Analysis of nephritogenic antigens in human glomerular basement membrane by two-dimensional gel electrophoresis

Collagenase digests of GBM were partially purified by column chromatography and analyzed by 2-D gel electrophoresis. Silver staining of 2-D gels showed charge- and size-related heterogeneity of proteins in the 45 to 50 kDa and 25 to 27 kDa regions. These components were transferred to nitrocellulose sheets and reacted with 10 human anti-GBM autoantibodies. Detection of bound anti-GBM autoantibodies to blotted proteins was carried out with peroxidase-labeled goat anti-human IgG and revealed binding predominantly to the cationic (pI 8 to 9.0) 45 to 50 kDa and 25 to 27 kDa components. Positive-staining patterns of blotted proteins were similar with all anti-GBM autoantibodies except that three sera additionally identified neutral (pI 5.5 to 6.5) protein components. One anti-GBM autoantibody, which developed following renal transplantation, lacked reactivity with the most cationic components in the 25 to 27 kDa region. These findings suggest heterogeneity of nephritogenic GBM antigens. The cationic 45 to 50 kDa components were sensitive to reduction, while one neutral 45 to 50 kDa component was resistant; a complex array of 25 to 30 kDa proteins (pI 5.5 to 7.5) were observed by silver staining postreduction. None of the reduced protein components reacted with anti-GBM antibodies, suggesting that epitopes on nephritogenic GBM antigens may be related to disulfide-bonded regions. Although there is variable immunohistochemical reactivity of anti-GBM autoantibodies with the GBM of infant kidneys, 2-D gels of collagenase-digested human infant GBM blotted and reacted with anti-GBM autoantibodies and showed staining patterns similar to that of adult GBM. These studies demonstrate the presence of nephritogenic antigens in the GBM of immature human kidney which are not detectable by immunohistochemical analysis.     

Factors controlling phytoplankton primary productivity in Byram Lake,Mt. Kisco,N.Y., summer 1977

Phytoplankton productivity was measured in Byram Lake Reservoir during summer 1977. Depth integrated productivity (0–5 gC m^{– 2} d^–1) increased with station depth, which together with visibility measurements indicated that light did not limit deep station productivity (C1 and S2). Macrophytes at station C5 (shallow) reduced the euphotic zone to 0 in June.On a unit depth basis, C5 was the most productive station. Apparently changes in macrophyte growth, regulated by light and temperature, controlled phytoplankton production. At C1, productivity was related to levels of different nutrients at different depths, the thermocline influencing nutrient availability at mid-depth. At S2, NH₃-N controlled mid-depth productivity. Surface and mid-depth productivity appeared influenced by factors not measured in this study.     

Evidence for separate networks of classical and novel basement membrane collagen. Characterization of alpha 3(IV)-alport antigen heterodimer.

The COOH-terminal non-collagenous domains (NC1) of type IV collagen from glomerular basement membranes (GBM), lens capsule basement membranes, and Descemet's membrane varied in the distribution of their NC1 subunits. All of these basement membranes (BMs) contained both classical (alpha 1(IV) and alpha 2(IV)) and novel collagen chains (alpha 3(IV), alpha 4(IV) and the Alport antigen). Whereas GBM had a predominance of disulfide-bonded subunits, the lens capsule and Descemet's membrane were primarily monomeric, differences that are likely related to the functional and structural diversity of collagen in various tissues. A heterodimer formed from monomeric subunits of alpha 3(IV) and the Alport antigen exists in human and bovine GBM. This dimer represents an important cross-link of the NC1 domain of novel collagen. Additionally, immunoaffinity methodology showed that the novel BM collagen hexamers segregate into populations containing only novel BM subunits without the participation of the classical subunits (alpha 1(IV) and alpha 2(IV)). These data provided evidence for the presence of two separate networks of BM collagen: one containing alpha 1(IV) and alpha 2(IV), and the other consisting of the novel collagen chains.     

The fate of the carotenoid pigment fucoxanthin during passage through the copepod gut: pigment recovery as a function of copepod species, season and food concentration

The amount of fucoxanthin, a taxonomically diagnostic carotenoid,recovered after passage through the guts of the copepods Acartiacalifomiensis and Calanus pacificus, was determined after thecopepods had fed on low (50 µg Cl^–1) and high (350µg C1^–1 for Acartia; 500 ug C H for Calanus) concentrationsof the diatom Thalassiosira weissftogii, during spring (May)and winter (December). Changes in pigment concentrations andcell abundances were assessed in experimental (with copepods)and control (without copepods) samples by standard incubationexperiments. Pigment recovery was assessed by (i) comparingthe amount of ingested pigment recovered in the experimentalgroups with that predicted to have been ingested from cell countdata and (ii) comparing fuco-xanthin/cell ratios in controland experimental samples. Both techniques suggested that pigmentloss is substantial (usually 60–100%), regardless of species,food availability or season. Patterns of pigment conservationdiffered between species, although pigment recovery was alwayshigher at high, than at low, food concentrations. Pigment recoveryin Acartia was higher (9.4–28.0%) in the spring than duringthe winter (0 recovery), regardless of food concentration. InCalanus, however, pigment recovery was always higher at high(34.9–67.8%) than at low (0 recovery) food concentrations,regardless of season.     

Comparison of non-collagenous type IV collagen components in the human glomerulus and EHS tumor

A method for the isolation of the NC1 domain of type IV collagen has been developed using the EHS sarcoma, a basement membrane-producing mouse tumor. This NC1 domain has been compared to the NC1 of human glomerular basement membrane (hGBM) to assess its usefulness in the biochemical characterization of the Goodpasture antigen which is associated with NC1. Both NC1 isolates appeared to migrate by gel filtration as hexamers (Mr 160,000) and in SDS-polyacrylamide gel electrophoresis as dimers and monomers (Mr 54,000 and 26,000), and were shown to share biochemical identity by amino acid analysis. The hGBM NC1 showed greater complexity in the monomer region, and when compared by two-dimensional gel electrophoresis was found to contain more components in both regions than EHS NC1. Anti-GBM autoantibodies from patients with Goodpasture's syndrome reacted with the EHS NC1 by immunoblotting of two-dimensional gels. The EHS NC1 isolated by reverse phase HPLC partially inhibited the reactivity of the anti-GBM autoantibodies against hGBM NC1 by inhibition ELISA assay. Reverse phase HPLC elution of EHS and hGBM NC1 showed differences in subunit composition and interaction; complete dissociation of the EHS monomers and dimers in 0.1% trifluoroacetic acid was observed, whereas hGBM monomers and dimers eluted together. Rotary shadowing of hGBM NC1 domains revealed size heterogeneity of globular domains, compared with greater uniformity of EHS NC1 hexamers. We conclude that EHS NC1 contains an epitope(s) that is reactive with human autoantibodies to hGBM NC1. However, the immune response in Goodpasture's syndrome may involve antibodies directed against epitopes which are present in greater density and on a more complex array of peptides in the hGBM NC1 than in EHS NC1.     

Land-use proximity as a basis for assessing stream water quality in New York State (USA)

The influence of the proximity of urbanization and agriculture to stream water quality is often difficult to quantify. The objectives of this study were to (1) compare the influence of far-field land-use, encompassing a watershed drainage area, to a near-field, 200-m buffer on each side of the stream in an attempt to determine on which zone of influence land-use has the largest impact on water quality, and (2) incorporate the EPA's Rapid Habitat Assessment Protocol (Barbour et al., 1999) to characterize the riparian and channel characteristics of a stream that influence water quality, which can improve New York State's monitoring protocols. Impacts were assessed through biological, chemical, and physical-habitat data from 29 streams located within a variety of land-use categories. Land-use was identified through USGS National Land Cover Data (NLCD). Principal components analysis (PCA) indicated that land-use and water quality variables were associated with non-point source contaminants (e.g. nutrients and specific conductance). Using Spearman's rank correlation coefficient, significant relationships between all three land-use types and stream water quality were determined at the 200-m buffer zone of influence. At the watershed zone of influence, water quality indicators did not correlate significantly with land cover type. DO and BAP values within the 200-m buffer zone varied inversely with the percentage of urban-land cover. The stronger correlation between land cover and stream water quality at the 200-m proximity than that of the watershed suggests that the presence of a riparian buffer zone between streams and agricultural and urban areas is a significant factor in reducing contamination from non-point source loading.     

Antibody specificity of human glomerular basement membrane type IV collagen NC1 subunits. Species variation in subunit composition

NC1 subunits were purified from gel filtration pools of acid-extracted, collagenase-digested human glomerular basement membranes (hGBM). This methodology, which enriches 28-kDa monomers (M28) in the total digest, allowed purification of these monomers and 24-kDa (M24) and 26-kDa (M26) monomers free from dimers. Reactivity of these subunits with Goodpasture autoantibodies using immunoblotting of sodium dodecyl sulfate-polyacrylamide gel electrophoresis and two-dimensional nonequilibrium pH gradient electrophoresis gels showed strong reactivity with the purified M28 subunits. Inhibition enzyme-linked immunosorbent assay, used to quantitate the reactivity of the purified NC1 subunits, indicated that M28 had a greater than 10-fold increase in ability to inhibit binding to NC1 than NC1 itself. Comparison of hGBM NC1 components were made with those obtained from collagenase digests of salt and acid-extracted bovine and sheep GBM and Englebreth-Holm-Swarm tumor similarly purified by gel filtration and reverse-phase high performance liquid chromatography. Two-dimensional gel analysis of these NC1 isolates revealed absence of the very cationic M28 monomers. Reactivity with antibodies eluted from diseased kidneys of sheep immunized with hGBM (Steblay nephritis) was compared with Goodpasture autoantibody reactivity by immunoblotting two-dimensional gels of hGBM NC1. We conclude that a very cationic M28 monomer (M28 ) found only in hGBM is the probable target in Goodpasture syndrome, that the epitope is present on most NC1 components from extracted and unextracted hGBM, and is exposed by urea denaturation which is enhanced by acid treatment. A weakly cationic M28 monomer (M28+) is present in GBM from other species and is the probable target in Steblay nephritis. Differential recognition of the two M28 components by these antibodies points to different genetic origins or possibly distinct post-translational modifications for these components. This is supported by their presence or absence in different species and tissues, as well as biochemical differences from the M24/26 monomers which presumably are derived from alpha 1(IV) and alpha 2(IV) collagen chains.     

Copepod abundance in the western Irish Sea: relationship to physical regime, phytoplankton production and standing stock

The distinct patterns of stratification in the North Channeland stratified region of the western Irish Sea influence theseasonal abundance of phytoplankton. The 3–4 month productionseason in the stratified region was characterized by productionand biomass peaks in the spring (up to 2378 mg C m² day^–1and 178.4 mg chlorophyll m^–2) and autumn (up to 1280 mgC m^–2 day^–1 and 101.9 mg chlorophyll m^–2).Phytoplankton in the North Channel exhibited a short, late productionseason with a single summer (June/July) peak in production (4483mg Cm^–2 day^–1) and biomass (–160.6 mg chlorophyllm^–2). These differences have little influence on copepoddynamics. Both regions supported recurrent annual cycles ofcopepod abundance with similar seasonal maxima (182.8–241.810³ind. m^–2) and dominant species (Pseudocalanus elongatusand Acartia clausi). Specific rates of population increase inthe spring were 0.071 and 0.048 day¹ for the North Channel andstratified region, respectively. Increased copepod abundancein the stratified region coincided with the spring bloom, andwas significantly correlated with chlorophyll standing stock.Increased copepod abundance preceded the summer production peakin the North Channel. This increase was not correlated withchlorophyll standing crop, suggesting that a food resource otherthan phytoplankton may be responsible for the onset of copepodproduction prior to the spring bloom. Hetero-trophic microplanktonas an alternative food source, and advection of copepods fromthe stratified region, are proposed as possible explanationsfor copepod abundance increasing in advance of the summer peakin primary production.     

Population growth patterns of Skeletonema costatum and nutrient levels in the lower East River,New York,U.S.A.

The population growth patterns of Skeletonema costatum and nutrient levels in the lower East River were examined through field measurements and laboratory experimentation. Maximum growth rates of this diatom (approximately 1.8 divisions per day) were obtained in water samples from the late winter-early spring months. Summer water samples supported little or no growth of this diatom. Measurements of NH₃-N, PO₄,-P, and Si in water samples from the lower East River indicated that nutrient saturated conditions exist year round in this area. The effect of toxic substances in the water column may be responsible for limiting S. costatum growth during the summer months.     

Are copepods important grazers of the spring phytoplankton bloom in the western Irish Sea?

The spring phytoplankton bloom and copepod grazing were studied at a coastal and offshore station in the western Irish Sea during 1997. Maximum chlorophyll standing stocks of 132.8 mg m^-2 inshore and 199.4 mg m^-2 offshore were measured in late April. At that time, mean water column temperatures were 10 and 8C at the coastal and offshore station, respectively. Spring bloom production at the coastal station was estimated as 31.2 g C m^-2 and was dominated by the diatom Guinardia delicatula. Offshore, production was 28.2 g C m^-2 and the bloom was composed of small (10 m) phytoflagellates and the silicoflagellate Dictyocha speculum. Maximum copepod abundance (189 and 544 x 10³ individuals m^-2, inshore and offshore, respectively) coincided with the spring bloom. Pseudocalanus and Temora ingestion rates were derived from measurements of gut pigment fluorescence, and were found to vary during the course of the spring bloom as a result of changes in gut content. Grazing by late copepodite and adult Pseudocalanus and Temora was variable inshore, but overall accounted for 17% of bloom production. Offshore, 22% of bloom production was grazed with maximum grazing (76% of daily production) occurring at the end of the bloom. Large copepod species were not major grazers of the spring bloom. Greater utilization of spring bloom production by copepods in the western Irish Sea compared to regions of the North Sea is attributed to differences in population size at the time of the bloom.