when the leaves are still below ground and achlorophyllous, microbodies are present as small organelles (e.g., 0.3 microm in diameter) associated with endoplasmic reticulum, and that after the leaves have turned green and expanded fully, the microbodies occur as much larger organelles (e.g., 1.5 microm in diameter) associated with chloroplasts. Specific activities of the peroxisomal enzymes increase 3- to 10-fold during this period. A second study showed that when etiolated seedlings are transferred to light, the microbodies do not appear to undergo any immediate morphological change, but that by 72 h they have attained approximately the size and enzymatic activity possessed by microbodies in the mature primary leaves of light-grown plants. It is concluded from the ultrastructural observations that leaf microbodies form as small particles and gradually develop into larger ones through contributions from smooth portions of endoplasmic reticulum. In certain aspects, the development of peroxisomes appears analogous to that of chloroplasts. The possibility is examined that microbodies in green leaves may be relatively long-lived organelles.

Alcohol Oxidoreductases↗

Microbodies (Glyoxysomes and Peroxisomes) in Cucumber Cotyledons: Correlative Biochemical and Ultrastructural Study in Light- and Dark-grown Seedlings.

The changes in activities of glyoxysomal and peroxisomal enzymes have been correlated with the fine structure of microbodies in cotyledons of the cucumber (Cucumis sativus L.) during the transition from fat degradation to photosynthesis in light-grown plants, and in plants grown in the dark and then exposed to light. During early periods of development in the light (days 2 through 4), the microbodies (glyoxysomes) are interspersed among lipid bodies and contain relatively high activities of glyoxylate cycle enzymes involved in lipid degradation. Thereafter, these activities decrease rapidly as the cotyledons expand and become photosynthetic, and the activity of glycolate oxidase rises to a peak (day 7); concomitantly the microbodies (peroxisomes) become preferentially associated with chloroplasts.In seedlings grown in the dark for 10 days, the reserve lipid and the glyoxylate cycle enzyme activities persist for a longer time than in the light; correlated with this, there is a continued association of the microbodies with the lipid bodies. When these dark-grown seedlings are then exposed to 51 hours of the light-dark cycle, peroxisomal marker enzymes increase rapidly in activity, and the microbodies become appressed to chloroplasts. We conclude that the characteristic association observed between glyoxysomes and lipid bodies reflects their mutual involvement in net gluconeogenesis through the conversion of fatty acids to carbohydrate, while the close spatial relationship observed between peroxisomes and chloroplasts at later stages of development reflects their mutual involvement in glycolate metabolism.Although glyoxysomal enzyme activities are dropping rapidly while peroxisomal enzyme activities are increasing rapidly during the transition period in the light, the electron microscopic evidence does not indicate that glyoxysomes are being degraded or peroxisomes are being formed. Since in the dark-grown seedlings the activities of peroxisomal enzymes remain low and do not increase as they do in the light, an opportunity is afforded to compare quantitatively any changes in numbers of microbodies per cell with the changes in activities of glyoxysomal enzymes. It is found that the magnitude of the decrease in numbers of microbodies is considerably less than that of the decrease in glyoxysomal enzyme activities between days 4 and 10. When the cotyledons are exposed to light, peroxisomal enzyme activities increase greatly, but again there is no ultrastructural evidence for the synthesis of a new population of microbodies to accommodate this increase. These results allow us to conclude that the developmental transition from glyoxysomal to peroxisomal function almost certainly does not involve the actual replacement of one population of microbodies by another. Rather, the transition probably occurs within existing particles, either by a sequential functioning of two different kinds of microbodies or by a change in enzyme complement within a single population. Our findings with both light- and dark-grown cotyledons favor the latter possibility. The cytoplasmic invaginations into microbodies seen during greening of both light-grown cotyledons and etiolated cotyledons exposed to light may be morphological manifestations of the mechanism by which the microbodies lose or gain enzymes.

Journal Article↗

A study of sieve element starch using sequential enzymatic digestion and electron microscopy.

The fine structure of plastids and their starch deposits in differentiating sieve elements was studied in bean (Phaseolus vulgaris L.). Ultrastructural cytochemistry employing two carbohydrases specific for different linkages was then used to compare the chemical nature of "sieve tube starch" (the starch deposited in sieve elements) with that of the ordinary starch of other cell types. Hypocotyl tissue from seedlings was fixed in glutaraldehyde, postfixed in osmium tetroxide, and embedded in Epon-Araldite. Treatment of thin sections on uncoated copper grids with alpha-amylase or diastase at pH 6.8 to cleave alpha-(1 --> 4) bonds resulted in digestion of ordinary starch grains but not sieve element grains, as determined by electron microscopy. Since alpha-(1 --> 6) branch points in amylopectin-type starches make the adjacent alpha-(1 --> 4) linkages somewhat resistant to hydrolysis by alpha-amylase, other sections mounted on bare copper or gold grids were treated with pullulanase (a bacterial alpha-[1 --> 6] glucosidase) prior to digestion with diastase. Pullulanase did not digest sieve element starch, but rendered the starch digestible subsequently by alpha-amylase. Diastase followed by pullulanase did not result in digestion. The results provide evidence that sieve element starch is composed of highly branched molecules with numerous alpha-(1 --> 6) linkages.

Aldehydes↗

Cytochemical localization of catalase in leaf microbodies (peroxisomes).

Segments of mature tobacco leaves were fixed in glutaraldehyde, incubated in medium containing 3,3'-diaminobenzidine (DAB) and hydrogen peroxide, and postfixed in osmium tetroxide. Electron microscopic observation of treated tissues revealed pronounced deposition of a highly electron-opaque material in microbodies but not in other organelles. The coarsely granular reaction product is presumably osmium black formed by reaction of oxidized DAB with osmium tetroxide. Reaction of the microbodies with DAB was completely inhibited by 0.02 M 3-amino-1,2,4-triazole and was considerably reduced by 0.01 M potassium cyanide. These results, when considered in light of recent biochemical studies, strongly suggest that catalase is responsible for the reaction. Sharp localization of this enzyme in microbodies establishes that they are identical to the catalase-rich "peroxisomes" recently isolated from leaf cell homogenates. A browning reaction that occurred in leaves during the incubation step was inhibited by cyanide but not by aminotriazole and therefore could not have been caused by the same enzyme. This reaction and a slight deposition of dense material within primary and secondary walls are ascribed to oxidation of DAB by soluble and wall-localized peroxidases.

Catalase↗

An atypical crista resembling a "tight junction" in bean root mitochondria.

The conformation and structure of an atypical crista found in a small percentage of the mitochondria in root tip cells of Phaseolus vulgaris L. have been studied electron microscopically in material fixed in glutaraldehyde followed by osmium tetroxide. In its transformation into an atypical crista, a normal crista elongates, broadens, and flattens, and the inner leaflets of its apposed unit membranes appear to fuse in a manner analogous to the formation of "tight junctions" between certain animal cells. The result is a large platelike, quintuple-layered structure, 240-260 A thick, whose long axis parallels that of the mitochondrion. The outer layers of the "plate," bordering on the mitochondrial matrix, are thickened and exhibit striking patterns in the micrographs. The structure of the plate is compared with that previously described for tight junctions between animal cells.

Membranes↗

Fine structure of protein-storing plastids in bean root tips.

The fine structure of leucoplasts in root tip cells of Phaseolus vulgaris L. has been studied in material fixed in glutaraldehyde followed by osmium tetroxide and poststained in uranyl acetate and lead citrate. Plastid development has been followed from the young stages in and near the meristematic region, through an ameboid stage, to the larger forms with more abundant storage products in the outermost cells. The plastids contain a dense stroma penetrated by tubules and cisternae arising from the inner membrane of the plastid envelope. Also located in the stroma are lamellae, ribosome-like particles, phytoferritin granules, and fine fibrils in less dense regions. In some elongate plastids microfilaments run lengthwise in the stroma near the surface. The same plastids store both starch and protein, but in a strikingly different manner. The starch is deposited in the stroma, while the protein always is accumulated within membrane-bounded sacs. These sacs arise as outgrowths from a complex of interconnected tubules which in turn appears to originate by coalescence and proliferation of tubules and cisternae arising from the inner plastid membrane. This "tubular complex" bears a strong resemblance to the prolamellar body of etiolated chloroplasts, but is smaller and ordinarily less regularly organized, and is apparently light-insensitive. Crystallization of the protein commonly occurs in the sacs and occasionally takes place within the tubules of the complex as well. The fine structure of the leucoplasts is discussed in relation to that of etiolated chloroplasts. Suggestions are made concerning the function of the tubular complex, role of the ameboid plastid forms, and manner of accumulation of the storage protein in the plastids.

Cell Differentiation↗