Evidence for a cooperative role of gelatinase A and membrane type-1 matrix metalloproteinase during Xenopus laevis development

Matrix metalloproteinases (MMPs) are a large family of extracellular or membrane-bound proteases. Their ability to cleave extracellular matrix (ECM) proteins has implicated a role in ECM remodeling to affect cell fate and behavior during development and in pathogenesis. We have shown previously that membrane-type 1 (MT1)-MMP [corrected] is coexpressed temporally and spatially with the MMP gelatinase A (GelA) in all cell types of the intestine and tail where GelA is expressed during Xenopus laevis metamorphosis, suggesting a cooperative role of these MMPs in development. Here, we show that Xenopus GelA and MT1-MMP interact with each other in vivo and that overexpression of MT1-MMP and GelA together in Xenopus embryos leads to the activation of pro-GelA. We further show that both MMPs are expressed during Xenopus embryogenesis, although MT1-MMP gene is expressed earlier than the GelA gene. To investigate whether the embryonic MMPs play a role in development, we have studied whether precocious expression of these MMPs alters development. Our results show that overexpression of both MMPs causes developmental abnormalities and embryonic death by a mechanism that requires the catalytic activity of the MMPs. More importantly, we show that coexpression of wild type MT1-MMP and GelA leads to a cooperative effect on embryonic development and that this cooperative effect is abolished when the catalytic activity of either MMP is eliminated through a point mutation in the catalytic domain. Thus, our studies support a cooperative role of these MMPs in embryonic development, likely through the activation of pro-GelA by MT1-MMP.     

Application of two-dimensional electrophoresis for monitoring gastrointestinal digestion of milk

Summary. Two-dimensional electrophoresis (2-DE) was used for tracing in vivo gastrointestinal digestion of milk proteins in a rapid model system with rats. Contents of stomach and small intestine from digestion trials with rats given a single dose of milk powder were recovered after 1 hour. They were then subjected to 2-DE (IEF and SDS-PAGE). 2-DE showed undigested proteins in a MW range 13.0–66.0 kDa in stomach and 13.0–25.0 kDa in the small intestine, thus indicating that milk proteins are slowly digested. This approach may shed light on pattern of protein digestion and mechanism of amino acid and peptide assimilation.     

Transport of L-proline,L-proline-containing peptides and related drugs at mammalian epithelial cell membranes

Summary. Membrane transport of L-proline has received considerable attention in basic and pharmaceutical research recently. Of the most recently cloned members of the solute carrier family, two are “proline transporters”. The amino acid transporter PAT1, expressed in intestine, kidney, brain and other organs, mediates the uptake of proline and derivatives in a pH gradient-dependent manner. The Na⁺-dependent proline transporter SIT1, cloned in 2005, exhibits the properties of the long-sought classical IMINO system. Proline-containing peptides are of interest for several reasons. Many biologically important peptide sequences contain highly conserved proline residues. Xaa-Pro peptides are very often resistant to enzymatic hydrolysis and display, in contrast to Pro-Xaa peptides, a high affinity to the H⁺/peptide cotransporter PEPT1 which is expressed in intestinal, renal, lung and biliary duct epithelial cells. Furthermore, several orally available drugs are recognized by PEPT1 as Xaa-Pro analogues due to their sterical resemblance to small peptides.     

Comparison of cell wall regeneration on maize protoplasts isolated from leaf tissue and suspension cultured cells

Summary The cell wall regeneration on protoplasts derived from maize mesophyll cells was compared with wall regeneration on protoplasts derived from suspension cultured cells using light microscopy, transmission electron microscopy, and mass spectrometry. The time course of cell wall regeneration has shown that the mesophyll protoplasts regenerated walls much slower than the protoplasts derived from cultured cells. Moreover, cell wall materials on the mesophyll protoplasts were often unevenly distributed. Electron microscopy has further demonstrated that the mesophyll protoplasts have less organized and compact walls than the protoplasts from cultured cells. Chemical analysis revealed that the mesophyll protoplasts had a lower ratio ofβ-(1–3)-glucan toβ-(1–4)-glucan than protoplasts from cultured cells. The significance of these results for the viability and development of protoplasts in culture is discussed. National Research Council of Canada paper no. 32458.     

Membrane type-1 matrix metalloproteinases and tissue inhibitor of metalloproteinases-2 RNA levels mimic each other during Xenopus laevis metamorphosis

Matrix metalloproteinases (MMPs) and their endogenous inhibitors TIMPs (tissue inhibitors of MMPs), are two protein families that work together to remodel the extracellular matrix (ECM). TIMPs serve not only to inhibit MMP activity, but also aid in the activation of MMPs that are secreted as inactive zymogens. Xenopus laevis metamorphosis is an ideal model for studying MMP and TIMP expression levels because all tissues are remodeled under the control of one molecule, thyroid hormone. Here, using RT-PCR analysis, we examine the metamorphic RNA levels of two membrane-type MMPs (MT1-MMP, MT3-MMP), two TIMPs (TIMP-2, TIMP-3) and a potent gelatinase (Gel-A) that can be activated by the combinatory activity of a MT-MMP and a TIMP. In the metamorphic tail and intestine the RNA levels of TIMP-2 and MT1-MMP mirror each other, and closely resemble that of Gel-A as all three are elevated during periods of cell death and proliferation. Conversely, MT3-MMP and TIMP-3 do not have similar RNA level patterns nor do they mimic the RNA levels of the other genes examined. Intriguingly, TIMP-3, which has been shown to have anti-apoptotic activity, is found at low levels in tissues during periods of apoptosis.     

Gene expression of β–catenin is up-regulated in inner dental epithelium and enamel knots during molar tooth morphogenesis in the mouse

Beta–catenin is a multi–functional molecule that is involved in both cell–cell adhesion and signaling. We analyzed changes in β–catenin gene expression during mouse molar tooth development by in situ hybridization. Prominent up–regulation of the expression of this gene was evident exclusively in the enamel knot at the early cap stage. During the cap and bell stages, the enamel knot, inner dental epithelium, and differentiating stratum intermedium expressed the β–catenin gene more strongly than other parts of the enamel organ. During these stages, the strength of the gene expression changed heterogeneously within the inner dental epithelium and stratum intermedium. However, the heterogeneity was not evident at the late bell stage, when the cells in the inner dental epithelium had differentiated into ameloblasts at the cusp tip. No spatiotemporal change in β–catenin gene expression was apparent in the dental papilla except for the cells that differentiated into odontoblasts, which became negative for the expression of the gene after their differentiation. Thus, the up-regulated expression of the β–catenin gene was strongly associated with epithelial morphogenesis. These findings raise the possibility that the up–regulation of the gene expression and the stabilization of the protein by Wnt signaling play a role in the regulation of the activities of β–catenin in tooth morphogenesis.     

靶向膜型1基质金属蛋白酶反义肽的虚拟筛选与分子模拟

膜型1基质金属蛋白酶(Membrane type-1 matrix metalloproteinase,MT1-MMP,MMP14)在肿瘤的发生发展及转移中起着重要作用,是肿瘤潜在理想的药物靶标。为了获得MT1-MMP结合肽,我们首先采用生物信息学方法分析MMPs序列,获得MT1-MMP差异大且特异的序列。以此为正义肽靶标,设计反义肽库,然后通过分子对接、分子动力学模拟以及体外细胞实验等多种方法,进行靶向MT1-MMP反义肽的筛选与活性研究。多序列比对确定了位于MT1-MMP环区的特异序列AYIREGHE(简称MT1-loop),并构建1 536条反义肽。经两轮虚拟筛选,选取打分位于前五的反义肽用于后续研究。该五条反义肽与MT1-MMP存在较强的相互作用且能很好地对接于正义肽区域。进一步分析其与MMPs其他家族成员(MMP1-3,MMP7-13,MMP14HPX,MMP16)的亲和力,发现反义肽FVTFPYIR对MT1-MMP具有更强的特异性。分子动力学模拟表明,反义肽FVTFPYIR可能是通过影响受体MT1-MMP的构象稳定性,进而影响其功能活性。体外细胞实验初步确定反义肽FVTFPYIR可选择性地抑制表达MT1-MMP的人成骨肉瘤细胞MG63和乳腺癌MDA-MB-231细胞的增殖。本研究为抗肿瘤反义肽先导药物的研发提供了一种新的思路与途径。     

Effects of resistance training and protein plus amino acid supplementation on muscle anabolism,mass, and strength

Summary. This study examined 10 wks of resistance training and the ingestion of supplemental protein and amino acids on muscle performance and markers of muscle anabolism. Nineteen untrained males were randomly assigned to supplement groups containing either 20 g protein (14 g whey and casein protein, 6 g free amino acids) or 20 g dextrose placebo ingested 1 h before and after exercise for a total of 40 g/d. Participants exercised 4 times/wk using 3 sets of 6–8 repetitions at 85–90% of the one repetition maximum. Data were analyzed with two-way ANOVA (p < 0.05). The protein supplement resulted in greater increases in total body mass, fat-free mass, thigh mass, muscle strength, serum IGF-1, IGF-1 mRNA, MHC I and IIa expression, and myofibrillar protein. Ten-wks of resistance training with 20 g protein and amino acids ingested 1 h before and after exercise is more effective than carbohydrate placebo in up-regulating markers of muscle protein synthesis and anabolism along with subsequent improvements in muscle performance.     

Amphibian organ remodeling during metamorphosis: insight into thyroid hormone-induced apoptosis

During amphibian metamorphosis, the animal body dramatically remodels to adapt from the aquatic to the terrestrial life. Cell death of larval organs/tissues occurs massively in balance with proliferation of adult organs/tissues, to ensure survival of the individuals. Thus, amphibian metamorphosis provides a unique and valuable opportunity to study regulatory mechanisms of cell death. The advantage of this animal model is the absolute dependence of amphibian metamorphosis on thyroid hormone (TH). Since the 1990s, a number of TH response genes have been identified in several organs of Xenopus laevis tadpoles such as the tail and the intestine by subtractive hybridization and more recently by cDNA microarrays. Their expression and functional analyses, which are still ongoing, have shed light on molecular mechanisms of TH‐induced cell death during amphibian metamorphosis. In this review, I survey the recent progress of research in this field, focusing on the X. laevis intestine where apoptotic process is well characterized at the cellular level and can be easily manipulated in vitro. A growing body of evidence indicates that apoptosis during the intestinal remodeling occurs not only via a cell‐autonomous pathway but also via cell–cell and/or cell–extracellular matrix (ECM) interactions. Especially, stromelysin‐3, a matrix metalloproteinase, has been shown to alter cell–ECM interactions by cleaving a laminin receptor and induce apoptosis in the larval intestinal epithelium. Here, I emphasize the importance of TH‐induced multiple apoptotic pathways for massive and well‐organized apoptosis in the amphibian organs and discuss their conservation in the mammalian organs.     

1-(<Emphasis Type="Italic">N</Emphasis>-Trifluoroacetylamino)alkylphosphonic acids: synthesis and properties

Summary. The 1-(N-trifluoroacetylamino)alkylphosphonic acids (TFA-AA^P) – sub-products in the synthesis of O,O-dialkyl 1-(N-trifluoroacetylamino)alkylphosphonates and O,O-diethyl 1-aminoalkylphosphonates, were synthesized in two-stage transformations of 1-aminoalkylphosphonic acids including: trifluoroacetylation of 1-aminoalkylphosphonic acids (AA^P) using a trifluoroacetic anhydride/trifluoroacetic acid reagent (AA^P + TFAA/TFA→2) and subsequent hydrolysis of the intermediary compounds 2 into desired TFA-AA^P (2→TFA-AA^P). These intermediates 2 presented mixtures of the type of mixed anhydrides of TFAA and 1-(N-trifluoroacetylamino)alkylphosphonic, pyrophosphonic and polyphosphonic acids, which underwent rapid and quantitative conversion to corresponding TFA-AA^P during treatment with an excess of water. The title acids were isolated by direct evaporation of the corresponding post-reaction mixtures, and their physicochemical proprieties, including deacylation abilities, were determined. TFA-AA^P compounds can be re-converted into the starting amino acids AA^P under respectively mild conditions (AA^P→TFA-AA^P→AA^P).     

Intraprotoplasmic and wall-localised formation of arabinoxylan-bound diferulates and larger ferulate coupling-products in maize cell-suspension cultures

Maize (Zea mays L.) cell cultures incorporated radioactivity from [¹⁴C]cinnamate into hydroxycinnamoyl-CoA derivatives and then into polysaccharide-bound feruloyl residues. Within 5–20 min, the CoA pool had lost its ¹⁴C by turnover and little or no further incorporation into polysaccharides then occurred. The system was thus effectively a pulse–chase experiment. Kinetics of radiolabelling of diferulates (also known as dehydrodiferulates) varied with culture age. In young (1–3 d) cultures, polysaccharide-bound [¹⁴C]feruloyl- and [¹⁴C]diferuloyl residues were both detectable within 1 min of [¹⁴C]cinnamate feeding. Thus, feruloyl residues were dimerised <1 min after their attachment to polysaccharides. For at least the first 2.3 h after [¹⁴C]cinnamate feeding, polysaccharide-bound [¹⁴C]diferuloyl residues remained almost constant at ≈7% of the total polysaccharide-bound [¹⁴C]ferulate derivatives. Since feruloyl residues are attached to polysaccharides <1 min after the biosynthesis of the latter, and >10 min before secretion, the data show that extensive feruloyl coupling occurred intra-protoplasmically. Exogenous H₂O₂ (1 mM) caused little additional feruloyl coupling; therefore, wall-localised coupling may have been peroxidase-limited. In older (e.g. 4 d) cultures, less intraprotoplasmic coupling occurred: during the first 2.5 h, polysaccharide-bound [¹⁴C]diferuloyl residues were a steady 1.4% of the total polysaccharide-bound [¹⁴C]ferulate derivatives. In contrast to the situation in younger cultures, exogenous H₂O₂ induced a rapid 4- to 6-fold increase in all coupling products, indicating that coupling in the walls was H₂O₂-limited. In both 2- and 4-d-old cultures, polysaccharide-bound ¹⁴C-trimers and larger coupling products exceeded [¹⁴C]diferulates 3- to 4-fold, but followed similar kinetics. Thus, although all known dimers of ferulate can now be individually quantified, it appears to be trimers and larger products that make the major contribution to cross-linking of wall polysaccharides in cultured maize cells. We argue that feruloyl arabinoxylans that are cross-linked before and after secretion are likely to loosen and tighten the cell wall, respectively. The consequences for the control of cell expansion and for the response of cell walls to an oxidative burst are discussed. Received: 19 January 2000 / Accepted: 13 April 2000     

The renal type H+/peptide symporter PEPT2: structure-affinity relationships

Summary. The H⁺/peptide cotransporter PEPT2 is expressed in a variety of organs including kidney, lung, brain, mammary gland, and eye. PEPT2 substrates are di- and tripeptides as well as peptidomimetics, such as β-lactam antibiotics. Due to the presence of PEPT2 at the bronchial epithelium, the aerosolic administration of peptide-like drugs might play a major role in future treatment of various pulmonary and systemic diseases. Moreover, PEPT2 has a significant influence on the in vivo disposition and half-life time of peptide-like drugs within the body, particularly in kidney and brain. PEPT2 is known to have similar but not identical structural requirements for substrate recognition and transport compared to PEPT1, its intestinal counterpart. In this review we compiled available affinity constants of 352 compounds, measured at different mammalian tissues and expression systems and compare the data whenever possible with those of PEPT1.     

Selective determination of mimosine and its dihydroxypyridinyl derivative in plant systems

Summary. Our observations on the growth stimulatory nature of mimosine, (β-(3-hydroxy-4-pyridon-1-yl)-L-alanine), the toxic non-protein plant amino acid, in some model experimental systems, warranted sensitive and selective routine estimations. For the determination of both mimosine and DHP, an indirect spectrophotometric method was developed based on their individual reaction with known excess of DZSAM and by estimating the remaining DZSAM with N-(1-naphthyl)ethylene-diamine (NEDA). The resultant decrease in the secondary coupled product was measured at 540 nm. On equimolar basis, DHP had 40% of the reactivity of mimosine while interference from other relevant compounds was 15–35%. The determination of mimosine and DHP in tissue samples under different physiological conditions was effected after paper chromatographic separation of mimosine and DHP with distinctly differing R_f, from other compounds. The indirect method is superior in terms of absolute selectivity, sensitivity and ease of applicability with linear decreases in absorbance, proportional to increasing concentrations of mimosine from 0.1 to 0.75 μM or DHP from 0.2 to 1.5 μM and with recoveries of 99.2 to 100.5%.     

MT1‐MMP modulates melanoma cell dissemination and metastasis through activation of MMP2 and RAC1

Metastatic melanoma remains the deadliest of all skin cancers with a survival rate at five years of less than 15%. MT1‐MMP is a membrane‐associated matrix metalloproteinase that controls pericellular proteolysis and is an important, invasion‐promoting, pro‐tumorigenic MMP in cancer. We show that deregulation of MT1‐MMP expression happens as early as the transition from nevus to primary melanoma and continues to increase during melanoma progression. Furthermore, MT1‐MMP expression is associated with poor melanoma patient outcome, underscoring a pivotal role of MT1‐MMP in melanoma pathogenesis. We demonstrate that MT1‐MMP is directly required for melanoma cells to metastasize, as cells deprived of MT1‐MMP fail to form distant metastasis in an orthotopic mouse melanoma model. We show that MT1‐MMP affects cell invasion by activating its target MMP2. Importantly, we demonstrate, for the first time, that activation of MMP2 by MT1‐MMP is required to sustain RAC1 activity and promote MT1‐MMP‐dependent cell motility. These data highlight a novel MT1‐MMP/MMP2/RAC1 signaling axis in melanoma that may represent an intriguing molecular target for the treatment of invasive melanoma.     

High-efficiency induction of soybean hairy roots and propagation of the soybean cyst nematode

Cotyledon explants of 10 soybean [Glycine max (L.) Merr.] cultivars were inoculated with Agrobacterium rhizogenes strain K599 with and without binary vectors pBI121 or pBINm-gfp5-ER possessing both neomycin phosphotransferase II (nptII) and β-glucuronidase (gus) or nptII and green fluorescent protein (gfp) genes, respectively. Hairy roots were produced from the wounded surface of 54–95% of the cotyledon explants on MXB selective medium containing 200 μg ml⁻¹ kanamycin and 500 μg ml⁻¹ carbenicillin. Putative individual transformed hairy roots were identified by cucumopine analysis and were screened for transgene incorporation using polymerase chain reaction. All of the roots tested were found to be co-transformed with T-DNA from the Ri-plasmid and the transgene from the binary vectors. Southern blot analysis confirmed the presence of the 35S-gfp5 gene in the plant genomes. Transgene expression was also confirmed by histochemical GUS assay and Western blot analysis for the GFP. Attempts to induce shoot formation from the hairy roots failed. Infection of hairy roots of the soybean cyst nematode (Heterodera glycines Ichinohe)-susceptible cultivar, Williams 82, with eggs of H. glycines race 1, resulted in the development of mature cysts about 4–5 weeks after inoculation. Thus the soybean cyst nematode could complete its entire life cycle in transformed soybean hairy-root cultures expressing GFP. This system should be ideal for testing genes that might impart resistance to soybean cyst nematode. Received: 13 July 1999 / Accepted: 8 August 1999     

Substrate specificity of the amino acid transporter PAT1

Summary. The proton coupled amino acid transporter PAT1 expressed in intestine, brain, and other organs accepts L- and D-proline, glycine, and L-alanine but also pharmaceutically active amino acid derivatives such as 3-amino-1-propanesulfonic acid, L-azetidine-2-carboxylic acid, and cis-4-hydroxy-D-proline as substrates. We systematically analyzed the structural requirements for PAT1 substrates by testing 87 amino acids, proline homologs, indoles, and derivatives. Affinity data and effects on membrane potential were determined using Caco-2 cells. For aliphatic amino acids, a blocked carboxyl group, the distance between amino and carboxyl group, and the position of the hydroxyl group are affinity limiting factors. Methylation of the amino group enhances substrate affinity. Hetero atoms in the proline template are well tolerated. Aromatic α-amino acids display low affinity. PAT1 interacts strongly with heterocyclic aromatic acids containing an indole scaffold. The structural requirements of PAT1 substrates elucidated in this study will be useful for the development of prodrugs.     

Solid-phase S-3CR generates N-substituted α-aminonitriles for the synthesis of α-phenyl-α-(1-piperazinyl) substituted amino acids

Summary. Structurally diverse amino acids were prepared as versatile synthons for combinatorial chemistry. Using an optimized solid-phase synthesis by Strecker-three-component-reaction (S-3CR), two different polymer linker constructs carrying piperazine were investigated. (a) Acrylate derived base-labile linker yielded α-aminonitriles with N-alkylated piperazines via Hofmann elimination after quarternisation with an alkyl halide. The crude product purities were in the range of 54–87%. (b) A urethane type linker yielded α-aminonitriles with the free piperazine nitrogen when cleaved with acid and the product purities were 72–93%. The α-aminonitriles were easily converted to novel N^ɛ – Fmoc-protected α-amino acids with α-(1-piperazinyl) and α-phenyl substituents.     

Convenient synthesis of GOLD and MOLD and identification of their oxidation products in vitro and in vivo

Summary. Two Lys–Lys crosslinks, 1,3-bis-(5-amino-5-carboxypentyl)-1H-imidazolium (GOLD) and 1,3-bis(5-amino-5-carboxypentyl)-4-methyl-1H-imidazolium (MOLD) salts, have been synthesized by the reaction of imidazole or 4(5)-methyl imidazole with 5-(4-bromobutyl)-hydantoin followed by the hydrolysis of 1,3-substituted imidazolium derivatives by 6.0 N HCL at 110 °C. Treatment of GOLD and MOLD with hydrogen peroxide in acetic acid leads to MOLD oxidation only. The oxidation product of MOLD was detected in cataractous lens proteins.     

Immunolocalization of LeAGP-1, a modular arabinogalactan-protein, reveals its developmentally regulated expression in tomato

 Arabinogalactan-proteins (AGPs) are highly glycosylated cell surface proteins that are thought to function in plant growth and development. The developmentally regulated expression of LeAGP-1, a novel and major AGP in tomato, was examined in different organs and tissues of tomato (Lycopersicon esculentum Mill. cv. UC82B) plants with an anti-peptide antibody (i.e. the PAP antibody) directed specifically against the lysine-rich subdomain of the LeAGP-1 core protein. During cell differentiation in tomato plants, LeAGP-1 was associated with cell wall thickening and lignification of particular cell types. Specifically, LeAGP-1 was detected in secondary wall thickenings of maturing metaxylem and secondary xylem tracheary elements in roots and stems, and in thickened cell walls of phloem sieve elements. However, LeAGP-1 was also present in thin-walled, cortical parenchyma cells of seedling roots as well as thick-walled collenchyma cells in young stems, both of which are not lignified. Based on these observed patterns, possible roles for LeAGP-1 in plant growth and development are discussed. Received: 17 August 1999 / Accepted: 7 October 1999     

Enhancing effect of taurine on CYP7A1 mRNA expression in Hep G2 cells

Summary. Taurine has been reported to enhance cholesterol 7α-hydroxylase (CYP7A1) mRNA expression in animal models. However, no in vitro studies of this effect have been reported. The Hep G2 human hepatoma cell line has been recognized as a good model for studying the regulation of human CYP7A1. This work characterizes the effects of taurine on CYP7A1 mRNA levels of Hep G2 cells in a dose- and time-dependent manner. In the dose-dependent experiment, Hep G2 cells were treated with 0, 2, 10 or 20 mM taurine in the presence or absence of cholesterol 0.2 mM for 48 h. In the time-dependent experiment, Hep G2 cells were treated with 0 or 20 mM taurine for 4, 24 and 48 h with and without cholesterol 0.2 mM. Our data revealed that taurine showed time- and dose-response effects on CYP7A1 mRNA levels in Hep G2 cells. However, glycine – a structural analogue of taurine – did not have an effect on CYP7A1 gene expression. These results show that, in agreement to previous studies on animal models, taurine induces the mRNA levels of CYP7A1 in Hep G2 cells, which could enhance cholesterol conversion into bile acids. Also, Hep G2 cell line may be an appropriate model to study the effects of taurine on human cholesterol metabolism.