Spectral, Physical, and Electron Transport Activities in the Photosynthetic Apparatus of Mesophyll Cells and Bundle Sheath Cells of Digitaria sanguinalis (L.) Scop

Isolated mesophyll cells and bundle sheath cells of Digitaria sanguinalis were used to study the light-absorbing pigments and electron transport reactions of a plant which possesses the C₄-dicarboxylic acid cycle of photosynthesis. Absorption spectra and chlorophyll determinations are presented showing that mesophyll cells have a chlorophyll a-b ratio of about 3.0 and bundle sheath cells have a chlorophyll a-b ratio of about 4.5. The absorption spectrum of bundle sheath cells has a greater absorption in the 700 nm region at liquid nitrogen temperature, and there is a relatively greater amount of a pigment absorbing at 670 nm in the bundle sheath cells compared to the mesophyll cells. Fluorescence emission spectra, at liquid nitrogen temperature, of mesophyll cells have a fluorescence 730 nm-685 nm ratio of about 0.82 and bundle sheath cells have a ratio of about 2.84. The reversible light-induced absorption change in the region of P₇₀₀ absorption is similar in both cell types but bundle sheath cells exhibit about twice as much total P₇₀₀ change as mesophyll cells on a total chlorophyll basis. The delayed light emission of bundle sheath cells is about one-half that of mesophyll cells. Both mesophyll cells and bundle sheath cells evolve oxygen in the presence of Hill oxidants with the mesophyll cells exhibiting about twice the activity of bundle sheath cells, and both activities are inhibited by 1 μM 3-(3,4-dichlorophenyl)-1, 1-dimethylurea. Ferredoxin nicotinamide adenine dinucleotide phosphate reductase is present in both cells although it is about 3- or 4-fold higher in mesophyll cells than in bundle sheath cells. Glyceraldehyde 3-P dehydrogenases, both nicotinamide adenine dinucleotide and nicotinamide adenine dinucleotide phosphate, are equally distributed in the two cell types on a chlorophyll basis. Malic enzyme is localized in the bundle sheath cells.     

Monoclonal antibody against chicken type IX collagen: preparation, characterization, and recognition of the intact form of type IX collagen secreted by chondrocytes

A series of monoclonal antibodies was prepared against the pepsin-resistant fragment of type IX collagen designated HMW. One of these antibodies (called 2C2) was selected for further analysis. Antibody 2C2 showed no cross-reactivity with other collagen types by inhibition enzyme-linked immunosorbent assays. It recognized an epitope present in native HMW, but failed to recognize any of the three chains of HMW fractionated after denaturation followed by reduction and alkylation of interchain disulfide bridges. Electron microscopic observations after rotary shadowing showed that the location of the epitope for antibody 2C2 was close to the carboxy-terminus of HMW. Immunofluorescent staining of sections of embryonic and adult cartilage with antibody 2C2 after removal of proteoglycans by testicular hyaluronidase digestion showed that type IX collagen is distributed throughout the cartilage matrix, and is not present in other connective tissues or skeletal muscle. The intact type IX collagen molecule, which was secreted by a suspension culture of freshly isolated embryonic chick chondrocytes, was recognized by rotary shadowing in the presence of antibody 2C2 after first precipitating the procollagens from the culture medium with ammonium sulfate (30%). Two different collagenous molecules were present in the precipitate: a longer molecule of type II procollagen (average length, 335 nm) with both amino- and carboxy-propeptides still remaining uncleaved, and a shorter molecule (average length, 190 nm) which was identified as type IX collagen. Antibody 2C2 consistently bound to the shorter molecules at a site located 136 nm from a distinctive knob at one end of the molecule, and did not bind to any specific site on the type II procollagen molecules. The structure of the intact type IX collagen molecule with the location of both collagenous and noncollagenous domains was as predicted after converting the nucleotide sequence of a cDNA clone encoding for one of the chains of type IX collagen to an amino acid sequence (Ninomiya, Y., and B. R. Olsen, 1984, Proc. Natl. Acad. Sci. USA, 81:3014-3018).     

Azolla-Anabaena Relationships: VII. Distribution of Ammonia-assimilating Enzymes, Protein, and Chlorophyll between Host and Symbiont

The N₂-fixing Azolla-Anabaena symbiotic association is characterized in regard to individual host and symbiont contributions to its total chlorophyll, protein, and levels of ammonia-assimilating enzymes. The phycocyanin content of the association and the isolated blue-green algal symbiont was used as a standard for this characterization. Phycocyanin was measured by absorption and fluorescence emission spectroscopy. The phycocyanin content and total phycobilin complement of the symbiotic algae were distinct from those of Anabaena cylindrica and a free-living isolate of the Azolla endophyte. The algal symbiont accounted for less than 20% of the association's chlorophyll and protein. Acetylene reduction rates in the association (based solely on the amount of algal chlorophyll) were 30 to 50% higher than those attained when the symbiont was isolated directly from the fern. More than 75% of the association's glutamate dehydrogenase and glutamine synthetase activities are contributed by the host plant. The specific activity of glutamate dehydrogenase is greater than that of glutamine synthetase in the association and individual partners. Both the host and symbiont have glutamate synthase activity. The net distribution of these enzymes is discussed in regard to the probable roles of the host and symbiont in the assimilation of ammonia resulting from N₂ fixation by the symbiont.     

Purification and initial characterization of phycobiliproteins from the endophytic cyanobacterium of Azolla

The endophytic cyanobacterium, Anabaena azollae, isolated from laboratory cultures of Azolla caroliniana Willd., contains three spectroscopically distinct biliproteins. About 70% of the biliprotein is c-phycocyanin (_max 610 nm) and 13% is allophycocyanin (_max 647 nm, shoulder 620 nm). A third pigment corresponds to phycoerythrocyanin (_max 570 nm, shoulder 590 nm). In very dilute solutions of allophycocyanin, at constant pH and buffer strength, the 647 nm maximum disappears and a single _max occurs at 615–620 nm. The 647 nm absorption maximum reappears upon concentrating the dilute solution. Very dilute solutions of phycoerythrocyanin exhibit a broad peak between 570 and 590 nm. Absorption spectra of c-phycocyanin are not significantly altered upon dilution. Fluorescence emission maxima of phycoerythrocyanin, c-phycocyanin, and allophycocyanin occur at 630 nm, 643 nm and 660 nm respectively, using 540 nm excitation. Two subunits, of molecular weight 16,500 () and 20,600 (), are seen in c-phycocyanin upon dissociation with SDS. Dissociation of allophycocyanin and phycoerythrocyanin with SDS yields one sizeclass of subunits, with a molecular weight of about 17,500 for allophycocyanin and 18,000 for phycoerythrocyanin.Contribution No. 684 Offprint requests to: G. A. Peters     

Dual-action hypoglycemic and hypocholesterolemic agents that inhibit glycogen phosphorylase and lanosterol demethylase

Diabetic dyslipidemia requires simultaneous treatment with hypoglycemic agents and lipid-modulating drugs. We recently described glycogen phosphorylase inhibitors that reduce glycogenolysis in cells and lower plasma glucose in ob/ob mice (J. Med. Chem., 41: 2934, 1998). In evaluating the series prototype, CP-320626, in dogs, up to 90% reduction in plasma cholesterol was noted after 2 week treatment. Cholesterol reductions were also noted in ob/ob mice and in rats. In HepG2 cells, CP-320626 acutely and dose-dependently inhibited cholesterolgenesis without affecting fatty acid synthesis. Inhibition occurred together with a dose-dependent increase in the cholesterol precursor, lanosterol, suggesting that cholesterolgenesis inhibition was due to lanosterol 14alpha-demethylase (CYP51) inhibition. In ob/ob mice, acute treatment with CP-320626 resulted in a decrease in hepatic cholesterolgenesis with concomitant lanosterol accumulation, further implicating CYP51 inhibition as the mechanism of cholesterol lowering in these animals. CP-320626 and analogs directly inhibited rhCYP51, and this inhibition was highly correlated with HepG2 cell cholesterolgenesis inhibition (R2 = 0.77). These observations indicate that CP-320626 inhibits cholesterolgenesis via direct inhibition of CYP51, and that this is the mechanism whereby CP-320626 lowers plasma cholesterol in experimental animals. Dual-action glycogenolysis and cholesterolgenesis inhibitors therefore have the potential to favorably affect both the hyperglycemia and the dyslipidemia of type 2 diabetes.     

Lack of geographic variation in anonymous nuclear polymorphisms in the American oyster, Crassostrea virginica

Comparing geographic variation of noncoding nuclear DNA polymorphisms, which presumably are neutral to natural selection, with geographic variation of allozymes is potentially a good way to detect the effects of selection on allozyme polymorphisms. A previous study of four anonymous nuclear markers in the American oyster, Crassostrea virginica, found dramatic differences in allele frequency between the Gulf of Mexico and the Atlantic Ocean. In contrast, 14 allozyme polymorphisms were fairly uniform in frequency between the two areas. This led to the conclusion that all of the allozyme polymorphisms were kept uniform in frequency by balancing selection. To test the robustness of this pattern, six additional anonymous nuclear DNA polymorphisms were surveyed in oysters from Panacea, Fla, and Charleston, S.C. on the Gulf and Atlantic coasts, respectively. Unlike the previously studied DNA markers, the six DNA polymorphisms examined here show geographic variation that is not significantly greater than that of allozymes. The reason for the discrepancy between the two sets of DNA polymorphisms is unclear.      

Stimulation by insulin of the formation of ribonucleic acid and protein by mammary tissues in vitro

1. Insulin is one of the hormones that are essential for successful tissue culture of explants of the mammary glands of pregnant mice. We report here effects of insulin on RNA and protein formation by mammary tissue from pregnant mice and rats incubated in tissue-culture medium 199. 2. The incorporation of [(14)C]adenine over 3hr. into the RNA of explants of the mammary glands of pregnant mice was increased by an average of 68% when the medium contained 5mug. of insulin/ml. Under similar conditions the incorporation into the RNA of slices of the glands of pregnant rats was increased by an average of 61%. Incorporation into the RNA of slices from lactating rats was stimulated to a smaller extent. 3. Adipose tissue was separated from the glands of pregnant mice and the effect of insulin on the incorporation of adenine into its RNA was studied. In whole explants the incorporation of adenine, both with and without insulin, is almost entirely into the RNA of the mammary parenchyma and not of the adipose tissue. 4. Insulin also stimulated by 38% the incorporation of [(14)C]leucine over 3hr. into the proteins of slices of the glands of pregnant rats. It had no significant effect on slices from lactating rats. 5. Actinomycin D (10mug./ml.) decreased the incorporation of [(14)C]adenine into the RNA of slices of the glands of pregnant rats by an average of 97%. Though it also decreased the incorporation of [(14)C]leucine into the proteins by an average of 25%, the percentage stimulation by insulin of this incorporation remained unchanged.     

Fibroblasts promote the formation of a continuous basal lamina during myogenesis in vitro

Analyses were made of the requirements for the formation of a continuous basal lamina during myogenesis of quail muscle in vitro. A culture system was developed in which mass cultures of differentiating muscle cells were embedded in a native gel of rat tail collagen. Fibroblastic cells, which were also present in the cultures, migrated into the gel and within a few days surrounded the newly formed myotubes. In this environment, a continuous basal lamina was formed at the surface of the myotubes as demonstrated by immunofluorescent staining with monoclonal antibodies against type IV collagen, laminin, and heparan sulfate, as well as by electron microscopic immunolocalization. To distinguish between the role of the fibroblasts and the collagen gel in promoting basal lamina formation, clones of quail muscle cells lacking fibroblasts were subsequently embedded in a native rat tail collagen gel. Under these conditions, only very limited fluorescent staining for basement membrane components was observed associated with the myotubes. However, the introduction of chick muscle or skin fibroblasts into the clonal cultures just before gel formation resulted in the formation of an extensive basal lamina on the surface of the myotubes. Conditioned medium from fibroblast cultures by itself was not effective in promoting basal lamina formation. These results clearly show that during myogenesis in vitro fibroblasts must be in close proximity to the myotubes for a continuous basal lamina to form. These results probably relate closely to the interactions that must occur during myogenesis in vivo between the muscle cells and the surrounding connective tissue including the developing tendons.     

Location of the integrin complex and extracellular matrix molecules at the chicken myotendinous junction

Summary The distribution of several extracellular matrix macromolecules was investigated at the myotendinous junction of adult chicken gastrocnemius muscle. Localization using monoclonal antibodies specific for 3 basal lamina components (type IV collagen, laminin, and a basement membrane form of heparan sulfate proteoglycan) showed strong fluorescent staining of the myotendinous junction for heparan sulfate proteoglycan and laminin, but not for type IV collagen. In addition, a strong fluorescent stain was observed at the myotendinous junction using a monoclonal antibody against the subunit of the chicken integrin complex (antibody JG 22). Neither fibronectin nor tenascin were concentrated at the myotendinous junction, but instead were present in a fibrillar staining pattern throughout the connective tissue which was closely associated with the myotendinous junction. Tenascin also gave bright fluorescent staining of tendon, but no detectable staining of the perimysium or endomysium. Type I collagen was observed throughout the tendon and in the perimysium, but only faintly in the endomysium. In contrast, type III collagen was present brightly in the endomysium and in the perimysium, but could not be detected in the tendon except when associated with blood vessels and in the epitendineum, which stained intensely. Type VI collagen was found throughout the tendon and in all connective tissue partitions of skeletal muscle. The results indicate that one or more molecules of the integrin family may play an important role in the attachment of muscle to the tendon. This interaction does not appear to involve extensive binding to fibronectin or tenascin, but may involve laminin and heparan sulfate proteoglycan.     

Acquisition of type IX collagen by the developing avian primary corneal stroma and vitreous

Previous investigations from our laboratory and others have demonstrated that type II collagen, once thought to be a cartilage-specific molecule, is also a component of both the primary corneal stroma and the vitreous of embryonic chickens. In the present immunohistochemical study we have examined the expression in these embryonic matrices of another "cartilage-specific" collagen, type IX, along with type II. In the cornea, type IX collagen is in the primary stroma, but is not detectable in the mature, secondary stroma. Even within the primary stroma this collagen has a brief, transitory existence. It first appears in the peripheral stroma at the time the endothelial cells begin to migrate along its posterior surface, and spreads throughout the stroma during the following 24-36 hr. The epitopes on type IX collagen then suddenly become undetectable just before this matrix swells and becomes populated by the periocular mesenchymal cells (future keratocytes). In comparison, collagen type II (along with type I) is present in the stroma before and long after these events. Deposition of immunodetectable type IX collagen in the developing corneal stroma thus seems to be independent of type II. In the vitreous, we observed type IX collagen along with type II as soon as authentic vitreous could be identified and at all subsequent stages of development. In this tissue, therefore, the expression of collagen types IX and II appears to be coordinate.