(65/1076) Matrix metalloproteinases (MMPs) in fresh human prostate tumour tissue and organ-cultured prostate tissue: levels of collagenolytic and gelatinolytic MMPs are low, variable and different in fresh tissue versus organ-cultured tissue.
Prostate tissue was obtained from 22 radical prostatectomies (performed for clinical management of prostate carcinoma) immediately after surgery. A small piece of tissue was fixed immediately in formalin and used for routine histology while a second piece was frozen in OCT and used for immuno-histochemistry. Another small piece was used for isolation of epithelial and stromal cells. The remainder of the tissue was cut into 2 x 2 mm pieces and incubated in organ culture for 8 days. In organ culture, non-malignant, basal epithelial cells underwent a proliferative response. This was accompanied by de-differentiation of glandular structures and by migration of epithelial cells across the surface of the tissue. Erosion of the basement membrane could also be seen in places, but was not widespread. Invasion of epithelial cells into the adjacent stroma was not evident. Production of matrix metalloproteinases (MMPs) with gelatinolytic activity or collagenolytic activity was assessed in organ culture and compared to expression patterns in fresh tissue. MMP-1 (interstitial collagenase) and MMP-9 (92-kDa gelatinase B) were undetectable or low in fresh tissue specimens. Both enzymes were detected in organ culture and both increased over time. Even after 6 days, however, there was only a low level of gelatin-hydrolytic activity and no measurable collagen-hydrolytic activity. In past studies we used organ cultures of normal skin and malignant skin tumours (basal cell carcinomas) to help elucidate the role of collagenolytic and gelatinolytic MMPs in epithelial cell invasion (Varani et al, 2000). Compared to MMP levels observed in skin, levels of these enzymes in prostate are low. The low level of collagenolytic and gelatinolytic MMPs in fresh prostate tissue and in organ-cultured prostate tissue may help explain why there is little tissue destruction in many primary prostate tumours and why the majority of such tumours remain confined to the prostate for extended periods. (+info)
(66/1076) Localization of polycyclic hydrocarbon carcinogens in the lung following intratracheal instillation in gelatin solution.
The deposition and localization of benzo(a)pyrene (BP) suspended in a gelatin-0.9 percent NaCl solution was studied in hamster lungs by ultraviolet fluorescence microscopy. BP was deposited primarily in the alveolar region of the lung. Although large numbers of BP-filled macrophages were seen in the upper airways by 24 hr after an instillation, little BP appeared to penetrate into the bronchial epithelium. The intratracheal instillation of polycyclic hydrocarbons in a gelatin-0.9 percent NaCl solution appears to be a useful model when it is desired to deliver the carcinogen dose to the peripheral lung. (+info)
(67/1076) Gelatin-binding region of human matrix metalloproteinase-2: solution structure, dynamics, and function of the COL-23 two-domain construct.
Human matrix metalloproteinase-2 (MMP-2) contains an array of three fibronectin type II (FII) modules postulated to interact with gelatin (denatured collagen). Here, we verify that the NMR solution structure of the third FII repeat (COL-3) is similar to that of the second FII repeat (COL-2); characterize its ligand-binding properties; and derive dynamics properties and relative orientation in solution for the two domains of the COL-23 fragment, a construct comprising COL-2 and COL-3 in tandem, with each domain possessing a putative collagen-binding site. Interaction of the synthetic gelatin-like octadecapeptide (Pro-Pro-Gly)(6) (PPG6) with COL-3 is weaker than with COL-2. We found that a synthetic peptide comprising segment 33-42 (peptide 33-42) from the MMP-2 prodomain interacts with COL-3 and, albeit with lower affinity, with COL-2 in a way that mimics PPG6 binding. COL-3 strongly prefers peptide 33-42 over PPG6, which suggests that intramolecular interactions with the prodomain could modulate binding of pro-MMP-2 to its gelatin substrate. In COL-23, the two modules retain their structural individuality and tumble independently. Overall, the NMR data indicate that the relative orientation of the modules in COL-23 is not fixed in solution, that the modules do not interact with one another, and that COL-23 is rather flexible. The binding sites face opposite each other, and their responses to, and normalized affinities for, the longer ligand PPG12 are virtually identical to those of the individual domains for PPG6, thus precluding co- operativity, although they may interact simultaneously with multiple sites of the extracellular matrix. (+info)
(68/1076) nm23-H1 suppresses invasion of oral squamous cell carcinoma-derived cell lines without modifying matrix metalloproteinase-2 and matrix metalloproteinase-9 expression.
nm23-H1 is a candidate gene for the suppression of cancer metastasis. Several studies on human breast, hepatocellular, gastric, ovarian, and colon carcinomas and melanomas have shown that reduced nm23-H1 expression was closely related to metastatic progression with poor prognosis. However, the biochemical mechanism by which nm23-H1 suppresses the metastasis has yet to be elucidated. In this study, we analyzed the correlation between nm23 expression, cell motility, and the invasive abilities of six different oral squamous cell carcinoma cell lines (HSC2, HSC3, HSC4, KB, OSC19, and OSC20). Reduced mRNA/protein expression of the nm23-H1 was observed in three cell lines (HSC2, HSC3, and HSC4). These cell lines exhibited increased cell motility and an invasive character on organotypic raft culture. On the other hand, the cell lines (KB, OSC19, and OSC20) that showed a higher expression of nm23-H1 exhibited a threefold to fivefold reduced motility and also reflected fewer invasions compared to the former three cell lines. Because the HSC3 cells demonstrated the lowest nm23-H1 expression with the highest cell motility and invasive character, we established nm23-H1-transfected HSC3 cell lines to investigate whether exogenous nm23-H1 protein could inhibit cell migration and invasive activity. These transfectants showed a significant reduction in cell motility with exogenous nm23-H1 in a dose-dependent manner, and exhibited a noninvasive character. An immunofluorescence study demonstrated a distinct stress-fiber distribution at peripheral region of these transfectants. However, no significant difference of matrix metalloproteinase (MMP)-2 and MMP-9 expression was observed between mock transfectant and nm23-H1-transfected cells. These findings suggest that nm23-H1 inhibits the invasive activity of oral squamous cell carcinoma by suppression of cell motility without altering the MMP-2 and MMP-9 status. (+info)
(69/1076) An investigation of the action of porcine pancreatic alpha-amylase on native and gelatinised starches.
The action of pancreatic alpha-amylase (EC 220.127.116.11) on various starches has been studied in order to achieve better understanding of how starch structural properties influence enzyme kinetic parameters. Such studies are important in seeking explanations for the wide differences reported in postprandial glycaemic and insulinaemic indices associated with different starchy foodstuffs. Using starches from a number of different sources, in both native and gelatinised forms, as substrates for porcine alpha-amylase, we showed by enzyme kinetic studies that adsorption of amylase to starch is of kinetic importance in the reaction mechanism, so that the relationship between reaction velocity and enzyme concentration [E0] is logarithmic and described by the Freundlich equation. Estimations of catalytic efficiencies were derived from measurements of kcat/Km performed with constant enzyme concentration so that comparisons between different starches were not complicated by the logarithmic relationship between E0 and reaction velocity. Such studies reveal that native starches from normal and waxy rice are slightly better substrates than those from wheat and potato. After gelatinisation at 100 degrees C, kcat/Km values increased by 13-fold (waxy rice) to 239-fold (potato). Phosphate present in potato starch may aid the swelling process during heating of suspensions; this seems to produce a very favourable substrate for the enzyme. Investigation of pre-heat treatment effects on wheat starch shows that the relationship between treatment and kcat/Km is not a simple one. The value of kcat/Km rises to reach a maximum at a pre-treatment temperature of 75 degrees C and then falls sharply if the treatment is conducted at higher temperatures. It is known that amylose is leached from starch granules during heating and dissolves. On cooling, the dissolved starch is likely to retrograde and become resistant to amylolysis. Thus the catalytic efficiency tends to fall. In addition, we find that the catalytic efficiency on the different starches varies inversely with their solubility and we interpret this finding on the assumption that the greater the solubility, the greater is the likelihood of retrogradation. We conclude that although alpha-amylase is present in high activity in digestive fluid, the enzymic hydrolysis of starch may be a limiting factor in carbohydrate digestion because of factors related to the physico-chemical properties of starchy foods. (+info)
(70/1076) Intramyocardial delivery of basic fibroblast growth factor-impregnated gelatin hydrogel microspheres enhances collateral circulation to infarcted canine myocardium.
The present study examined whether basic fibroblast growth factor (bFGF)-impregnated acidic gelatin hydrogel microspheres (AGHM) would enhance collateral development to the infarct area in dogs with coronary occlusion. Studies were conducted in 28 dogs with a 2-week occlusion of the proximal left anterior descending coronary artery (LAD). The dogs were divided into 3 groups according to treatment: Group A treated with bFGF-impregnated AGHM in the infarct area; Group B with free-form bFGF; Group C with AGHM alone. Coronary angiography (n=15; Group A, 7 dogs; Group B, 5 dogs; Group C, 3 dogs) and a regional myocardial blood flow study (n=13; Group A, 6 dogs; Group B, 4 dogs; Group C, 3 dogs) were repeated at a 2-week interval. Coronary angiography revealed that in Group A, antegrade flow in the LAD distal to the occlusion, which was assessed by Thrombolysis in Myocardial Infarction (TIMI) grade, was significantly increased after treatment. In contrast, in Groups B and C, the treatment did not change the flow grade in the LAD. In Group A, the regional myocardial blood flow in the collateral dependent area was significantly increased after treatment, and the regional myocardial blood flow reserve after adenosine injection was also significantly increased. These measurements remained after treatment in Groups B and C. The immunohistochemical study with factor VIII-related antigen revealed an increase of vascular density in the ischemic region in Group A. Intramyocardial delivery of bFGF-impregnated AGHM, but not free-form bFGF, improves the collateral circulation to the infarct area of a coronary occlusion in dogs. (+info)
(71/1076) Rapid differential endogenous plasminogen activator expression after acute middle cerebral artery occlusion.
BACKGROUND AND PURPOSE: During focal cerebral ischemia, the microvascular matrix (ECM), which participates in microvascular integrity, is degraded and lost when neurons are injured. Loss of microvascular basal lamina antigens coincides with rapid expression of select matrix metalloproteinases (MMPs). Plasminogen activators (PAs) may also play a role in ECM degradation by the generation of plasmin or by MMP activation. METHODS: The endogenous expressions of tissue-type plasminogen activator (tPA), urokinase (uPA), and PA inhibitor-1 (PAI-1) were quantified in 10-microm frozen sections from ischemic and matched nonischemic basal ganglia and in the plasma of 34 male healthy nonhuman primates before and after middle cerebral artery occlusion (MCA:O). RESULTS: Within the ischemic basal ganglia, tissue uPA activity and antigen increased significantly within 1 hour after MCA:O (2P<0.005). tPA activity transiently decreased 2 hours after MCA:O (2P=0.01) in concert with an increase in PAI-1 antigen (2P=0.001) but otherwise did not change. The transient decrease in free tPA antigen was marked by an increase in the tPA-PAI-1 complex (2P<0.001). No significant relations to neuronal injury or intracerebral hemorrhage were discerned. CONCLUSIONS: The rapid increase in endogenous PA activity is mainly due to significant increases in uPA, but not tPA, within the ischemic basal ganglia after MCA:O. This increase and an increase in PAI-1 coincided with latent MMP-2 generation and microvascular ECM degeneration but not neuronal injury. (+info)
(72/1076) Evaluation of basement membrane degradation during TNF-alpha-induced increase in epithelial permeability.
We evaluated whether tumor necrosis factor (TNF)-alpha induces an increase in permeability of an alveolar epithelial monolayer via gelatinase secretion and basement membrane degradation. Gelatinase secretion and epithelial permeability to radiolabeled albumin under unstimulated and TNF-alpha-stimulated conditions of an A549 human epithelial cell line were evaluated in vitro. TNF-alpha induced both upregulation of a 92-kDa gelatinolytic activity (pro form in cell supernatant and activated form in extracellular matrix) and an increase in the epithelial permeability coefficient compared with the unstimulated condition (control: 1.34 +/- 0.04 x 10(-6) cm/s; 1 microg/ml TNF-alpha: 1.47 +/- 0.05 x 10(-6) cm/s, P < 0.05). The permeability increase in the TNF-alpha-stimulated condition involved both paracellular permeability, with gap formation visualized by actin cytoskeleton staining, and basement membrane permeability, with an increase in the basement membrane permeability coefficient (determined after cell removal; control: 2.58 +/- 0.07 x 10(-6) cm/s; 1 microg/ml TNF-alpha: 2.82 +/- 0.02.10(-6) x cm/s, P < 0.05). Because addition of gelatinase inhibitors [tissue inhibitor of metalloproteinase (TIMP)-1 or BB-3103] to cell supernatants failed to inhibit the permeability increase, the gelatinase-inhibitor balance in the cellular microenvironment was further evaluated by cell culture on a radiolabeled collagen matrix. In the unstimulated condition, spontaneous collagenolytic activity inhibited by addition to the matrix of 1 microg/ml TIMP-1 or 10(-6) M BB-3103 was found. TNF-alpha failed to increase this collagenolytic activity because it was associated with dose-dependent upregulation of TIMP-1 secretion by alveolar epithelial cells. In conclusion, induction by TNF-alpha of upregulation of both the 92-kDa gelatinase and its inhibitor TIMP-1 results in maintenance of the gelatinase-inhibitor balance, indicating that basement membrane degradation does not mediate the TNF-alpha-induced increase in alveolar epithelial monolayer permeability. (+info)