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Results for “Chain Migration”

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PubMed1999

Ultraviolet B radiation upregulates the production of macrophage migration inhibitory factor (MIF) in human epidermal keratinocytes.

Human epidermal cells are capable of secreting various cytokines with immunologic, inflammatory, and proliferative properties. In a previous study, by reverse transcription-polymerase chain reaction and immunohistochemical analysis, we have shown that human epidermal keratinocytes express macrophage migration inhibitory factor and identified its presence in the cytoplasm. In this study, we detected an increased serum macrophage migration inhibitory factor level by enzyme-linked immunosorbent assay after a single total-body ultraviolet B exposure in vivo, indicating that human keratinocytes respond and release this cytokine in response to ultraviolet B irradiation. Moreover, we evaluated the effect of ultraviolet B on migration inhibitory factor production in cultured human epidermal keratinocytes and epidermal sheets. The results of enzyme-linked immunosorbent assay and northern blot analyses showed that migration inhibitory factor production of cultured keratinocytes was increased by ultraviolet B exposure. During the past few years, migration inhibitory factor was found to have a variety of biologic functions, such as being essential for T cell activation and induction of inflammatory cytokines. In this context, these results should encourage further investigation on the pathophysiologic role of migration inhibitory factor in cutaneous inflammatory reactions and immune responses.

Cell Survival↗

PubMed1998

The effects of exogenous free fatty acids on lipoprotein migration in serum high-resolution electrophoresis: addition of free fatty acids improves visualization of normal and abnormal alpha1-antitrypsin.

Abnormalities in the alpha1 region are occasionally difficult to interpret on high-resolution electrophoresis (HRE) gels because alpha1-antitrypsin (A1AT) can be obscured by elevated levels of lipoprotein. The addition of saturated free fatty acids (SFFAs) to serum samples causes a selective anodal migration of lipoproteins when examined by HRE. This "clearing" of the alpha1 region improves visualization of A1AT. In this study, we evaluated 6- to 24-carbon SFFAs for their ability to cause anodal migration of serum lipoproteins; identified the optimal SFFA and determined its effects on other proteins; and applied the SFFA to problematic serum samples, including those containing dense alpha lipoprotein regions. When added to serum samples, a mixture of medium-chain (12-18 carbon) SFFAs caused a dose-dependent anodal migration of the alpha and beta lipoproteins and albumin. Lauric acid (C(12:0); 3.2-6.5 mmol/L) caused an optimal effect--maximal anodal migration of alpha lipoproteins and clearing of the alpha1 region, facilitating inspection of that area for A1AT variants. At the optimal C(12:0) concentration, the migration and resolution of A1AT were minimally affected, anodal slurring of beta lipoproteins and albumin were slight, and there was no effect on interpretation of monoclonal proteins. At higher concentrations, C(12:0) increased anodal migration of beta lipoproteins and decreased the resolution of A1AT. C(12:0) at 3.2 mmol/L in serum samples of heparinized patients showed an exaggerated effect similar to higher concentrations of C(12:0) in nonheparinized serum samples. Hyperbilirubinemia did not alter the effect of C(12:0) on serum proteins. C(12:0) treatment of serum samples displaying dense alpha lipoprotein bands on HRE dramatically improved visualization of A1AT. We report the incidental identification of A1AT variants in two such samples treated with C(12:0). The addition of C(12:0) to serum samples markedly improves visualization of normal and abnormal A1AT in the alpha1 region in HRE.

Dose-Response Relationship, Drug↗

PubMed1991

Migration patterns of sympathetic preganglionic neurons in embryonic rat spinal cord.

The displacement of immature neurons from their place of origin in the germinal epithelium toward their adult positions in the nervous system appears to involve migratory pathways or guides. While the importance of radial glial fibers in this process has long been recognized, data from recent investigations have suggested that other mechanisms might also play a role in directing the movement of young neurons. We have labeled autonomic preganglionic cells by microinjections of horseradish peroxidase (HRP) into the sympathetic chain ganglia of embryonic rats in order to study the migration and differentiation of these spinal cord neurons. Our results, in conjunction with previous observations, suggest that the migration pattern of preganglionic neurons can be divided into three distinct phases. In the first phase, the autonomic motor neurons arise in the ventral ventricular zone and migrate radially into the ventral horn of the developing spinal cord, where, together with somatic motor neurons, they form a single, primitive motor column (Phelps P. E., Barber R. P., and Vaughn J. E. (1991). J. Comp. Neurol. 307:77-86). During the second phase, the autonomic motor neurons separate from the somatic motor neurons and are displaced dorsally toward the intermediate spinal cord. When the preganglionic neurons reach the intermediolateral (IML) region, they become progressively more multipolar, and many of them undergo a change in alignment, from a dorsoventral to a mediolateral orientation. In the third phase of autonomic motor neuron development, some of these cells are displaced medially, and occupy sites between the IML and central canal. The primary and tertiary movements of the preganglionic neurons are in alignment with radial glial processes in the embryonic spinal cord, an arrangement that is consistent with a hypothesis that glial elements might guide autonomic motor neurons during these periods of development. In contrast, during the second phase, the dorsal translocation of preganglionic neurons occurs in an orientation perpendicular to radial glial fibers, indicating that glial elements are not involved in the secondary migration of these cells. The results of previous investigations have provided evidence that, in addition to glial processes, axonal pathways might provide a substrate for neuronal migration. Logically, therefore, it is possible that the secondary dorsolateral translocation of autonomic preganglionic neurons could be directed along early forming circumferential axons of spinal association interneurons, and this hypothesis is supported by the fact that such fibers are appropriately arrayed in both developmental time and space to guide this movement.

Animals↗

PubMed1986

Extracellular matrix components and testicular peritubular cells influence the rate and pattern of Sertoli cell migration in vitro.

We report the patterns of migration of Sertoli cells plated on specific substrata, and the influences of testicular peritubular cells on these processes. Data presented indicate that while peritubular cells readily spread when explanted onto Type I collagen, Sertoli cells do not. A delay of 4 to 6 days occurs after Sertoli cells are plated before they begin to migrate randomly to form plaque-like monolayers on Type I collagen. These processes are dependent upon the synthesis and subsequent deposition of laminin and/or Type IV collagen by Sertoli cells, and are independent of fibronectin. A different behavior occurs when reconstituted mixtures of purified Sertoli cells and pertiubular cells are sparsely plated onto Type I collagen. Peritubular cells rapidly spread to form chains of cells between Sertoli cell aggregates. Sertoli cells then migrate on the surfaces of the peritubular cells, culminating in the formation of cable-like structures between aggregates. Evidence is presented that the Sertoli cell migration to form "cables" under these conditions is dependent upon fibronectin synthesized by peritubular cells, and is independent of the presence of laminin or Type IV collagen. We discuss the possible relevance of these data to the role which precursors of peritubular cells may play in determining the behavior of Sertoli cell precursors in vivo during tubulogenesis, or in the remodelling of the seminiferous tubule which occurs during different stages of the cycle of the seminiferous epithelium in spermatogenesis.

Animals↗

PubMed1977

Purification of immunuglobulin light chain messenger RNA by immunoprecipitation from mouse myeloma tumor, MOPC-31C.

Polysomes producing IgGl(kappa) myeloma protein were specifically selected by an immunoprecipitation method, and immunoglobulin light chain mRNA was purified from the precipitated polysomes. The purified mRNA migrated predominantly as a single band and the molecular weight of this mRNA was calculated to be 410.000 by polyacrylamide gel electrophoresis in 98% formamide. A protein possessing a molecular weight of 25,000, which is the size of the light chain precursor, was synthesized as a major product of translation in a wheat germ cell-free system. DNA complementary to the mRNA (cDNA) was prepared with avian myeloblastosis virus RNA-dependent DNA polymerase. This cDNA had an average size of 8.3S as determined by sedimentation through an alkaline sucrose gradient. Using this cDNA, Crt 1/2 values of template RNA and RNA from various preparations were calculated from the results of molecular hybridization. The relative content of the mRNA increased 4,4-fold during the immunoprecipitation of polysomes.

Animals↗

PubMed2005

[The effect of short-chain fatty acids on expression of endothelial adhesion molecules stimulated by oxidatively modified LDL].

INTRODUCTION: Adhesion and migration of mononuclear leukocytes into the vascular wall is a crucial step in atherogenesis. Expression of cell adhesion molecules by endothelial cells plays a leading role in this process. Natural short-chain fatty acids produced by bacterial fermentation of dietary fiber have been attributed with antiatherogenic properties. MATERIAL AND METHODS: This in vitro study has been conceived in an attempt to shed light on the mechanisms responsible for the modulatory effect of short-chain fatty acids in atherogenesis. The following conclusions have been drawn. Butyrate and propionate are selective inhibitors of ox-LDL-stimulated VCAM-1 and ICAM-1 expression with a mechanism of action involving reduced protein and specific mRNA production through inhibition of NF-kappaB activation. Suppressed expression of VCAM-1 and ICAM-1 was associated with reduced adherence of monocytes and lymphocytes to ox-LDL stimulated endothelial cells. RESULTS: Butyrate and propionate may have anti-inflammatory and antiatherogenic properties partly attributable to an inhibitory effect on NF-kappaB activation.

Atherosclerosis↗

PubMed1995

Isolation of unstable myosins and the analysis of light chains by capillary electrophoresis.

A rapid method for the isolation of unstable fish myosins by Sepharose Q ion-exchange chromatography is described which yields a pure protein essentially free of contamination or breakdown products in less than 6 h. A protocol was developed for determining the molar ratios of myosin light chains (LC) by capillary electrophoresis. The method is quantitative, rapid (<20 min), highly reproducible (<2.1% variation in relative peak migration time), and only uses Femtomole quantities of protein. It was able to separate myosin light chains which could previously only be resolved by 2-D sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Capillary electrophoresis in the presence of SDS gave similar apparent relative molecular masses (M(r)) for most proteins, but an anomalously high M(r) for myosin light chain 3(LC3), as has been reported previously for SDS-PAGE methods.

Animals↗

PubMed1997

Uterine natural killer cells do not require interleukin-2 for their differentiation or maturation.

PROBLEM: Natural Killer lymphocytes (NK cells) from the pregnant uterus and from other tissues in pregnant and nonpregnant mammals can be stimulated by interleukin-2 (IL-2) during culture to become Lymphokine Activated Killer (LAK) cells. The susceptibility of cultured trophoblast cells to lysis by LAK cells raises the enigma of why uterine (u) NK cells that are characterized by morphology and by surface phenotyping as "activated," and thus potentially damaging to the placenta, become localized to implantation sites during normal rodent gestation. METHOD: uNK cells migrating from explant cultures of the metrial gland were assessed for expression (mRNA and protein) of each chain of the IL-2 receptor (IL-2R). Implantation sites from transgenic mice lacking a functional IL-2 gene were examined histologically for the differentiation of mature, granulated uNK cells. RESULTS AND CONCLUSION: Early post-implantation, mRNA from migrating uNK cells contains transcripts for all three chains of the IL-2R. Only IL-2Rgamma was expressed at day 12 of gestation; expression of this gene was also lost by day 16. Loss of IL-2R transcription did not result in loss of protein expression; however, it did coincide with loss of uNK cell viability in vivo. Apparently normal differentiation of uNK cells occurred in IL-2(-/-) mice and in doubly mutant IL-2(-/-).beta2m(-/-) mice. Thus, despite uNK cell expression of the full IL-2R at day 8 of gestation, IL-2 is not required for the maturation of uNK cells to their fully granulated form or for normal placental development.

Animals↗

PubMed1989

Peyer's patch-specific lymphocyte homing receptors consist of a VLA-4-like alpha chain associated with either of two integrin beta chains, one of which is novel.

Lymphocytes home to various lymphoid organs by adhering to and migrating through specialized high endothelial venules (HEV). The murine cell surface heterodimer LPAM-1 is involved in the homing of lymphocytes to mucosal sites (Peyer's patches). LPAM-1 has an alpha subunit (alpha 4m) analogous to the alpha chain of the human integrin molecule VLA-4. Here we show that the LPAM-1 beta subunit (beta p) is immunochemically and biochemically distinct from previously defined integrin beta subunits, suggesting that beta p represents a novel integrin beta subunit. Depending on the cellular source two alternative beta subunits, beta p and integrin beta 1, can be isolated in association with alpha 4m. Therefore, alpha 4m is the common subunit of the unique integrin LPAM-1 (alpha 4m beta p) and of the heterodimer LPAM-2 (alpha 4m beta 1), which is analogous to VLA-4. Antibody-blocking experiments suggest that, in addition to LPAM-1, LPAM-2 is also involved in the organ-specific adhesion of lymphocytes to Peyer's patch HEV.

Animals↗

PubMed2005

Paclitaxel impairs endothelial cell adhesion but not cytokine-induced cellular adhesion molecule expression.

Local delivery of antiproliferative agents using drug-eluting stents has become a productive area of research for preventing in-stent restenosis. Recently, the microtubule stabilizing drug paclitaxel has been used to coat stents. While the actions of paclitaxel on smooth muscle are well documented, effects on endothelial cells (ECs) are largely unknown. Nevertheless, restoration of EC function is a critical step in repairing the vascular lesion. We assessed the effects of paclitaxel by examining three events that are critical in controlling the severity of vascular injury: (1) adhesion of ECs to matrix proteins, (2) EC migration, and (3) cytokine-stimulated cellular adhesion molecule (CAM) expression on the surface of ECs. Paclitaxel inhibited both EC adhesion and migration of ECs; however, it had no effect on tumor necrosis-stimulated CAM expression on ECs. The mechanisms of paclitaxel action on matrix adhesion and migration are not clear, but protein kinase C and myosin light chain kinase do not appear to play a role as they are unaffected by treatment of the cells with paclitaxel. On the other hand, the MAP kinase ERK1/2 is modestly inhibited by paclitaxel. While paclitaxel-coated endovascular stents may prevent smooth muscle proliferation, their attenuation of EC migration and adhesion to the lesion coupled with an inability to reduce cytokine-induced CAM expression on ECs may limit their effectiveness.

Antineoplastic Agents↗

PubMed2003

ZO-1 redistribution and F-actin stress fiber formation in pulmonary endothelial cells after thermal injury.

BACKGROUND: In response to isolated inflammatory stimuli, changes in endothelial cell morphology that enhance paracellular flow of solutes result from F-actin stress fiber formation, myosin phosphorylation, and actin anchoring protein (ZO-1) modifications. We hypothesized that myosin light chain kinase inhibition would diminish burn-enhanced endothelial monolayer permeability by secondarily preventing F-actin and actin anchoring protein rearrangements. METHODS: Human pulmonary microvascular endothelial cells were treated for 4 hours with 20% human burn serum (isolated from patients with > 45% total body surface area thermal injury or healthy volunteers). Select cultures were pretreated with myosin light chain kinase inhibitors (ML-9). Permeability was assessed by migration of bovine serum albumin across cell monolayers. Cells were stained with rhodamine-phalloidin and anti-ZO-1 antisera and examined by means of confocal microscopy. RESULTS: Burn serum significantly enhanced monolayer permeability to albumin, whereas pretreatment with ML-9 limited this effect. Control cells maintained cortical F-actin and peripheral ZO-1 distributions (1a, b), whereas burn serum induced transcellular F-actin stress fiber formation and a diffuse ZO-1 staining (2a, b). ML-9 prevented burn-induced actin rearrangements, but not the diffuse redistribution of ZO-1. CONCLUSION: These data demonstrate that endothelial F-actin stress fiber formation and ZO-1 redistribution contribute to postburn loss of pulmonary endothelial monolayer integrity. Although myosin phosphorylation appears to be required for endothelial F-actin stress fiber formation, redistribution of actin-membrane anchoring proteins appears to be regulated independently after thermal injury.

Actins↗

PubMed2006

Evidence of association of macrophage migration inhibitory factor gene polymorphisms with systemic lupus erythematosus.

The aim of this study was to evaluate the potential association of functional polymorphisms of macrophage migration inhibitory factor with systemic lupus erythematosus. Our study includes 711 systemic lupus erythematosus (SLE) patients and 755 healthy controls. We genotyped the migration inhibitory factor (MIF) -173G/C using a polymerase chain reaction (PCR) system with predeveloped TaqMan allelic discrimination assay and the MIF -794 CATT(n) microsatellite polymorphism using a PCR-fluorescent method. A statistically significant difference in the distribution of the MIF -173(*)C allele between SLE patients and controls (P=0.004, OR=1.34, 95% CI=1.05-1.27) was observed. In addition, the frequency of the MIF -173(*)C/C genotype was higher in SLE patient (P=0.002, OR=2.58, 95% CI=1.32-5.10). No differences in the distribution of CATT(n) were found. However, the haplotypes analyses showed that only the CATT(7)-MIF -173(*)C haplotype was associated with a higher susceptibility to SLE (P=0.001, OR 1.84, 95% CI 1.35-2.79). No association with clinical features was detected in any case. These results suggest that both, MIF -173(*)C allele and CATT(7)-MIF -173(*)C haplotype, confer susceptibility to SLE in our population.

Adult↗

PubMed1980

A fully active, two-active-site, single-chain sucrase.isomaltase from pig small intestine. Implications for the biosynthesis of a mammalian integral stalked membrane protein.

Detergent-solubilized pig intestinal sucrase . isomaltase (EC 3.2.1.48-EC 3.2.1.10) was purified 40 to 100 times with a yield of 10 to 20% by a rapid immunoadsorbent technique. The purified enzyme was shown to be homogeneous by immunoelectrophoresis and was essentially free of other known brush border peptidases and disaccharidases. Intestinal sucrase . isomaltase isolated from pigs with intact pancreatic ducts consisted of two polypeptide chains with apparent molecular weights of 140,000 and 150,000, respectively. In contrast, the enzyme isolated from pigs in which the pancreas was completely disconnected from the duodenum 3 days before killing migrated in polyacrylamide gel electrophoresis in dodecyl sulfate as a single polypeptide chain with an apparent molecular weight of 260,000. Treatment with pancreatic proteases in vitro converted the large polypeptide chain into bands with molecular weights equal to or somewhat larger than those of sucrase . isomaltase purified from normal pigs. No increase of enzymatic activity could be detected during this transformation. It is suggested that the single-chain sucrase . isomaltase represents a precursor, which is converted to the final sucrase . isomaltase in vivo by pancreatic proteolytic enzymes. This is one of the few examples in vertebrates of a single polypeptide chain carrying two enzymatically active sites. The significance of the result for the mechanism of the biosynthesis of sucrase . isomaltase is discussed.

Animals↗

PubMed1994

Herpes simplex virus type 1 (HSV-1) UL56 gene is involved in viral intraperitoneal pathogenicity to immunocompetent mice.

A comparison of the pathogenicity in mice of the recombinant herpes simplex virus type 1 (HSV-1) strain HSV-1-M-LacZ, in which the UL56 gene has been deleted, was made with its parental strain F, following infection in different mouse strains. The polymerase chain reaction (PCR) technique was used to study the migration of virus DNA in the mouse model. Tissues from adult mice infected intraperitoneally (IP) with one of three HSV-1 strains (F, HFEM or HSV-1-LacZ) were examined for the presence of viral DNA. DNA of the pathogenic strain F was detected in the adrenal glands, spinal cord, brain, liver and pancreas. DNA of HSV-1-M-LacZ was detected in the same tissues. However, DNA of the apathogenic strain HFEM was detected transiently (on days 2 and 3 p.i., but not days 1, 5 or 7), only in the adrenal glands and no viral DNA was detected in any of the other tissues. HSV-1 pathogenic strains injected intraperitoneally into newborn mice (7 days old) killed most of the mice. In the surviving mice viral DNA of the three virus strains was found in peritoneal exudate cells (PEC), adrenal glands, spinal cord, liver and spleen. It was found that HSV-1-M-LacZ, which lacks the UL56 gene, resembled in pathogenicity to the newborn mice the pathogenic HSV-1 strains F and KOS. The PCR technique was used to trace viral DNA in tissues of the mice which survived HSV-1 infection at 7 weeks of age. Only HSV-1 (KOS) DNA was detected in the pancreas. The brains of these mice did not contain viral DNA. It is suggested that HSV-1 DNA may reside in surviving HSV-1- infected newborn mice in a "latent" state in nonneural tissues.

Adrenal Glands↗

PubMed1995

An alpha tubulin mutation suppresses nuclear migration mutations in Aspergillus nidulans.

Microtubules and cytoplasmic dynein, a microtubule-dependent motor, are required for nuclei to move along the hyphae of filamentous fungi. Nuclear migration in Aspergillus nidulans is blocked by heat-sensitive (hs-) mutations in the nudA gene, which encodes dynein heavy chain, and the nudF gene, which encodes a G protein beta-subunit-like protein. Hs- mutations in the nudC and nudG genes also prevent nuclear migration. We have isolated extragenic suppressor mutations that reverse the hs- phenotypes caused by these mutations. Here we show that one nudF suppressor also suppresses hs- mutations in nudA, nudC, and nudG and deletions in nudA and nudF. This suppressor mutation is in the tubA alpha tubulin gene, and its characteristics suggest that it destabilizes microtubules. The mutation alters microtubule staining and confers sensitivity to cold and benomyl, two treatments that destabilize microtubules. Treatment with low concentrations of benomyl also suppresses the hs- nudA, nudC, nudF, and nudG mutations and the nudA and nudF deletions. Suppression of the hs- nudA mutation and the nudA deletion is especially interesting because these strains lack active dynein heavy chain. Together, these results suggest that microtubule destabilization allows nuclei to migrate even in the absence of cytoplasmic dynein motor function.

Aspergillus nidulans↗

PubMed1986

Partial characterization of heparan and dermatan sulfate proteoglycans synthesized by normal rat glomeruli.

Rat glomerular heparan sulfate (HS) and dermatan sulfate (DS) proteoglycan synthesis was studied in vitro and in vivo. Incorporation of [35S]sulfate into macromolecules was linear over 16 h in vitro, and DS was the predominant glycosaminoglycan (GAG), while HS dominated in vivo incubations. Proteoglycans were found in the bottom 2/5 (high density) CsCl gradient fractions and eluted as two overlapping peaks from DEAE-Sephacel columns. The proportion of low density 35S-glycoproteins and 35S-proteoglycans increased with time. Two high buoyant density HS proteoglycans were extracted from glomeruli and eluted in DEAE peak I. The first, HS-tIA, had an Mr of 130 X 10(3) with Mr 12.5 X 10(3) GAG chains. This proteoglycan was released from the tissue by trypsin and was partially displaced by heparin treatment. In addition, it was rapidly released into the medium of label-chase experiments after which it migrated slightly more rapidly than HS-tIA in gels, with HS chains similar in length to its tissue counterpart. The second, HS-tIB, had an Mr of 8.6 X 10(3) with little or no attached protein. This proteoglycan was characterized as intracellular as it resisted release by trypsin treatment or heparin extraction in medium and was not detected in the medium of label-chase experiments. Two tissue DS proteoglycans were characterized. The first, DS-tIA, co-purified with HS-tIA and was the predominant proteoglycan synthesized during 4-h in vitro incubations. Like HS-tIA, it was rapidly released into medium and displaced from cell surfaces or tissue "receptors" by heparin or trypsin treatments. A second, Sepharose CL-6B-excluded DS proteoglycan from DEAE peak II, DS-tII, accumulated in tissue over 16 h in vitro. This proteoglycan was self-associating and contained clusters of iduronic acid residues along its Mr 26 X 10(3) DS chains. It resisted extraction from the tissue with heparin, trypsin, and detergent. No DS-tII was detected in the incubation medium. Instead, medium proteoglycans eluted as single Sepharose CL-6B-included peaks. DS chains from medium proteoglycans were shorter (Mr 18 X 10(3)) and had more regularly spaced iduronic acid residues than GAGs from DS-tII. The length and sulfation patterns of DS-mII GAG were similar to GAG from DS-tIA. Thus, glomeruli rapidly synthesized and released Sepharose CL-6B-included heparin-displaceable DS and HS proteoglycans while retaining a Sepharose CL-6B-excluded self-associating DS proteoglycan and an intracellular HS.

Animals↗

PubMed2005

Novel enhanced lung-colonizing variant of murine MBT-2 bladder cancer cells.

OBJECTIVES: To develop an enhanced lung-colonizing variant of murine bladder cancer that will allow the mechanism of metastasis to be studied more readily. METHODS: We implanted murine bladder tumor cells (MBT-2) into the leg muscles of C3H mice. We developed variant cells from a lung metastasis nodule. We compared the MBT-2 cells and variant cells (MBT-2V) in vivo by evaluating lung nodule formation, survival, in vitro adhesion, and migration-invasion assays. Zymography and semiquantitative reverse transcriptase-polymerase chain reaction analyses were also performed to characterize the metastatic ability of both cells. RESULTS: MBT-2 and MBT-2V cells were tumorigenic when injected intramuscularly into C3H mice, but MBT-2 cells had little potential to metastasize compared with MBT-2V cells. Metastases were observed in the lungs of mice injected in the tail vein with MBT-2 and MBT-2V cells. Mice receiving MBT-2V cells had significantly shorter survival (P <0.01) and more lung nodules (245 versus 106, P <0.0001) than those receiving MBT-2 cells. In vitro study revealed that MBT-2V cells exhibited more adhesion, greater migration, and more invasiveness than did MBT-2 cells. Pathologic examination revealed the tumors from MBT-2V cells to be more aggressive than those from MBT-2 cells. MBT-2V also showed significantly greater matrix metalloproteinase-9 expression. CONCLUSIONS: We generated an enhanced lung-colonizing variant of MBT-2 cells. Our MBT-2V cells showed more aggressive and invasive metastatic ability than that of the MBT-2 cells. Zymography and reverse transcriptase-polymerase chain reaction analyses indicated that matrix metalloproteinase-9 might be associated with the metastatic ability of MBT-2V cells.

Animals↗

PubMed2000

DDRT-PCR: use of agarose gels for detection of amplified products.

The present study focuses on the detection of differentially expressed genes in migrating (healing) and nonmigrating (normal) corneal epithelium on agarose gel using a modified procedure of differential display reverse transcriptase-polymerase chain reaction (DDRT-PCR). Rabbit corneal epithelial organ cultures were used to obtain nonmigrating and migrating samples. RNA was extracted using Trizol LS reagent. PCR was modified in order to allow detection of amplified products on 3% agarose gel with ethidium bromide staining. Products were also resolved on 6% denaturing polyacrylamide-urea gels and observed by silver staining. Agarose gels showed two prominent bands that were heavily expressed in the 458 bp and 587 bp region of the nonmigrating samples. In addition light bands were visible in the region corresponding to 234 bp and 450 bp. In the migrating samples, two light bands were visible in the region of 267 bp and 300 bp. Eight amplicons, six in the nonmigrating corneal epithelial sample and two in the migrating corneal epithelial samples, were also found to be differentially expressed when products were run on 6% denaturing polyacrylamide-urea gels. Thus, DDRT-PCR products can be detected on agarose gels and prove very helpful and economical in the initial studies of DDRT-PCR.

Animals↗