Peripheral distribution of dermatan sulfate proteoglycans (decorin) in amyloid-containing plaques and their presence in neurofibrillary tangles of Alzheimer's disease.

We used a polyclonal antibody and a mixture of three monoclonal antibodies (MAb), all recognizing the protein core of the small dermatan sulfate proteoglycan (DSPG) (known as PG-II or decorin) derived from human skin fibroblasts, to immunolocalize this molecule in the characteristic lesions in Alzheimer's brain. All antibodies demonstrated positive decorin immunostaining in both the amyloid deposits of neuritic plaques (NPs) and the filamentous structures within neurofibrillary tangles (NFTs). Unlike heparan sulfate proteoglycans (HSPGs), which tend to be evenly distributed throughout NPs containing amyloid fibrils, decorin was primarily localized to the periphery of the spherically shaped amyloid plaques and to the edges of amyloid fibril bundles within the plaque periphery. Decorin was also immunolocalized to the paired helical and straight filaments within NFTs and to collagen fibrils surrounding blood vessels. The unusual distribution of decorin confined to the periphery of amyloid plaques in AD brain suggests that this particular PG may play an important role in the development of the amyloid plaque.     

A monoclonal antibody that detects a specific human neutrophil antigen involved in phorbol myristate acetate- and formyl-methionyl-leucyl-phenylalanine-triggered respiratory burst.

A mouse IgM mAb termed P1E3 was raised against resting human peripheral blood neutrophils and has been shown to recognize a cell-surface Ag with an apparent molecular mass of 155 kDa, as assessed by immunoprecipitation analysis. In addition to the main 155-kDa protein, an additional band of about 210 kDa was also recognized by P1E3 in Western blot analysis. Sequential immunoprecipitation assays showed that the Ag recognized by P1E3 differed from the CD29 and CD45 Ag. However, sequential immunoprecipitation assays carried out with two distinct anti-CD15 mAb and P1E3 showed that P1E3 reacted with CD15 or with a CD15-like Ag. P1E3 stained strongly resting human peripheral blood neutrophils, hardly reacted with peripheral blood monocytes and did not react with PBL and platelets, as assessed by immunofluorescence flow cytometry. P1E3 inhibited the respiratory burst induced by PMA or FMLP, but not the oxidative response induced by Con A or the calcium ionophores A23187 or ionomycin. Furthermore, P1E3 inhibited the activation of the Na+/H+ antiporter in response to PMA or FMLP and the phosphorylation of a protein of about 50 kDa in response to PMA. However, preincubation of neutrophils with P1E3 did not affect the increase in cytosolic free calcium concentration induced by FMLP. These data suggest that the Ag recognized by P1E3 may play a role in modulating the activation of the respiratory burst induced by PMA or FMLP, and that P1E3 seems to affect protein kinase C-mediated signal transduction mechanisms coupled to the induction of the respiratory burst.     

Uridine transport in basolateral plasma membrane vesicles from rat liver

Summary The characteristics of uridine transport were studied in basolateral plasma membrane vesicles isolated from rat liver. Uridine was not metabolized under transport measurement conditions and was taken up into an osmotically active space with no significant binding of uridine to the membrane vesicles. Uridine uptake was sodium dependent, showing no significant stimulation by other monovalent cations. Kinetic analysis of the sodium-dependent component showed a single system with Michaelis-Menten kinetics. Parameter values were K _M 8.9 m and V _max 0.57 pmol/mg prot/sec. Uridine transport proved to be electrogenic, since, firstly, the Hill plot of the kinetic data suggested a 1 uridine: 1 Na⁺ stoichiometry, secondly, valinomycin enhanced basal uridine uptake rates and, thirdly, the permeant nature of the Na⁺ counterions determined uridine transport rates (SCN^– > NO ₃ ^– > Cl^– > SO ₄ ^2– ). Other purines and pyrimidines cis-inhibited and trans-stimulated uridine uptake.This work has been partially supported by grant PM90-0162 from D.G.I.C.Y.T. (Ministerio de Educación y Ciencia, Spain). B.R.-M. is a research fellow supported by the Nestlé Nutrition Research Grant Programme.     

Characterization by photoaffinity labeling of a steroid binding protein in rat liver plasma membrane

Summary The mechanism of steroid uptake by the cell remains controversial. [³H]R5020 was utilized to characterize by photoaffinity labeling the steroid binding site in plasma membrane. This binding was saturable, reversible and had one type of binding site (K _d = 33 ± 4 nm, B _max = 32 ± 2 pmol/mg). [³H]R5020 could be prevented from binding by a variety of steroids (cortisol, progesterone, deoxycorticosterone, and levonorgestrel); estradiol did not have affinity for this binding site. The kinetics of R5020 photoactivation was time dependent and saturable. SDS-PAGE showed a specific band which corresponded to a 53-kDa peptide. The sucrose density gradient analysis has revealed the existence of a protein with a sedimentation coefficient of 3.6 ± 0.2 S. This polypeptide shows different characteristics than cytosolic steroid receptor or serum steroid binding proteins. This binding protein could correspond to the steroid binding site previously found in the plasma membrane.This work was supported by grants PB85-0461 from the Comisión Asesora de Investigatión Científica y Técnica and PGV-8612 from the Departamento de Educatión, Universidades e Investigation del Gobierno Vasco. We thank Roussel-Uclaf (France) for the nonradioactive RU-steroids kindly provided.     

Genetic and functional analysis of the basic replicon of pPS10, a plasmid specific for Pseudomonas isolated from Pseudomonas syringae patovar savastanoi.

The sequence of a 1823 base-pair region containing the replication functions of pPS10, a narrow host-range plasmid isolated from a strain of Pseudomonas savastanoi, is reported. The origin of replication, oriV, or pPS10 is contained in a 535 base-pair fragment of this sequence that can replicate in the presence of trans-acting function(s) of the plasmid. oriV contains four iterons of 22 base-pairs that are preceded by G+C-rich and A+T-rich regions. A dnaA box located adjacent to the repeats of the origin is dispensable but required for efficient replication of pPS10; A and T are equivalent bases at the 5' end of the box. repA, the gene of a trans-acting replication protein of 26,700 Mr has been identified by genetic and functional analysis. repA is adjacent to the origin of replication and is preceded by the consensus sequences of a typical sigma 70 promoter of Escherichia coli. The RepA protein has been identified, using the minicell system of E. coli, as a polypeptide with an apparent molecular mass of 26,000. A minimal pPS10 replicon has been defined to a continuous 1267 base-pair region of pPS10 that includes the oriV and repA sequences.     

Proliferation index in bone marrow cells from severely malnourished rats during lactation.

The aim of this study was to determine if severe malnutrition affects the proportion of proliferating bone marrow cells in malnourished rats during lactation. Sister chromatid staining of metaphases was used as a parameter, as well as the morphology, size and color of bromodeoxyuridine labeled interphase nuclei. The BrdU proliferation labeling index was statistically lower in malnourished rats (20.4%), than in well-nourished controls (35.1%). A difference was also found between the two groups in the proportion of metaphases in first, second and third or successive cell cycle. The average generation time was longer in the malnourished group: 15.3 h, against 11.8 h for the controls. These results indicate that severe malnutrition affects both the proportion of bone marrow proliferating cells and their cell kinetics.     

Germination response to temperature,salinity, light and depth of sowing of ten tropical dune species

This study describes the germination responses of ten tropical dune species to several factors to which their seeds are exposed in the field. Species studied were: three sand dune endemics (Amaranthus greggii, Palafoxia lindenii, and Trachypogon gouini), three pantropical coastal species (Sesuvium portulacastrum, Sporobolus virginicus and Ipomoea stolonifera) and four cosmopolitan grasses also found inland (Panicum repens, Panicum maximum, Pappophorum vaginatum and Andropogon glomeratus). Six species germinated when exposed to different constant temperatures. Four required temperature fluctuation (S. portulacastrum, P. lindenii, S. virginicus, P. repens). Light promoted germination of three species (S. Portulacastrum, P. vaginatum, P. lindenii). Species varied in their degree of sensitivity to salinity. Seeds of T. gouini, I. stolonifera, S. portulacastrum, P. repens, P. Maximum and P. vaginatum were able to germinate under some of the salinity concentrations. Not all species were able to recover after being transferred to distilled water. Seedling emergence was inhibited when seeds were buried. This response was related to depth and to seed size. S. portulacastrum and S. virginicus were the most affected. Nitrates only affected germination response of two species. Seed age promoted germination under a wider range of conditions. P. lindenii showed very heterogeneous responses depending on seed cohort. No dormancy mechanisms were found, other than a thermoperiod and/or light requirement for some of the species. Cosmopolitan grasses tolerated both sand burial and salinity, although the endemics were the most successful in emerging from sand burial; coastal pantropicals were very tolerant to high saline concentrations and recovered completely.     

Purification and Properties of Glyoxysomal Cuprozinc Superoxide Dismutase from Watermelon Cotyledons (Citrullus vulgaris Schrad)

A glyoxysomal copper,zinc-containing superoxide dismutase (EC 1.15.1.1) was purified to homogeneity, for the first time, from watermelon cotyledons (Citrullus vulgaris Schrad.). The stepwise purification procedure consisted of acetone precipitation, batch anion-exchange chromatography, anion-exchange Fast Protein Liquid Chromatography and gel-filtration column chromatography. Pure copper,zinc-superoxide dismutase (Cu,Zn-SOD II) had a specific activity of 1211 units per milligram protein and was purified 400-fold, with a yield of 8 micrograms enzyme per gram cotyledon. The glyoxysomal Cu,Zn-SOD had a relative molecular weight of about 33,000 and was composed of two equal subunits of 16,500 Daltons. Metal analysis showed that the enzyme, unlike other Cu,Zn-SODs, contained 1 gram-atom Cu and 1 gram-atom Zn per mole dimer. No iron and manganese were detected. Ultraviolet and visible absorption spectra were reminiscent of other copper,zinc-superoxide dismutases.     

Effect of the VAM fungus Glomus sp. on the growth and yield of soybean inoculated with Bradyrhizobium japonicum

The effect of two Bradyrhizobium japonicum strains (D344 and Urbana), on the frequency and intensity of infection by a VAM fungal Glomus sp. and the effect of VAM on biomass production by nodulating plants were tested in soybean growing in a soil containing low levels of accessible P and N. During the initial stage of vegetative growth, mycorrhiza frequency in roots inoculated with the two rhizobial strains did not differ. However, during flowering it was 178% higher in roots with the strain D344 than in the presence of the strain Ubrana. At final harvest (green pods) the VAM frequency did not differ in the presence of either strain. VAM positively affected biomass production, foliar concentrations of P, Zn and Cu, and number and dry matter yield of pods, but did not increase concentrations of total N and K. In nonmycorrhizal plants total nitrogenase activity (not nodule mass) and growth were higher with the rhizobial strain Urbana. The greatest nitrogenase activity, growth and yield occurred in the presence of the VAM fungus, and did not differ for plants with different strains of rhizobia.     

Evaluation of host and pathogen responses for screening soil amendments to control Sclerotium rolfsii

Three strains of Bradyrhizobium, 280A, 2209A and 32H1, that nodulated peanuts (Arachis hypogaea L.), were tested for their ability to grow and survive at elevated temperatures of up to 42°C in laboratory culture. Strain 32H1 was unable to grow at 37°C and was more sensitive to elevated temperatures than the other two strains. All three produced heat-shock proteins of molecular weights 17 kDa and 18 kDa. Two greenhouse experiments were conducted to determine the effect of high root temperature on nodulation, growth and nitrogen fixation of peanut. Two peanut varieties (Virginia cv NC7 and Spanish cv Pronto) were inoculated and exposed to root temperatures of 30°, 37° and 40°C. Nodulation and nitrogen fixation were strongly affected by root temperature but there was no variety × temperature interaction. At a constant 40°C root temperature no nodules were formed. Nodules were formed when roots were exposed to this temperature with diurnal cycling but no nitrogen fixation occurred. Highest plant dry weight, shoot nitrogen content and total nitrogen were observed at a constant root temperature of 30°C. Increasing root temperature to 37°C reduced average nitrogen content by 37% and total nitrogen by 49% but did not reduce nodulation. The symbiotic performance of the strains corresponded to their abilities to grow and survive at high temperature in culture.