A defective seed coat pattern (Net) is correlated with the post-transcriptional abundance of soluble proline-rich cell wall proteins

The pigmented seed coats of several soybean (Glycine max (L.) Merr.) plant introductions and isolines have unusual defects that result in cracking of the mature seed coat exposing the endosperm and cotyledons. It has previously been shown that the T (tawny) locus that controls the color of trichomes on stems and leaves also has an effect on both the structure and pigmentation of the seed coat. Distribution of pigmentation on the seed coat is controlled by alleles of the I (inhibitor) locus. It was also found that total seed coat proteins were difficult to extract from pigmented seed coats with i T genotypes because they have procyanidins that exhibit tannin properties. We report that the inclusion of poly-L-proline in the extraction buffer out-competes proteins for binding to procyanidins. Once this problem was solved, we examined expression of the proline-rich cell wall proteins PRP1 and PRP2 in pigmented genotypes with the dominant T allele. We found that both homozygous i T and i t genotypes have reduced soluble PRP1 levels. The epistatic interaction of the double recessive genotype at both loci is necessary to produce the pigmented, defective seed coat phenotype characteristic of seed coats with the double recessive i and t alleles. This implies a novel effect of an enzyme in the flavonoid pathway on seed coat structure in addition to its effect on flavonoids, anthocyanidins, and proanthocyanidins. No soluble PRP1 polypeptides were detectable in pigmented seed coats (i T genotypes) of isolines that also display a net-like pattern of seed coat cracking, known as the Net defect. PRP2 was also absent in one of the these lines. However, both PRP1 and PRP2 cytoplasmic mRNAs were found in the Net-defective seed coats. Together with in vitro translation studies, these results suggest that the absence of soluble PRP polypeptides in the defective Net lines is post-translational and could be due to a more rapid or premature insolubilization of PRP polypeptides within the cell wall matrix.     

Genetic polymorphism of human salivary proline-rich proteins: Further genetic analysis

Electrophoresis of concentrated parotid saliva on slab polyacrylamide gels negatively stained with 3,3-dimethoxybenzidine and hydrogen peroxide (DMB stain) showed nine phenotypes among the proline-rich proteins. These phenotypes are the expression of four autosomal codominant alleles. Gene frequencies are, for Caucasians, Pr ¹=0.640, Pr ¹=0.005, Pr ²=0.080, Pr ²=0.275; for Negroes, Pr ¹=0.700, Pr ¹=0.050, Pr ²=0.080, Pr ²=0.170; for Chinese, Pr ¹=0.770, Pr ¹=0, Pr ²=0, Pr ²=0.230. The presence or absence of another pair of proteins giving the same negative staining is inherited as an autosomal dominant trait (Db). Homozygous Db + + and heterozygous Db + – individuals could not be distinguished. The genetic determinant (Db) for this pair of proteins is either closely linked to or part of the Pr locus. Gene frequencies are, for Caucasians, Db ⁺=0.12, Db ^–=0.88; for Negroes, Db ⁺=0.56, Db ^–=0.44; for Chinese, Db ⁺=0.07, Db ^–=0.93.This study was supported by a grant from the National Institutes of Dental Research (9-R01-DE-03685-08) and in part by grant GM 15422 from the National Institutes of Health.     

Localization of repetitive proline-rich proteins in the extracellular matrix of pea root nodules

Summary Early responses of legume roots toRhizobium inoculation include new cell wall synthesis and induction of some putative wall protein genes. Although the predicted amino acid sequences of several early nodulins indicate that they encode proline-rich proteins (PRPs), the proteins have been neither isolated nor has their presence been demonstrated in cell walls. We have used polyclonal antibodies against PRP2 from soybean to identify and localize proline-rich proteins in pea nodules. On immunoblots, several PRPs were detected, ranging from less than 20 kDa to 110 kDa. Immunocytochemistry revealed that tissues of the vascular cylinder contained abundant PRPs, particularly in the secondary cell walls of xylem elements and phloem fibers. PRPs were also found within the primary wall of the nodule endodermis and within Casparian strips of the vascular endodermis. Of symbiotic importance, PRPs were a prominent component of the infection thread matrix in newly infected root cells and in nodules. PRPs were also secreted by cells in the uninfected nodule parenchyma, where they were found occluding intercellular spaces outside the middle lamella. Despite structural conservation among members of this class of cell wall proteins, PRPs were targeted to distinct layers of the extracellular matrix dependent upon cell type, and may thus play separate roles in the biology of plant cells. The putative functions and the potential for interactions between PRPs and other wall polymers are discussed.Abbreviations DTT dithiothreitol - EDTA ethylenediamine tetraacetate - GRP glycine-rich protein - PCR polymerase chain reaction - PGA polygalacturonic acid - PMSF phenylmethylsulfonyl fluoride - PRP proline-rich protein - SDS-PAGE sodium dodecylsulfate-polyacrylamide gel electrophoresis - Tris tris(hydroxylmethyl) aminomethane - Tween 20 polyoxyethylene sorbitan monolaurate Dedicated to the memory of Professor John G. Torrey     

Heat shock induced proteins in plant cells

Tobacco (Nicotiana tabacum) and soybean (Glycine max) tissue culture cells were exposed to a heat shock and protein synthesis studied by SDS-polyacrylamide gel electrophoresis after labeling with radioactive amino acids. A new pattern of protein synthesis is observed in heat-shocked cells compared to that in control cells. About 12 protein bands, some newly appearing, others synthesized in greatly increased quantities in heat-shock cells, are seen. Several of the heat-shock proteins (HSPs) in both tobacco and soybean are similar in size. One of the HSPs in soybean (76K) shares peptide homology with its presumptive 25°C counterpart, indicating that the synthesis of at least some HSPs may not be due to activation of new genes. The optimum temperature for maximal induction of most HSPs is 39–40°C. Total protein synthesis decreases as heat-shock temperature is increased and is barely detectable at 45°C. The heat-shock response is maintained for a relatively short time in tobacco cells. After 3 hr at 39°C, a decrease is seen in the synthesis of the HSPs, and after 4 hr practically no HSPs are synthesized. After exposure to 39°C for 1 hr, followed by a return of tobacco cells to 26°C, recovery to the control pattern of synthesis requires greater than 6 hours. These results indicate that cells of flowering plants exhibit a heat-shock response similar to that observed in animal cells.     

Involvement of a maize proline-rich protein in secondary cell wall formation as deduced from its specific mRNA localization

A clone encoding a proline-rich protein (ZmPRP) has been obtained from maize root by differential screening of a maturing elongation root cDNA library. The amino acid sequence deduced from the full-length cDNA contains a putative signal peptide and a highly repetitive sequence containing the PEPK motif, indicating that the ZmPRP mRNA may code for a cell wall protein. The PEPK repeat is also found in a previously reported wheat sequence but differs from the repeated sequences found in hydroxyproline-rich glycoproteins (HRGP) and in dicot proline-rich proteins (PRP). In the maize genome, the ZmPRP protein is encoded by a single gene that is expressed in maturing regions of the root, in the hypocotyl and in the pericarp. In these organs, the ZmPRP mRNA accumulates in the xylem and surrounding cells, and in the epidermis. No ZmPRP mRNA was found in the phloem. The pattern of mRNA accumulation is very similar to the one observed for genes coding for proteins involved in lignin biosynthesis and, like most cell wall proteins, ZmPRP synthesis is also induced by wounding. These data support the hypothesis that ZmPRP is a member of a new class of fibrous proteins involved in the secondary cell wall formation in monocot species.     

Regulation of cell growth and the expression of extracellular matrix proteins in colorectal adenocarcinoma: a fibroblast-tumor cell coculture model to study tumor-host interactions in vitro

The production of abundant connective tissue within malignant tumors, the so-called desmoplastic stromal reaction, is a hallmark of colorectal adenocarcinomas. This stroma is produced to a large extent by myofibroblasts and contains various amounts of collagens (type I, III, and V), chondroitin sulfate proteoglycan, hyaluronic acid, fibronectin, and tenascin-C. In this study we have established a monolayer coculture model between two different colorectal adenocarcinoma cell lines (HRT-18, and CX-2) and colonic fibroblasts (CCD-18) to investigate the mechanisms regulating (i) the production of extracellular matrix (ECM) components, (ii) the induction of myofibroblastic differentiation, and (iii) cellular proliferation. We found that TGFbeta1 and FGF-2 stimulated ECM synthesis of fibroblasts. Myofibroblastic differentiation was stimulated by TGFbeta1 but suppressed by FGF-2. There was a mutual stimulation of proliferation between fibroblasts and carcinoma cells. The analogies with ECM components expressed in cocultures and colorectal adenocarcinoma samples suggest that the coculture model used in this study is useful to study tumor cell-fibroblast interactions.     

The matrilins--adaptor proteins in the extracellular matrix

The matrilins form a four-member family of modular, multisubunit matrix proteins, which are expressed in cartilage but also in many other forms of extracellular matrix. They participate in the formation of fibrillar or filamentous structures and are often associated with collagens. It appears that they mediate interactions between collagen-containing fibrils and other matrix constituents, such as aggrecan. This adaptor function may be modulated by physiological proteolysis that causes the loss of single subunits and thereby a decrease in binding avidity. Attempts to study matrilin function by gene inactivation in mouse have been frustrating and so far not yielded pronounced phenotypes, presumably because of the extensive redundancy within the family allowing compensation by one family member for another. However, mutations in matrilin-3 in humans cause different forms of chondrodysplasias and perhaps also hand osteoarthritis. As loss of matrilin-3 is not critical in mouse, these phenotypes are likely to be caused by dominant negative effects.     

Simulated weightlessness changes the cytoskeleton and extracellular matrix proteins in papillary thyroid carcinoma cells

Studies of astronauts, experimental animals, and cells have shown that, after spaceflights, the function of the thyroid is altered by low-gravity conditions. The objective of this study was to investigate the cytoskeleton and extracellular matrix (ECM) protein synthesis of papillary thyroid cancer cells grown under zero g. We investigated alterations of ONCO-DG 1 cells exposed to simulated microgravity on a three-dimensional random-positioning machine (clinostat) for 30 min, 24 h, 48 h, 72 h, and 120 h (n=6, each group). ONCO-DG 1 cells grown under microgravity exhibited early alterations of the cytoskeleton and formed multicellular spheroids. The cytoskeleton was disintegrated, and nuclei showed morphological signs of apoptosis after 30 min. At this time, vimentin was increased. Vimentin and cytokeratin were highly disorganized, and microtubules (α–tubulin) did not display their typical radial array. After 48 h, the cytoskeletal changes were nearly reversed. The formation of multicellular spheroids continued. In parallel, the accumulation of ECM components, such as collagen types I and III, fibronectin, chondroitin sulfate, osteopontin, and CD44, increased. The levels of both transforming growth factor beta-1 (TGF-β₁) and TGF-β receptor type II proteins were elevated from 24 h until 120 h clinorotation. Gene expression of TGF-β₁ was clearly enhanced during culture under zero g. The amount of E-cadherin was enhanced time-dependently. We suggest that simulated weightlessness rapidly affects the cytoskeleton of papillary thyroid carcinoma cells and increases the amount of ECM proteins in a time-dependent manner.The work of Augusto Cogoli was supported by ETH Zurich, Switzerland.     

Dynamic continuity of nuclear and mitotic matrix proteins in the cell cycle

The eukaryotic cell nucleus is a membrane-enclosed compartment containing the genome and associated molecules supported by a highly insoluble filamentous network known as the nucleoskeleton or nuclear matrix. The nuclear matrix is believed to play roles in maintaining nuclear architecture and organizing nuclear metabolism. Recently, advances in microscopic techniques and the availability of new molecular probes have made it possible to localize functional domains within the nuclear matrix and demonstrate dynamic interactions between both soluble and insoluble components involved in the control of multiple nuclear transactions. Like the cytoplasm and its skeleton, the nucleoplasm is highly structured and very crowded with an equally complex skeletal framework. In fact, there is growing evidence that the two skeletal systems are functionally contiguous, providing a dynamic cellular matrix connecting the cell surface with the genome. If we impose cell cycle dynamics upon this skeletal organization, it is obvious that the genome and associated nuclear matrix must undergo a major structural transition during mitosis, being disassembled and/or reorganized in late G2 and reassembled again in daughter nuclei. However, recent evidence from our laboratory and elsewhere suggests that much of the nuclear matrix is used to form the mitotic apparatus (MA). Indeed, both facultative and constitutive matrix-associated proteins such as NuMA, CENP-B, CENP-F, and the retinoblastoma protein (Rb) associate within and around the MA. During mitosis, the nuclear matrix proteins may either become inert “passengers” or assume critical functions in partitioning the genome into newly formed G1 nuclei. Therefore, we support the view that the nuclear matrix exists as a dynamic architectural continuum, embracing the genome and maintaining cellular regulation throughout the cell cycle. © 1996 Wiley-Liss, Inc.     

Spatial interrelationships between proteoglycans and extracellular matrix proteins in cell cultures

Immunolabelling of cultured cells for chondroitin-sulfate proteoglycan (CSPG), in conjunction with antibodies to fibronectin, collagen and laminin, revealed the spatial interrelationships between the different matrix components. CSPG was organized in two major forms. Fibronectin-independent dotted patterns of CSPG were detected on the substrate and cell surfaces at early stages after plating. At later stages, however, significant overlapping was found between the two extracellular matrix components. Immunoelectron microscopic examination indicated that the CSPG was organized as granules of varying sizes which were associated with the cell surface, the substrate, or with the periphery of the fibronectin network.     

Allelic variants of acidic proline-rich proteins observed in Japanese,Chinese, and Malays

Three new variants of acidic proline-rich proteins (At, Au, Aw) were found in human parotid saliva by isoelectric focusing and basic gel electrophoresis. Electrophoretic comparison of the purified proteins and their tryptic peptides suggested minor charge and size differences from other acidic PRPs. Genetic and biochemical studies indicate that the At and Aw proteins are allelic products of thePRH1 locus. Gene frequencies of the At productive allele (PRH1 ⁶) in Japanese, Chinese, and Malays were 0.008, 0.012, and 0.004, respectively. The Au protein was also found in Japanese (2 in 746 samples), Chinese (1 in 215 samples), and Malays (1 in 220 samples), however, the Aw protein was found only in one Japanese (n=746). These three proteins were not found in 106 Indian subjects.This study was supported in part by a grant from International Scientific Research Program of the Ministry of Education, Science and Culture of Japan, No. 62043071 (representative: K.S.).     

Identification and characterization of a cell surface protein of Prevotella intermedia 17 with broad-spectrum binding activity for extracellular matrix proteins

Prevotella intermedia binds and invades a variety of host cells. This binding is most probably mediated through cell surface proteins termed adhesins. To identify proteins binding to the host extracellular matrix (ECM) component, fibronectin, and study the molecular mechanism underlying bacterial colonization, we applied proteomic approaches to perform a global investigation of P. intermedia strain 17 outer membrane proteins. 2-DE followed by Far Western Blot analysis using fibronectin as a probe revealed a 29-kDa fibronectin-binding protein, designated here AdpB. The molecular identity of the protein was determined using PMF followed by a search of the P. intermedia 17 protein database. Database searches revealed the similarity of AdpB to multiple bacterial outer membrane proteins including the fibronectin-binding protein from Campylobacter jejuni. A recombinant AdpB protein bound fibronectin as well as other host ECM components, including fibrinogen and laminin, in a saturable, dose-dependent manner. Binding of AdpB to immobilized fibronectin was also inhibited by soluble fibronectin, laminin, and fibrinogen, indicating the binding was specific. Finally, immunoelectron microscopy with anti-AdpB demonstrated the cell surface location of the protein. This is the first cell surface protein with a broad-spectrum ECM-binding abilities identified and characterized in P. intermedia 17.     

The expression of mRNA of cytokines and of extracellular matrix proteins in triiodothyronine-treated rat hearts

In various models of cardiac hypertrophy, e.g. treatment of rats with norepinephrine infusion or pressure overload, increased expression of cytokines together with increase in extracellular matrix proteins (ECMP) was reported. In this study the effect of triiodothyronine (T3) on the expression of mRNA for cytokines and ECMP was investigated. Female Sprague-Dawley rats were treated daily with T3 in a dose of 0.2 mg.kg^–1 of body weight s.c. Changes in the left (LV) and right (RV) ventricular function were measured 6, 24, 48, 72 h and 7 and 14 days after the first T3-injection using Millar ultraminiature pressure catheter transducers. RNA was isolated from LV and RV tissue, and the expression of cytokines and ECMP was measured using the ribonuclease protection assay. T3-treatment induced a significant increase in LV dP/dt_max and RV dP/dt_max, (p < 0.05) 24 h after the first injection of T3 together with an increase in heart rate (p < 0.01). The RV systolic pressure increased 48 h after the first T3 injection, whereas the LV systolic pressure remained unchanged. After 48 h the heart weight to body weight ratio was increased (p< 0.01). Hypertrophy of the RV was more prominent than that of the LV (155.9 vs. 137.7%).In all groups the expression of mRNA for interleukins (IL) IL-6, IL-1, IL-1 and tumour necrosis factor (TNF)- in both ventricles did not change (p > 0.05). There was a significant increase in the mRNA for colligin 24 h after the T3 injection in both LV (p < 0.01) and RV (p< 0.05). This was followed by an increase in the mRNA for collagen I and III 72 h after the first T3-dose (p < 0.05 in RV; p < 0.01 in LV). At this point, the mRNA for tissue inhibitor of matrix metalloproteinases-2 (TIMP-2) was increased (p < 0.01) in the LV only. Moreover, after 7 days also the mRNA for matrix metalloproteinase (MMP)-2 increased (p < 0.01) in the LV. Both, TIMP-2 and MMP-2 were increased in the RV only after 14 days (p < 0.05). The gelatinase activity of MMP-2, however, was unchanged in both ventricles. The T3-induced cardiac hypertrophy was not accompanied by fibrosis as measured by the Sirius red staining after 14-days of T3-treatment. The moderate increase in mRNA for ECMP and MMP may be attributed more to the increasing mass of the ventricles with the accompanying remodelling of the ECM than to increased fibrosis.     

Monocyte cytokine synthesis in response to extracellular cell stress proteins suggests these proteins exhibit network behaviour

Human peripheral blood monocytes were exposed to single or pairs of cell stress proteins (CSPs), specifically Hsp10, Hsp27, Hsp60 and Hsp70—the former two having anti-inflammatory actions while the latter pair being assumed to be pro-inflammatory in activity. This study was to test if these proteins exhibited any network behaviour. To control for possible lipopolysaccharide contamination, polymyxin B was used. Surprisingly, at concentrations higher than 1 μg/ml, polymyxin B itself could induce cytokine synthesis. A number of commercial sources of the molecular chaperones were tested, and marked variations in monocyte cytokine synthesis were found. All four CSPs stimulated the same profile of IL-1/IL-6 synthesis and IL-10/TNF-α synthesis although the kinetics of production of these two pairs of cytokines were very different. A key question was whether extracellular molecular chaperones exhibited network behaviour. To test this, monocytes were cultured with suboptimal concentrations of single CSP and pairs of CSP to look for additive, synergistic or antagonistic cell responses. The major finding was that pairs of molecular chaperones, including chaperones thought to stimulate monocyte cytokine synthesis, could produce significant antagonistic cellular responses. This demonstrates that extracellular CSPs constitute an additional potent layer within the complex cytokine network and furthermore suggests that monocytes have evolved to dampen their immune responses upon exposure to extracellular networks of CSPs—perhaps as a mechanism for protecting cells against detrimental cellular stress responses.     

Biochemical characterization of human kallikrein 8 and its possible involvement in the degradation of extracellular matrix proteins

Human kallikrein 8 (KLK8) is a member of the human kallikrein gene family of serine proteases, and its protein, hK8, has recently been suggested to serve as a new ovarian cancer marker. To gain insights into the physiological role of hK8, the active recombinant enzyme was obtained in a pure state for biochemical and enzymatic characterizations. hK8 had trypsin-like activity with a strong preference for Arg over Lys in the P1 position, and its activity was inhibited by typical serine protease inhibitors. The protease degraded casein, fibronectin, gelatin, collagen type IV, fibrinogen, and high-molecular-weight kininogen. hK8 also converted human single-chain tissue-type plasminogen activator (65 kDa) to its two-chain form (32 and 33 kDa) by specifically cleaving the peptide bond Arg275-Ile276. This conversion resulted in a drastic increase in the activity of the activator toward the fluorogenic substrate Pyr-Gly-Arg-MCA and plasminogen in the absence of fibrin. Our findings suggest that hK8 may be implicated in ECM protein degradation in the area surrounding hK8-producing cells.     

Platelet glycoprotein IIb-IIIa-like proteins mediate endothelial cell attachment to adhesive proteins and the extracellular matrix

The adherence of human umbilical vein endothelial (HUVE) cells to adhesive matrix proteins was examined to determine if cell attachment and spreading were mediated by the glycoprotein (GP) IIb-IIIa complex on endothelial cells. The HUVE cells adhered well to glass slides that had been coated with fibronectin, vitronectin, fibrinogen, or von Willebrand factor but failed to adhere to albumin-coated or to uncoated slides. The HUVE cell attachment and spreading on vitronectin, fibrinogen, and von Willebrand factor were greatly inhibited by a GP IIb-IIIa monoclonal antibody (7E3). In contrast, HUVE cell attachment to fibronectin was not inhibited by 7E3 but was inhibited by a fibronectin-receptor antibody (alpha GP140), which had no effect on cell attachment to the other adhesive proteins. The 7E3 antibody, but not alpha GP140, disrupted HUVE cell monolayers by detaching cells from their naturally occurring extracellular matrix. These data indicate that platelet GP IIb-IIIa-like proteins mediate the adherence of HUVE cells to specific adhesive proteins and to the extracellular matrix.     

Red blood cell lysate modulates the expression of extracellular matrix proteins in dermal fibroblasts

Trichinella spiralis is a zoonotic nematode and food borne parasite and infection with T. spiralis leads to suppression of the host immune response and other immunopathologies. The excretory/secretory (ES) products of T. spiralis play important roles in the process of immunomodulation. However, the mechanisms and related molecules are unknown. Macrophages, a target for immunomodulation by the helminth parasite, play a critical role in initiating and modulating the host immune response to parasite infection. In this study, we examined the effect of ES products from different stages of T. spiralis on modulating J774A.1 macrophage activities. ES products from different stages of T. spiralis reduced the capacity of macrophages to express pro-inflammatory cytokines (tumor necrosis factor α, interleukin-1β , interleukin-6 , and interleukin-12) in response to lipopolysaccharide (LPS) challenge. However, only ES products from 3-day-old adult worms and 5-day-old adult worms/new-born larvae significantly inhibited inducible nitric oxide synthase gene expression in LPS-induced macrophages. In addition, ES products alone boosted the expression of anti-inflammatory cytokines interleukin-10 and transforming growth factor-β and effector molecule arginase 1 in J774A.1 macrophages. Signal transduction studies showed that ES products significantly inhibited nuclear factor-κB translocation into the nucleus and the phosphorylation of both extracellular signal-regulated protein kinase 1/2 and p38 mitogen-activated protein kinase in LPS-stimulated J774A.1 macrophages. These results suggest that ES products regulate host immune response at the macrophage level through inhibition of pro-inflammatory cytokines production and induction of macrophage toward the alternative phenotype, which maybe important for worm survival and host health.