Matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) play a significant role in regulating angiogenesis, the process of new blood vessel formation. Interstitial collagenase (MMP-1), 72 kDa gelatinase A/type IV collagenase (MMP-2), and 92 kDa gelatinase B/type IV collagenase (MMP-9) dissolve extracellular matrix (ECM) and may initiate and promote angiogenesis. TIMP-1, TIMP-2, TIMP-3, and possibly, TIMP-4 inhibit neovascularization. A new paradigm is emerging that matrilysin (MMP-7), MMP-9, and metalloelastase (MMP-12) may block angiogenesis by converting plasminogen to angiostatin, which is one of the most potent angiogenesis antagonists. MMPs and TIMPs play a complex role in regulating angiogenesis. An understanding of the biochemical and cellular pathways and mechanisms of angiogenesis will provide important information to allow the control of angiogenesis, e.g. the stimulation of angiogenesis for coronary collateral circulation formation; while the inhibition for treating arthritis and cancer.
Gelatinase A (MMP-2) is considered to play a critical role in cell migration and invasion. The proteinase is secreted from the cell as an inactive zymogen. In vivo it is postulated that activation of progelationase A (proMMP-2) takes place on the cell surface mediated by membrane-type matrix metalloproteinases (MT-MMPs). Recent studies have demonstrated that proMMP-2 is recruited to the cell surface by interacting with tissue inhibitor of metalloproteinases-2 (TIMP-2) bound to MT1-MMP by forming a ternary complex. Free MT1-MMP closely located to the ternary complex then activates proMMP-2 on the cell surface. MT1-MMP is found in cultured invasive cancer cells at the invadopodia. The MT-MMP/TIMP-2/MMP-2 system thus provides localized expression of proteolysis of the extracellular matrix required for cell migration.
The matrix metalloproteinases (MMPs) are a family of zinc-dependent endopeptidases that play a key role in both normal and pathological processes involving tissue remodeling events. The expression of these proteolytic enzymes is highly regulated by a balance between extracellular matrix (ECM) deposition and its degradation, and is controlled by growth factors, cytokines, hormones, as well as interactions with the ECM macromolecules. Furthermore, the activity of the MMPs is regulated by their natural endogenous inhibitors, which are members of the tissue inhibitor of metalloproteinases (TIMP) family. In the normal mammary gland, MMPs are expressed during ductal development, lobulo-alveolar development in pregnancy and involution after lactation. Under pathological conditions, such as tumorigenesis, the dysregulated expression of MMPs play a role in tumor initiation, progression and malignant conversion as well as facilitating invasion and metastasis of malignant cells through degradation of the ECM and basement membranes.
The hedgehog-patched (hh-ptc) intercellular signaling pathway has recently been shown to control the proliferation of epithelial stem cells in both Drosophila and vertebrates. Mutant and ectopic expression analyses in Drosophila suggest that the HH protein diffuses from the signaling cells to promote the proliferation of nearby ovarian somatic stem cells by antagonizing the suppression of its receptor PTC towards the CI transcription factor in the stem cells. Consequently, the transcription of CI-dependent genes leads to stem cell proliferation. This regulatory pathway appears to function also in vertebrates, where defects in ptc cause basal cell carcinoma, tumors of epidermal stem cell origin. Basal cell carcinoma can also be induced by ectopic expression of Sonic hedgehog (shh) or Gli1, the vertebrate homolog of ci. These studies suggest the conservation of the hh signaling pathway in controlling epithelial stem cell divisions among different organisms.
Protein kinase RAF is strategically located in the "Ras-MAP-kinase signal transduction pathway", a principle system which transmits signals from growth factor receptors to the nucleus, resulting in cell proliferation. Growth factor responses are mediated in part by activation of Ras, which in turn activates RAF to phosphorylate MEK, its downstream substrate. MEK activates MAP-kinase to influence nuclear events. It is clear, however, that a network of signals other than those carried by Ras plays a role in RAF regulation. These orthogonal influences are mediated by: serine/threonine kinases, tyrosine kinases, and protein-protein interactions. As a further complication to the RAF network, three isoforms of RAF have been established which have divergent N-terminal regulatory domains. Whereas these divergent regulatory domains implicate isoform-specific functions, no clear evidence or hypothesis for distinct functions for individual isoforms has been presented. Recently, "isoform-specific protein interactions" have been identified among numerous proteins interacting with RAF. These studies may serve to delineate independent functions for RAF isoforms.
NO is now known to be an important messenger molecule in biology. It regulates a variety of functions within cells and tissues including vasodilation, neurotransmission and immunological process. This review will focus on the nitric oxide synthase gene family and recent progress on molecular genetic analysis of NOS1, NOS2 and NOS3 genes.
The thyroid hormones L-thyroxine and triiodo-L-thyronine have profound effects on postembryonic development of most vertebrates. Analysis of their action in mammals is vitiated by the exposure of the developing foetus to a number of maternal factors which do not allow one to specifically define the role of thyroid hormone (TH) or that of other hormones and factors that modulate its action. Amphibian metamorphosis is obligatorily dependent on TH which can initiate all the diverse physiological manifestations of this postembryonic developmental process (morphogenesis, cell death, re-structuring, etc.) in free-living embryos and larvae of most anurans. This article will first describe the salient features of metamorphosis and its control by TH and other hormones. Emphasis will be laid on the key role played by TH receptor (TR), in particular the phenomenon of TR gene autoinduction, in initiating the developmental action of TH. Finally, it will be argued that the findings on the control of amphibian metamorphosis enhance our understanding of the regulation of postembryonic development by TH in other vertebrate species.
The posterior gut of the Drosophila embryo, consisting of hindgut and Malpighian tubules, provides a simple, well-defined system where it is possible to use a genetic approach to define components essential for epithelial morphogenesis. We review here the advantages of Drosophila as a model genetic organism, the morphogenesis of the epithelial structures of the posterior gut, and what is known about the genetic requirements to form these structures. In overview, primordia are patterned by expression of hierarchies of transcription factors; this leads to localized expression of cell signaling molecules, and finally, to the least understood step: modulation of cell adhesion and cell shape. We describe approaches to identify additional genes that are required for morphogenesis of these simple epithelia, particularly those that might play a structural role by affecting cell adhesion and cell shape.
Interleukin-4 is a cytokine produced by activated T cells, mast cells, and basophils that elicits many important biological responses (see Tab 1). These responses range from the regulation of helper T cell differentiation and the production of IgE to the regulation of the adhesive properties of endothelial cells via VCAM-1. In keeping with these diverse biological effects, high-affinity binding sites for IL-4 (Kd 20 to 300 pM) have been detected on many hematopoietic and non-hematopoietic cell types at levels ranging from 50 to 5000 sites per cell. This review will focus on the discrete signal transduction pathways activated by the IL-4 receptor and the coordination of these individual pathways in the regulation of a final biological outcome.
Trichosanthin (TCS) is a potent allergen to mice. According to our previous experiments, it could bring out the IgE response to ovabumin (OVA) if TCS was given one day before OVA immunization, while OVA alone could not induce IgE to it. In this work, the kinetics of interleukin 4(IL-4) and interferon gamma(IFN-gamma) gene expression in the mesenteric lymph node (MLN) of TCS-immunized mice was investigated using a semi-quantitative RT-PCR method. It indicated that TCS induced significant IL-4 gene expression and the peaks of IL4 gene expression were on day one after TCS immunization in both primary and secondary response. In contrast, the IFN-gamma gene expression was suppressed. Furthermore, the IL-4 gene expression in the secondary response was lower than that in the primary response. Thus the presence of IgE memory B cells were studied. Results showed that the amount of mature IgE mRNA arose significantly and rapidly one day after TCS restimulation, while in the MLN of the mice primed 30 days before and without boost, it was almost as the same amount of the unimmunized control. These findings suggest the existence of the IgE memory B cells in the mice after the primary TCS immunization.
In present paper, one of the T-DNA insertional embryonic lethal mutant of Arabidopsis is identified and designated as acd mutant. The embryo development of this mutant is arrested in globular stage. The cell division pattern is abnormal during early embryogenesis and results in disturbed cellular differentiation. Most of mutant embryos are finally degenerated and aborted in globular stage. However, a few of them still can germinate in agar plate and produce seedlings with shorter hypocotyl and distorted shoot meristem. To understand the molecular basis of the phenotype of this mutant, the joint fragment of T-DNA/plant DNA is isolated by plasmid rescue and Dig-labeled as probe for cDNA library screening. According to the sequence analysis and similarity searching, a 936 bp cDNA sequence (EMBL accession #: Y12555) from selected positive clone shows a 99.8% (923/925bp) sequence homology with Alanyl-tRNA Synthetase (AlaRS) gene of Arabidopsis thaliana. Furthermore, the data of in situ hybridization experiment indicate that the expression of AlaRS gene is weak in early embryogenesis and declines along with globular embryo 'development' in this mutant. Accordingly, the reduced expression of AlaRS gene may be closely related to the morphological changes in early embryogenesis of this lethal mutant.
A simple method to create a chromosome-specific DNA library of rice, including microdissection, amplification, characterization and cloning, is described. Rice chromosome 4 from a metaphase cell has been isolated and amplified by the Linker Adapter PCR (LA-PCR). The PCR products were labeled as probes with DIG-11-dUTP using the random priming method. Southern blot analysis with rice genomic DNA and specific RFLP markers demonstrated that the PCR products were derived from rice chromosome 4. A large library comprising over 100,000 recombinant plasmid microclones from rice chromosome 4 was constructed. Colony hybridization showed that 58% of the clones contained single or low-copy sequences and 42% contained repetitive sequences. The size of inserts generated by PCR ranged from 140bp to 500bp. This method will facilitate cloning of the specific chromosome DNA markers and important genes of rice.
The effects of antisense oligonucleotide to insulin-like growth factor II (IGFII) to induce apoptosis in human ovarian cancer cells were evaluated. antiproliferation effects of antisense to IGFII in ovarian cancer AO cells were determined by 3H-thymidine incorporation. Apoptosis of the IGFII antisense-treated cells was quantitated by both nuclear condensation and flow cytometry after cells were stained with propidium iodide. IGFII antisense (4.5 microM) treatment of 48 h maximally inhibited proliferation of AO cells. More than 25% of IGFII antisense-treated cells (4.5 microM for 24 h) had undergone apoptosis, whereas less than 3% of the cells were apoptotic in either IGFII sense-treated cells or untreated cells. Antisense oligonucleotide to IGFII significantly inhibited cell proliferation and induced apoptosis in human ovarian cancer AO cell. These data suggest that IGFII may be a potential target in treatment of ovarian cancer and antisense oligonucleotide to IGFII may serve as a therapeutic approach.
The nuclear matrix attachment regions(MARs) and the binding nuclear matrix proteins in the 5'-flanking cis-acting elements of the human epsilon-globin gene have been examined. Using in vitro DNA-matrix binding assay, it has been shown that the positive stage-specific regulatory element (epsilon-PREII, -446 bp(-)-419 bp) upstream of this gene could specifically associate with the nuclear matrix from K562 cells, indicating that epsilon-PREII may be an erythroid-specific facultative MAR. In gel mobility shift assay and Southwestern blotting assay, an erythroid-specific nuclear matrix protein (epsilon-NMP kappa) in K562 cells has been revealed to bind to this positive regulatory element (epsilon-PREII). Furthermore, we demonstrated that the silencer (-392 bp(-)-177 bp) upstream of the human epsilon-globin gene could associate with the nuclear matrices from K562, HEL and Raji cells. In addition, the nuclear matrix proteins prepared from these three cell lines could also bind to this silencer, suggesting that this silencer element might be a constitutive nuclear matrix attachment region (constitutive MAR). Our results demonstrated that the nuclear matrix and nuclear matrix proteins might play an important role in the regulation of the human epsilon-globin gene expression.
It was the first time demonstrated by us that the number of newborn neurons was increased after making lesion in forebrain of adult ring dove (Streptopelia risoria) by means of autoradiography and immunohistochemistry. Neurogenesis in the adult avian is restricted to the telencephalon. In doves with bilateral electrolytic lesion of nucleus ectostriatum (E), the mean number of proliferating cells in the lateral ventricular zone (LVZ) and newborn neurons in the forebrain increased by 1.95 times and 2.38 times respectively as compared with that in intact doves. The most remarkable increase of neurogenesis induced by nucleus ectostriatum lesions was found at the anterior-posterior level 3 (L3), where the lesion site was located. These results showed that the electrolytic brain lesion altered the distribution pattern of proliferating cells in the LVZ and resulted in increase of the number of newborn neurons in the non-VZ areas of forebrain. The changes in number and distribution pattern of proliferating cells in LVZ and newborn neurons in forebrain may be dependent on site of lesion. Studies on the relationship between proliferating cells in LVZ and newly generated neurons in non-VZ areas may help to understand the mechanism of brain plasticity and development.
The effects of human EGFR to the malignant phenotype of human breast cancer cell line MDA-MB-231 were investigated experimentally. A retroviral vector containing a 5'1350bp fragment of the human EGFR cDNA in the antisense orientation was transfected into targeted cells by lipofectamine. The effects on cell proliferation, cell cycle and adherent ability to extracellular matrix (ECM) components were studied after the expression of antisense transcripts to EGFR 5'1350bp fragment in target cells. In vitro studies showed that the growth ability of the transfected cells was partially inhibited in comparison to parental cells and to cells transfected with the plasmid containing the neomycin resistance gene only. It was found that EGF (10 ng/ml) had an argumenation effect on the growth of transfected MDA-AS10 cells but not MDA-MB-231 cells. Flow cytometric analysis showed that the cell cycle of the transfected cells was abnormal with a decrease of cells in G2/M and S phases and an increase of cells in G1 phase, indicating a blockage in phase G1. Immunofluorescence of EGFR expression in transfectants stained with an anti-EGFR antibody was decreased and their growth in soft agarose was also severely impaired. The transfected cells showed less adherence to laminin (LN) and fibronectin (FN). In short, EGFR antisense RNA decreases the expression of EGFR on MDA-MB-231 cells and partially reverses their malignant phenotype as well.
The nonchromatin proteinous residue of the cell nucleus was revealed in our laboratory as early as in 1948 and then identified by light and electron microscopy as residual nucleoli, intranuclear network and nuclear envelope before 1960. This structure termed afterwards as "nuclear residue", "nuclear skeleton", "nuclear cage", "nuclear carcass" etc., was much later (in 1974) isolated, studied and entitled as "nuclear matrix" by Berezney and Coffey, to whom the discovery of this residual structure is often wrongly ascribed. The real history of nuclear matrix manifestation is reported in this paper.
The norepinephrine transporter(NET) is a member of the Na+/Cl- dependent neurotransmitter transporter family and constitutes the target of several clinically important antidepressants. To delineate the critical amino acid residues and the function of C-terminal in regulating transport activity of NET, here we constructed two site mutants (V70F, F72V; V70I, F72V) and one C-terminal truncated mutant (delta 611-617). The wild type and mutants of NET were expressed in Xenopus oocytes by injection of their cRNA. We found that all of these mutants lost their transport activity. These results indicate that the amino acid residues of V70 and F72, and the last seven amino acids of C-terminal are essential to the transport activity of NET.
In order to analyze the mechanism of immuno-modulation by LPS on murine peritoneal suppressor macrophages, we have, using RNase protection assay, checked the changes of mRNA expression pattern of several cytokine genes during the immuno-modulation. It has been found that, after treating peritoneal suppressor macrophages with LPS, mRNAs of IL-12 p35, IL-12 p40, IL-6 and IFN-gamma are newly appeared, while those of IL-1 alpha, IL-1 beta and IL-1Ra are increased and those of other cytokines, like TGF-beta 1 and MIF are not changed at all. It seems certain that those cytokines, whose expression is increased by LPS stimulation, may be responsible for the functional changes of suppressor macrophages during immuno-modulation. Among these changes, the appearance of IL-12 mRNA may play a critical role, and, in this regard, the synergetic effect between IFN-gamma and LPS on the increase of IL-12 p35 and Il-12 p40 mRNA expression is an interesting finding.