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.

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.

The occurrence of leghemoglobin protein in the uninfected interstitial cells of soybean root nodules.

The distribution of leghemoglobin (Lb) in resin-embedded root nodules of soybean (Glycine max (L.) Merr.) was investigated using immunogold labeling. Using anti-Lb immunoglobulin G and protein A-gold, Lb or its apoprotein was detected both in cells infected by Bradyrhizobium japonicum and in uninfected interstitial cells. Leghemoglobin was present in the cytoplasm, exclusive of the organelles, and in the nuclei of both cell types. In a comparison of the density of labeling in adjacent pairs of infected and uninfected cells, Lb was found to be about four times more concentrated in infected cells. This is the first report of Lb in uninfected cells of any legume nodule; it raises the possibility that this important nodule-specific protein may participate in mediating oxygen flow to host plant organelles throughout the infected region of the nodule.

Cellular compartmentation of ureide biogenesis in root nodules of cowpea (Vigna unguiculata (L.) Walp.).

Cowpea (Vigna unguiculata (L.) Walp.) nodules have been investigated by means of cytochemical and immunocytochemical procedures at the ultrastructural level in order to assess the role of the uninfected cells in ureide biogenesis. Uricase activity in the nodules was shown by cytochemical methods to be localized exclusively in the numberous large peroxisomes confined to the uninfected cells; the small peroxisomes in the infected cells did not stain for uricase. Uricase was also localized in the peroxisomes of uninfected cells by immunogold techniques employing polyclonal antibodies against nodule-specific uricase of soybean. There was no labeling above background of any structures in the infected cells. The results indicate that the uninfected cells are essential for ureide biogenesis in cowpea. Although tubular endoplasmic reticulum, the presumptive site of allantoinase, increases greatly in the uninfected cells during nodule development, it virtually disappears as the nodules mature. The inconsistency between the disappearance of the tubular endoplasmic reticulum from older nodules and the high allantoinase activity reported for older plants remains to be explained.

Immunogold localization of nodule-specific uricase in developing soybean root nodules.

Immunogold labeling was used to study the time of appearance and distribution of a nodule-specific form of uricase (EC 1.7.3.3) in developing nodules of soybean (Glycine max (L.) Merr.) inoculated with Bradyrhizobium japonicum. The enzyme was detected in thin sections of tissue embedded in either L R White acrylic resin or Spurr's epoxy resin, by employing a polyclonal antibody preparation active against a subunit of soybean nodule uricase. Antigenicity was better preserved in L R White resin, but ultrastructure was better maintained in Spurr's. Uricase was first detectable with protein A-gold in young, developing peroxisomes in uninfected cells, coincident with the release of Bradyrhizobium bacteroids from infection threads in adjacent infected cells. As the peroxisomes enlarged, labeling of the dense peroxisomal matrix increased. Gold particles were never observed over the paracrystalline inclusions of peroxisomes, however. Despite a close association between enlarging peroxisomes and tubular endoplasmic reticulum, uricase was not detectable in the latter. In mature nodules, labeling of uricase was limited to the large peroxisomes in uninfected cells. Small peroxisome-like bodies present in infected cells did not become labeled.

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.

Spatial relationships between uninfected and infected cells in root nodules of soybean.

In soybean (Glycine max (L.) Merr.) the uninfected cells of the root nodule are responsible for the final steps in ureide production from recently fixed nitrogen. Stereological methods and an original quantitative method were used to investigate the organization of these cells and their spatial relationships to infected cells in the central region of nodules of soybean inoculated with Rhizobium japonicum strain USDA 3I1B110 and grown with and without nitrogen (as nitrate) in the nutrient medium. The volume occupied by the uninfected tissue was 21% of the total volume of the central infected region for nodules of plants grown without nitrate, and 31% for nodules of plants grown with nitrate. Despite their low relative volume, the uninfected cells outnumbered the much larger infected cells in nodules of plants grown both without and with nitrate. The surface density of the interface between the ininfected and infected tissue in the infected region was similar for nodules in both cases also, the total range being from 24 to 26 mm(2)/mm(3). In nodules of plants grown without nitrate, all sampled infected cells were found to be in contact with at least one uninfected cell. The study demonstrates that although the uninfected tissue in soybean nodules occupies a relatively small volume, it is organized so as to produce a large surface area for interaction with the infected tissue.

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.

Uninfected cells of soybean root nodules: ultrastructure suggests key role in ureide production.

In soybean root nodules, which export recently fixed nitrogen mainly as the ureides allantoin and allantoic acid, cells uninfected by rhizobia undergo a pronounced ultrastructural differentiation not shown by the infected cells, including enlargement of the microbodies and proliferation of smooth endoplasmic reticulum. Since some of the enzymes contributing to ureide synthesis occur in these subcellular components in root nodule preparations, the uninfected cells may participate in ureide synthesis and thus play an essential role in the symbiosis between host and bacterium.

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.

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.

Microbodies and an anomalous "microcylinder" in the ultrastructure of plants with Crassulacean acid metabolism.

An ultrastructural study was made of the leaf tissues of four species of plants in three genera with Crassulacean acid metabolism ("CAM" plants): Kalanchoë daigremontiana Hamet et Perrier, K. verticillata Elliot, Sedum rubrotinctum clausen and Crassula tetragona L. Microbodies similar in appearance, with fibrillar or granular nucleoids but no crystalline deposits, were present in the mesophyll of all four species. The microbodies resembled in size and abundance those of C3 plants more closely than those of C4 plants, both under long-day and short-day conditions. The reaction for catalase activity employing 3,3'-diaminobenzidine produced a heavy deposit in the microbodies; the reaction was blocked by the catalase inhibitor, aminotriazole.Some of the plants of the two species of Kalanchoë studied contain in the epidermal and mesophyll cells of the leaves and plantlets an organelle-like structure consisting of a hollow cylinder, 90-160 nm in diameter and up to 2 µm or more in length, around which 18-20 or more minute tubules are wound in a steep helix. The tubules are only ca. 9 nm in diameter, hence are much smaller than conventional microtubules. The cylinder and surrounding tubules, herein tentatively assigned the term "microcylinder" for convenience, may represent a product of viral infection, or may be an organelle that appears at certain stages of growth or under particular environmental conditions. In any case it may prove to be of considerable importance for investigators of CAM plant physiology.

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.

The occurrence of microbodies and peroxisomal enzymes in achlorophyllous leaves.

Several types of leaves of leaf parts lacking chlorophyll were fixed and embedded according to conventional procedures and examined electron-microscopically for microbodies. Comparisons of relative abundance of microbodies, plastids and mitochondria were made by computing the average numbers of organelle profiles per cell section. Similar leaves were homogenized and assayed for three enzymes characteristic of leaf peroxisomes. The localization of these enzymes in microbodies was indicated for the achlorophyllous tissues by the positive result obtained when 3,3'-diaminobenzidine was used as an electron cytochemical stain for catalase activity.Microbodies were present in all non-photosynthetic leaves or leaf parts examined, including yellowish-white segments of variegated leaves, albino leaves, and etiolated leaves of two species. In several cases, the numbers of microbody profiles per cell section were as great in the achlorophyllous leaves as in the chlorophyllous. The levels of peroxisomal enzyme activity in the yellowish-white leaves were substantial, although often not as high as in the green leaves. It was concluded that enzymatically these microbodies are probably similar to the peroxisomes characterized from chlorophyllous leaves. In the absence of the photosynthetic product, glycolate, however, it seems unlikely that the organelle is performing the same functions as in green leaves. It is also apparent that the initial formation of peroxisomes in leaves can occur when neither light nor a photosynthate such as glycolate is present as an inducer.

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.

Ultrastructure and distribution of microbodies in leaves of grasses with and without CO2-photorespiration.

A comparative study was made of the ultrastructure, distribution and abundance of leaf microbodies in four species of "temperate" grasses with high and four "tropical" grasses with low CO2-photorespiration. The temperate grasses were all festucoid; the tropical grasses included two panicoid species and two chloridoid. Comparisons of relative abundance were made by computing the average numbers of microbody profiles per cell section.Although microbodies were present in the green parenchymatous leaf cells in all grasses examined, their average number per cell was in general severalfold greater in the grasses with high CO2-photorespiration than in those with low. Furthermore, whereas in the grasses with high CO2-photorespiration the microbodies were distributed through the mesophyll, in those with low CO2-photorespiration they were concentrated in the vascular-bundle-sheath cells and were smaller and relatively scarce in the mesophyll cells. The leaf microbodies of the eight grass species resembled one another in general morphology, but differed to some extent in regard to size and type of inclusion. Microbodies of all four festucoid species contained numerous fibrils with a discernible substructure. Those of the two panicoid species contained clusters of round bodies with transparent cores. The equivalence of the microbodies to peroxisomes as biochemically defined was shown cytochemically by employing 3,3'-diaminobenzidine for the localization of catalase, a marker enzyme for the peroxisome. This reaction was blocked by the catalase inhibitor, aminotriazole.The observations on the relative abundance and distribution of peroxisomes in leaves of grasses with high CO2-photorespiration versus those with low are consistent with the published biochemical data on the levels and distribution of peroxisomal enzymes in representatives of plants with high and low CO2-photorespiration, and may help explain the differences in apparent photorespiratory levels between these two groups of plants.

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.