Neutral Salt Extraction Research Articles

Determination of Available Lead in Greenhouse Soil by Solid Phase Dispersion Extraction Coupled with Atomic Absorption Spectrometry based on Primary Secondary Amine

In order to effectively evaluate the pollution status of available lead (Pb) in farmland environment, a method for the determination of available Pb in greenhouse soil by atomic absorption spectrometry with primary secondary amine (PSA) as solid phase dispersion adsorption material was established. In combination with neutral salt (such as 0.01 mol/L CaCl2) extraction technology, neutral lead-containing aqueous solution was obtained. Based on the rapid adsorption of Pb in neutral environment and the rapid desorption of Pb under acidic conditions, the pretreatment method of sample enrichment and purification based on PSA was established by optimizing the parameters. The samples after enrichment and purification were determined by atomic absorption spectrometry. Results showed that the linearity of the method was good in the range of 0.010 ∼ 0.050 mg/kg (R2 = 0.9990). The limit of detection was 0.0051 mg/kg and the limit of quantity was 0.017 mg/kg. The recoveries were 97.6% to 99.2% and the relative standard deviation was 0.79% ∼ 1.56% at the concentrations of 0.010, 0.030 and 0.050 mg/kg.

A comparison of physiologically based extraction test (PBET) and single-extraction methods for release of Cu, Zn, and Pb from mildly acidic and alkali soils

In vitro digestion test can be applied to evaluate the bioaccessibility of soil metals by measuring the solubility of the metals in synthetic human digestive tract. Physiologically based extraction test (PBET), composed of sequential digestion of gastric and intestinal phase, is one of the frequently used in vitro digestion tests. In this study, the PBET was chosen to determine the bioaccessibility of Cu, Zn, and Pb in 14 mildly acidic and alkali (pH 5.87-8.30) soils. The phytoavailability of Cu, Zn, and Pb in the same soils was also measured using six single-extraction methods (0.1 M HNO₃, 0.4 M HOAc, 0.1 M NaNO₃, 0.01 M CaCl₂, 0.05 M EDTA, and 0.5 M DTPA). The extraction efficiencies of the methods were compared. The PBET had a strong ability to extract metals from soil, which was much greater than neutral salt extraction and close to dilute acid and complex extraction in spite of the last 2 h neutral intestinal digestion. The amounts of bioaccessible Cu, Zn, and Pb in the gastric phase and in the gastrointestinal phase were both largely determined by the total content of soil Cu, Zn, and Pb. But the results of gastrointestinal digestion reflected more differences resulting from element and soil types than those of gastric digestion did. It was noticed that most of variations in the amounts of soil Cu, Zn, and Pb extracted by EDTA were well explained by the total soil Cu, Zn, and Pb, as same as the PBET. Moreover, the solubility of Cu, Zn, and Pb in the gastric phase and gastrointestinal phase were all positively linearly correlated with the results of EDTA. It was suggested that EDTA extraction can be used to predict the bioaccessibility of Cu, Zn, and Pb in mildly acidic and alkali (pH > 5.8) soils, and the PBET and EDTA could be applied to measure, in a certain extent, the bioaccessibility and phytoavailability of Cu, Zn, and Pb in mildly acidic and alkali (pH > 5.8) soils at the same time.

Type XIX Collagen Purified from Human Umbilical Cord Is Characterized by Multiple Sharp Kinks Delineating Collagenous Subdomains and by Intermolecular Aggregates via Globular, Disulfide-linked, and Heparin-binding Amino Termini

Type XIX collagen was discovered from the sequence of rhabdomyosarcoma cDNA clones. The chain is composed of a 268-residue amino terminus, an 832-residue discontinuous collagenous region, and a 19-residue carboxyl peptide. Light microscopy immunohistochemistry of adult human tissues demonstrated that type XIX is localized in vascular, neuronal, mesenchymal, and some epithelial basement membrane zones. It also appears to be involved in events linked to skeletal myogenesis. In this report, we have presented the first direct evidence for the molecular structure of type XIX collagen. Using human umbilical cord, native type XIX was purified by neutral salt extraction and by ion exchange and antibody affinity chromatography. Type XIX was found to represent only approximately 10(-6)% of the dry weight of tissue, making it by far the least abundant collagen ever isolated. Transmission electron microscopy after rotary shadowing revealed the appearance of rodlike structures with multiple sharp bends, a small nodule at one end of the molecule, and a total length of 240 nm. Domain-specific antibodies were used to identify the nodule as the noncollagenous amino terminus, whereas the location of most kinks corresponds to major interruptions separating the five collagenous subdomains. More than half of the type XIX molecules observed were present in oligomers of different size and complexity, resulting from association of the amino-terminal domains. Biochemical analysis demonstrated that these supramolecular aggregates are dependent upon and/or stabilized by intermolecular disulfide cross-links and that the globular amino terminus contains a high affinity, heparin-binding site. The polymorphic conformational states of this rare collagen, and its ability to self-assemble into a higher order structure provide focal points for future determination of biologically significant functions in cell-cell and/or cell-matrix interactions.

Comparison of decision support systems for an optimised application of compost and sewage sludge on agricultural land based on heavy metal accumulation in soil

Two different decision support systems (DSS) for the application of organic wastes on agricultural land were developed. Both DSS calculate the maximum application period of organic waste considering exhaustion of the uptake potential of soils for heavy metals. The definition of the uptake potential differs between the DSS alternatives. In the first DSS (DSS-AR), the uptake potential is derived from the difference of actual total heavy metal concentration in soil (according to aqua regia digestion) and the respective statutory limit value. The other DSS (DSS-SI) calculates the remaining sorption capacity of the soil for a heavy metal, i.e. the difference between the predefined maximum and the actual heavy metal concentration at the sorbent. The concentration of sorbed heavy metal is derived from pedotransfer functions (general purpose Freundlich isotherms) using predefined limit concentrations in soil solution (WHO drinking water quality standards) or the actual concentration of soluble heavy metal (according to neutral salt extraction), respectively. For evaluation of their individual characteristics, both DSS were tested in model scenarios using soil data (basic physicochemical properties; Cd, Pb, and Zn concentrations) from various agricultural regions and German guidelines for organic waste application. The DSS-SI showed a better performance than the DSS-AR in this context. The capacity of the soil for heavy metal uptake was used with higher efficiency, i.e. the potential was maximised while environmental limit values were still complied with. Furthermore, the DSS-SI offered a better approximation of the natural variability of soil conditions using an extended set of soil properties in comparison to the DSS-AR. Despite these indications of good DSS-SI performance, the approach requires improvement with regard to the pedotransfer functions implemented in order to consolidate and extend the range of predictions. Furthermore, it should be noted that assessment of the DSS-SI is influenced by the availability of data and is only valid under the current environmental and statutory framework at the regional scale observed. For this reason, transfer of the results of this study into regions with other administrative structures and/or environmental conditions is critical. Individual testing of the DSS-SI and the DSS-AR is required prior to the intended transfer, i.e. implementation of the approaches in the decision support process.

Matrix metalloproteinase 13 (collagenase 3) in human rheumatoid synovium

To show the eventual presence and extent of production of matrix metalloproteinase 13 (MMP-13, or collagenase 3) in rheumatoid synovial tissue samples and extracts, and to assess the inhibition characteristics of recombinant MMP-13. Immunohistochemical avidin-biotin-peroxidase complex staining/morphometry was used to analyze MMP-13-positive cells in situ. Neutral salt extraction of synovial tissue, electrophoresis of the extract in different buffer systems, and Western blotting were also used. The inhibitory properties of doxycycline, clodronate, pamidronate, and D-penicillamine for recombinant enzyme were determined with a soluble type II collagen assay. MMP-13 was detected in fibroblast- and macrophage-like mononuclear cells in the synovial lining and stroma and in vascular endothelial cells. The overall expression of MMP-13 in these cells in the synovial stroma was high in rheumatoid arthritis (86 +/- 12%) compared with osteoarthritis (17 +/- 5%) patient samples (P = 0.0027). In a high-pH native electrophoresis gel, immunoreactivity to anti-MMP-1 and anti-MMP-13 were clearly separated, with anti-MMP-13-immunoreactive material migrating faster than anti-MMP-1-immunoreactive material. Finally, in contrast to MMP-1 and MMP-8, MMP-13 was found to be relatively resistant to the inhibitory effects of doxycycline and clodronate in vitro. Due to its localization in synovial tissue, its substrate profile, increased expression, and relative resistance to known MMP inhibitors, MMP-13 is suggested to play a major role in the pathogenesis of tissue destruction in rheumatoid arthritis.

Ubiquitous expression of the alpha1(XIX) collagen gene (Col19a1) during mouse embryogenesis becomes restricted to a few tissues in the adult organism.

Type XIX collagen is a poorly characterized member of the fibril-associated collagens with an interrupted triple helices (FACIT) class of collagen molecules. As a first step toward elucidating its function, we have isolated full size cDNA clones from the mouse alpha1(XIX) collagen gene (Col19a1) and established its pattern of expression in the developing embryo and adult organism. Col19a1 transcripts can be detected as early as 11 days of gestation and in all embryonic tissues, except the liver, of an 18-day postcoitum mouse. In contrast, only a few adult tissues, brain, eye, and testis, seem to accumulate Col19a1 mRNA. Col19a1 transcripts are at least 10 times more abundant in adult than fetal brain and significantly less in adult than fetal muscle and skin. Consistent with the RNA data, polyclonal antibodies for alpha1(XIX) collagen reacted with a 150-kDa protein in the neutral salt extraction of adult mouse brain tissues. We therefore propose that type XIX collagen plays a distinct role from the other FACIT molecules, particularly in the assembly of embryonic matrices and in the maintenance of specific adult tissues.

Apparent Salt Sorption Reactions in an Unfertilized Acid Subsoil

AbstractThe equivalent retention of Ca2+ and SO2‐4 from gypsum solutions in acid subsoils has recently been reported. The term salt sorption has been coined to describe this process, yet little is known about the nature of the reactions involved. This study was conducted to determine if other cation‐anion combinations would exhibit similar behavior and to examine the extractability of the sorbed ions. An unfertilized acid Cecil (clayey, kaolinitic, thermic Typic Kanhapludult) subsoil was equilibrated using a saturated paste technique, with solutions of six different salts at a loading rate of ≈14.0 mmolc kg‐1. The salt solutions used were CaSO4, MgSO4, Na2SO4, CaCl2, MgCl2, and a deionized water control. This soil exhibited salt sorption with all six electrolyte solutions. The SO4 salts were almost completely sorbed with both cation and anion uptake on the order 13.8 mmolc kg‐1. Calcium and magnesium chloride were sorbed at ≈11.7 mmolc kg‐1, and NaCl was sorbed at 8.2 mmolc kg‐1. The treated soils were air dried and extracted with successive portions of deionized water or 0.5 M NH4NO3. A relatively large proportion of both cations and anions were removed by repeated extraction with water, suggesting that ionic strength played an important role in the salt sorption process. Nearly all of the sorbed cations and Cl‐ were recovered by neutral salt extraction, but <100% of the SO2‐4 could be accounted for by neutral salt extraction. Neutral salt extractable Al was significantly reduced by salt treatment. It appeared that salt sorption in this soil may have resulted from several mechanisms including precipitation, specific adsorption, ionic strength induced charging, and ion pair adsorption operating concurrently.

Increased degradation and altered tissue distribution of cartilage oligomeric matrix protein in human rheumatoid and osteoarthritic cartilage.

We investigated the degradation and tissue distribution of cartilage oligomeric matrix protein in normal, osteoarthritic, and rheumatoid arthritic articular cartilage of the human knee. Cartilage was subjected to sequential extractions with buffers containing neutral salt, with EDTA, and finally with guanidine/HCl and then was analyzed by Western blotting with a polyclonal antiserum to human cartilage oligomeric matrix protein. Western blots of the nine neutral salt extracts from normal cartilage revealed mostly intact pentameric molecules of cartilage oligomeric matrix protein, in contrast to the 13 osteoarthritic and five rheumatoid arthritic cartilage samples that demonstrated marked degradation of cartilage oligomeric matrix protein as noted by a predominance of reduction-sensitive bands at approximately 150 kDa and nonreduction-sensitive bands in the 67-94 kDa range. The EDTA and guanidine/HCl extracts from all groups were similar and showed mostly intact molecules of cartilage oligomeric matrix protein, with smaller amounts of degraded cartilage oligomeric matrix protein identical to those resolved by the Western blots of the neutral salt extracts. Western blots of matched pairs of synovial fluid and cartilage extracts demonstrated cartilage oligomeric matrix protein fragments of the same molecular mass. Competitive enzyme-linked immunosorbent assay revealed significantly less cartilage oligomeric matrix protein in rheumatoid articular cartilage than in either normal or osteoarthritic cartilage. In contrast to normal cartilage, where cartilage oligomeric matrix protein was predominantly localized to the interterritorial matrix throughout all zones of the matrix, with increased staining in the deeper cartilaginous zones, the most intense staining in osteoarthritic cartilage was in the superficial zones of fibrillated cartilage, with little to no immunostaining in the midzones and relatively poor staining in the deeper cartilaginous zones. This distribution was the inverse of that for proteoglycans, as demonstrated by toluidine blue staining, where proteoglycans were depleted primarily from the superficial fibrillated cartilage. In mild to moderately affected rheumatoid cartilage, the tissue distribution of cartilage oligomeric matrix protein was similar to the distribution of proteoglycans, with relatively uniform staining of the interterritorial and territorial matrics. In more severely affected rheumatoid cartilage, the superficial zones demonstrated punctate immunostaining for cartilage oligomeric matrix protein in the interterritorial and territorial matrics, and staining was restricted to the territorial matrix in the deep cartilaginous zones. It is evident from this study that (a) noncollagenous proteins such as cartilage oligomeric matrix protein are greatly affected in arthritis, (b) degradation fragments released from the matrix into the synovial fluid reflect the processes occurring within the matrix, and (c) different zones of the articular cartilage are susceptible to degradation of cartilage oligomeric matrix protein in the different disease processes.

Mast cell-derived tryptase in odontogenic cysts.

Inflammatory and developmental cysts of the jaws are relatively common bone destructive lesions in the human maxillofacial skeleton but their pathogenesis is still poorly understood. In this study the role of mast cells (MC), and mast cell tryptase in particular, was evaluated in the pathophysiology of bone resorption and jaw cyst formation in different types of cysts. The distribution of MC and the amount of tryptase in histological tissue sections were determined by immunohistochemistry using monoclonal antihuman tryptase antibodies and the results were quantitated by using an image analyzing system. The amount of tryptase was further studied by Western-blotting and measurement of trypsin-like activity from the neutral salt extracts obtained from different types of jaw cysts. In contrast to control tissue, high trypsin-like activities and abundant immunoreactive tryptase were observed in the extracts of all types of cysts studied (radicular, dentigerous and keratocyst). In tissue sections the highest amount of tryptase-positive staining was observed in radicular cysts (mean 6.2% of reference area) and the lowest amount in keratocysts (mean 2.1% of reference area, P < 0.01). MC were found to be located in inflammatory cell-rich tissue areas and just beneath the cyst epithelium. Importantly, MC located at the border of bone were observed to be degranulated, indicating high activity of MC and release of tryptase at the regions of early bone destruction. Based on previous findings addressing the role of mast cell tryptase in proteolytic cascades, and the known association of MC with osteoporosis, we suggest that mast cells and mast cell tryptase may contribute significantly to jaw cyst tissue remodelling during growth of a cyst, and to the destruction of the surrounding bone, resulting in jaw cyst expansion.

Characterization of interstitial collagenases in jaw cyst wall.

Neutral salt extracts of 14 specimens of jaw cysts were prepared. Histopathological analysis showed that the specimens consisted of 6 radicular cysts, 6 dentigerous cysts, 1 residual cyst, and 1 odontogenic keratocyst. One periapical granuloma, 1 dental follicle and a sample of clinically healthy oral mucosa were similarly processed and used as controls. Measurement of collagenase activity by monitoring the formation of specific degradation products of type I and II collagen in solution by SDS-PAGE demonstrated that all the cyst extracts contained collagenase, some of which was endogenously activated. Cyst wall collagenase preferably degraded type I over type II collagen, which suggests that the degradation was due to MMP-1 (matrix metalloproteinase-1) rather than the MMP-8 type. This was further supported by the doxycycline-inhibition profile of cyst collagenase, which was similar to that of MMP-1. Part of the cyst wall collagenase was in latent proenzyme form and probably derived, at least in part, from the newly synthesized intracellular collagenase pool. Latent cyst collagenase was efficiently activated with phenylmercuric chloride and to a lesser extent by gold (I) thioglucose and NaOCl. Western-blotting, using specific antibodies against collagenase from human polymorphonuclear neutrophilic leukocytes (MMP-8) and from fibroblasts (MMP-1), revealed a typical 55/45 kDa doublet; also MMP-8 in the latent 80 kDa form and fragmented to 65 kDa active species were found. These results suggest the presence of MMP-1 and, to a lesser extent, MMP-8 type collagenase in the cyst wall.(ABSTRACT TRUNCATED AT 250 WORDS)

Identification and characterization of gelatinases/type IV collagenases in jaw cysts.

The molecular mechanisms of jaw cyst expansion probably involve interactions of matrix metalloproteinases (MMPs) and the tissue inhibitors of MMPs (TIMPs). In this study, molecular species of gelatinases present in neutral salt extracts of cyst walls and cyst fluids were characterized by functional activity measurements (type I gelatin and alpha-casein zymography) and immunologically (Western-blotting). The effects of various protein thiol-group or cysteine-switch reactants involved in the activation of collagenases were studied on cyst gelatinases and a gelatinases purified from human gingival fibroblasts (72 kD MMP-2), gingival keratinocytes (92 kD MMP-9) and polymorphonuclear neutrophilic leukocytes (92 kD MMP-9). Western-blotting revealed the presence of both 92 kD (MMP-9) and 72 kD (MMP-2) gelatinases in cyst wall extracts and cyst fluids. Western-blot studies further suggested that jaw cyst gelatinases were only in part complexed with and thus inhibited by TIMP-1 or TIMP-2, suggesting that both MMP-9 and MMP-2 may participate in cyst expansion. MMP-2 was also partially fragmented to a 68 kD form and additional lower molecular weight proteinases (< 60 kD) were detected by alpha-casein zymography and by Western-blotting, suggesting proteolytic fragmentation. MMP-9 was at least partially activated by all protein-thiol group reactants and rather resistant to oxidative inhibition by hypochlorite (NaOCl); in contrast, MMP-2 was activated by APMA but not at all by gold thioglucose (GTG) and was clearly inactivated by hypochlorite (NaOCl).(ABSTRACT TRUNCATED AT 250 WORDS)

Identification of proteases from periodontopathogenic bacteria as activators of latent human neutrophil and fibroblast-type interstitial collagenases.

Activation of latent human fibroblast-type and neutrophil interstitial procollagenases as well as degradation of native type I collagen by supra- and subgingival dental plaque extracts, an 80-kDa trypsinlike protease from Porphyromas gingivalis (ATCC 33277), a 95-kDa chymotrypsinlike protease from Treponema denticola (ATCC 29522), and selected bacterial species commonly isolated in periodontitis was studied. The bacteria included were Prevotella intermedia (ATCC 25261), Prevotella buccae (ES 57), Prevotella oris (ATCC 33573), Porphyromonas endodontalis (ES 54b), Actinobacillus actinomycetemcomitans (ATCC 295222), Fusobacterium nucleatum (ATCC 10953), Mitsuokella dentalis (DSM 3688), and Streptococcus mitis (ATCC 15909). None of the bacteria activated latent procollagenases; however, both sub- and supragingival dental plaque extracts (neutral salt extraction) and proteases isolated from cell extracts from potentially periodontopathogenic bacteria P. gingivalis and T. denticola were found to activate latent human fibroblast-type and neutrophil interstitial procollagenases. The fibroblast-type interstitial collagenase was more efficiently activated by bacterial proteases than the neutrophil counterpart, which instead preferred nonproteolytic activation by the oxidative agent hypochlorous acid. The proteases were not able to convert collagenase tissue inhibitor of metalloproteinase (TIMP-1) complexes into active form or to change the ability of TIMP-1 to inhibit interstitial collagenase. None of the studied bacteria, proteases from P. gingivalis and T. denticola, or extracts of supra- and subgingival dental plaque showed any significant collagenolytic activity. However, the proteases degraded native and denatured collagen fragments after cleavage by interstitial collagenase and gelatinase. Our results indicate that proteases from periodontopathogenic bacteria can act as direct proteolytic activators of human procollagenases and degrade collagen fragments. Thus, in concert with host enzymes the bacterial proteases may participate in periodontal tissue destruction.

The determination of exchangeable cations in acid soils: Errors caused by weathering reactions during neutral salt extraction

To investigate pH-controlled chemical reactions associated with the neutral salt measurement of exchangeable cations in acid soils, and to estimate the potential error caused by such reactions, a series of experiments was performed which modify an established BaCl2 extraction method. Results showed that dissolution of amorphous silica, aluminum hydroxides and organic material occurs during neutral salt extraction, contributing cations to solution which are indistinguishable from exchangeable cations. As a result overestimates of exchangeable Al and Ca occur, but the error is not significant (&gt; 10%) except in certain Ae horizons which are low in total CEC and high in exchangeable acidity. The 2-h BaCl2 method is recommended over the 4-hour NaCl method to minimize interference from weathering reactions. Key words: Cation exchange, exchangeable acidity, Al, H, acid forest soils, weathering

Characterization of a novel calcium‐binding 90‐kDa glycoprotein (BM‐90) shared by basement membranes and serum

The protein BM-90 was solubilized from the mouse Engelbreth-Holm-Swarm tumor with neutral buffers in molar yields lower (15-30%) than found for other basement membrane proteins (e.g. laminin, BM-40). The purified protein was shown to be rich in cysteine (5 mol%) and to change in SDS electrophoresis from an 84-kDa position to a 95-kDa one upon reduction. BM-90 was also shown to be a calcium-binding protein. The N-terminal sequence of BM-90, as well as those of several internal peptides, showed no identity with any known protein sequences, indicating that it is a new protein. Specific radioimmunoassays showed no or only minor cross-reactions with other known basement membrane proteins. Immunological assays demonstrated BM-90 to be present in neutral salt extracts from mouse heart and kidney, in serum (20-40 micrograms/ml) and in the medium of various cultured cells (0.1-1 microgram/ml). The protein in these samples was identical in size to BM-90 purified from the tumor, indicating that negligible degradation occurs during purification. An extracellular matrix localization of BM-90 was shown by immunofluorescence for Reichert's membrane, lens capsules and other basement membranes. Thus, BM-90 appears to be a novel basement membrane protein whose functions remain to be studied.

A parallel between two techniques of extraction of connective tissue macromolecules

A method of extraction of the collagen and noncollagen proteins from deep dermis of young adult rabbits using a 0.1 m tartaric acid solution was set up. The tartaric acid extraction, together with the preliminary neutral salt extraction, solubilized 95% of the total collagen and 98% of the noncollagen proteins, far more than the 6 m guanidinium Cl solution used for comparison. Elastin was not extracted. Studies on the fibrillation of the extracted collagen in neutral solution at 25°C or on the results of pepsin digestion in acidic solution at +4°C showed that the tartaric acid-extracted collagen was in a nondenatured form, whereas that extracted by guanidinium Cl was largely denatured. Polyacrylamide gel electrophoresis (PAGE) indicated that most of the collagen was of type I and that many noncollagen proteins were present, mostly in the molecular weight range of 40 kDa. Bidimensional PAGE gave a reproducible pattern of these noncollagen proteins, showing that several additional proteins were present in tartaric acid extracts and not in guanidinium chloride extracts.

Initial characterization of neutral proteinases from oral spirochetes.

Intermediate size oral spirochetes were isolated and cultured from subgingival plaque of periodontitis patients utilizing a membrane separation and rifambin selection technique. Neutral salt extracts of the spirochetes were assayed for proteolytic activity against collagen types I and IV, gelatin, and synthetic elastase and trypsin substrates. Type IV collagen obtained from lens capsule basement membrane was degraded to small peptides by the spirochete proteinases. No marked degradation of type I collagen was found when incubations were performed at 25°C. The extracts were able, however, to activate latent collagenase obtained from human gingival fibroblasts and to degrade further the ¼ and ¾ fragments resulting from the collagenase cleavage. Denatured collagen was also degraded by the extracts. High trypsin‐Iike activity and relatively lower elastase‐like enzyme activity were also present in spirochete extracts. The results show that oral spirochetes have a potential for degradation of several proteins and that they may, therefore, have an active role in tissue destruction during periodontal disease.

Identification and interaction repertoire of large forms of the basement membrane protein nidogen.

Nidogen was purified in its genuine form with a mol. wt. of 150 000 (Nd-150) and as fragments with mol. wts. of 100 000 (Nd-100) and 80 000 (Nd-80) from a mouse tumor basement membrane by preventing activity of endogenous proteases with 6 M guanidine and protease inhibitors. The larger forms of nidogen were also identified in stable complexes with laminin in neutral salt extracts of the tumor and in cell culture medium. Purified Nd-150 and Nd-100, but not Nd-80, were shown to interact with laminin in various binding assays, albeit with lower potential than estimated for the genuine complexes formed in situ. Binding of Nd-150 and Nd-100 to fibronectin and to the globular domain of collagen IV was also observed, but not to heparan sulfate proteoglycan.

Evidence for a structural mutation of procollagen type I in a patient with the Ehlers-Danlos syndrome type VII.

We have found that the collagen from a patient with the Ehlers-Danlos syndrome type VII contained a polypeptide chain, pN alpha 2, not present in collagen prepared from normal tissue. Fibroblasts cultured from the patient's skin produced type I procollagen in which the NH2-terminal propeptide of pro alpha 2 was cleaved to about half of normal values by chick procollagen neutral protease which removes the NH2-terminal propeptides from procollagen (N-protease). The NH2-terminal propeptide on the pro alpha 2 chain of the patient's procollagen was also more resistant than procollagen from control fibroblasts to digestion by pepsin or alpha-chymotrypsin. assays for procollagen N-protease indicated that the patient's fibroblsts contained about the same level of enzymic activity as normal fibroblasts. These results suggest that the patient's fibroblasts synthesize both an abnormal pro alpha 2 chain and a normal pro alpha 2 chain. The abnormality probably consists of a structural mutation in or near the site at which procollagen N-protease cleaves the pro alpha 2 chain. The results presented here appear to provide the first example of a mutation in a structural gene for collagen. Since equal amounts of pN alpha 2 and alpha 2 are found in the protein in neutral salt extracts of the patient's tissue, as well as in newly synthesized collagen produced by cultured skin fibroblasts, and since both parents are phenotypically normal and express exclusively normal collagen chains, the patient is likely to be a sporadic heterozygote, arisen by new mutation, with one normal and one abnormal gene coding for pro alpha 2.

Changes in aggregate stability and associated organic matter properties after direct drilling and ploughing on some Australian soils

After 3-8 years' continuous cropping with conventional cultivation and direct drilling, five Australian soils were examined for changes in structural stability attributable to tillage methods. Some increase in organic carbon was found in four direct drilled soils, and the proportion of carbon in the greater than 2 �m fractions was also higher in these soils after short ultrasonic treatment. Direct drilled soils had significantly greater stability with at least one of the stability tests used, but no soil showed consistently greater stability to five different tests. One direct drilled soil was more stable than its ploughed equivalent after alkaline and neutral salt extraction of metal ions and associated humic substances. One direct drilled soil was more dispersed by polysaccharide extraction. Three direct drilled soils retained greater stability after selective extraction of trivalent metal ions complexed to humic fractions. Structural improvement takes place in Australian soils after several years of direct drilling, but may be at a slower rate and to a lesser extent than has been reported for wetter environments.

Isolation of salt-soluble elastin from ligamentum nuchae of copper-deficient calf.

This paper describes the isolation and amino acid analysis of un-cross-linked elastin obtained by neutral salt extraction from the ligamentum nuchae of a calf fed from birth to 9 months on a diet low in copper.