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The suitability of Atomic Layer Deposition for making corrosion protection coatings was examined. Thin films of Al2O3, TiO2, Ta2O5and Al2O3-TiO2multilayer were deposited on stainless steel substrates. The films were deposited at 150-400 °C. The corrosion behaviour of the samples was studied by Electrochemical Impedance Spectroscopy in 3.5 wt.% NaCl and 0.1 and 1 mol/l HCl solutions. Al2O3was found to protect the substrate material against corrosion for a limited time in NaCl. TiO2alone does not give good protection, but when combined with Al2O3in a multilayer structure better protection is achieved. Ta2O5films were tested in HCl and were found to protect the base material to some extent.

In Finland, Olkiluoto Island on the western coast has been selected as a repository site for spent nuclear fuel disposal. With approaching licensing steps, the biosphere assessment demonstrating the long-term safety of the repository is developed into more and more site specific. As the literature data on site-relevant crops and garden products on site-relevant soils are sparse or in some cases lacking for the elements of a high relevance to the biosphere assessment of the Olkiluoto spent fuel repository, crops and garden products were collected from farms nearby the site. The samples were then analysed for stable elements for initial contribution to the site-specific database in respect of these edibles.

This paper describes the design, manufacture and properties of membrane structures with capacitive detection of deflection. The purpose of the study is to find a sensing organ to be used in an integrated tactile sensor for medical applications. The designed structures are implemented in a commercial CMOS process with a small number of post-micromachining steps. The process used offers two possibilities to achieve capacitors with a moving membrane, namely sacrificial etching of aluminium or silicon dioxide. Both methods have their strengths, but also limitations. These issues, which are strongly affecting the design flow, are discussed together with measurement results of the fabricated structures. The membranes from sacrificial oxide etching showed reliable dielectric gap formation. However, the large variation in their stress properties together with limited available pitch rendered them inferior to membranes from sacrificial aluminium etching. Moreover, the aluminium release etch can be achieved with a simpler etch-opening formation procedure than the oxide release etch.

The use of numerical weather forecast model data as a source of data for soil moisture modelling was tested. Results show that the potential evaporation calculated using the Penman‐Monteith equation can be estimated accurately using data obtained from the output of a high resolution numerical atmospheric model (HIRLAM, High Resolution Limited Area Model). The mean bias error was 0.26 mm for a 36‐hour sum and the root mean square error was 2.14 mm. The evaporation obtained directly from HIRLAM was systematically smaller because this direct model output represents the real evaporation rather than the potential evaporation. The precipitation forecasts were less accurate. When the accuracy of parameters required for the calculation of potential evaporation were studied for one station, no serious bias was found. When two different irrigation models (AMBAV and SWAP) were run over one summer using either measured or HIRLAM data as the input, the results given by the models were quite similar regardless of input data source. The largest differences between the model outputs were caused by the formulation of crop and soil characteristics in the irrigation models. Copyright © 2005 Royal Meteorological Society

The use of numerical weather forecast model data as a source of data for soil moisture modelling was tested. Results show that the potential evaporation calculated using the Penman-Monteith equation can be estimated accurately using data obtained from the output of a high resolution numerical atmospheric model (HIRLAM, High Resolution Limited Area Model). The mean bias error was 0.26 mm for a 36-hour sum and the root mean square error was 2.14 mm. The evaporation obtained directly from HIRLAM was systematically smaller because this direct model output represents the real evaporation rather than the potential evaporation. The precipitation forecasts were less accurate. When the accuracy of parameters required for the calculation of potential evaporation were studied for one station, no serious bias was found. When two different irrigation models (AMBAV and SWAP) were run over one summer using either measured or HIRLAM data as the input, the results given by the models were quite similar regardless of input data source. The largest differences between the model outputs were caused by the formulation of crop and soil characteristics in the irrigation models.

Objectives. To evaluate ultrasonography (US) of salivary glands in primary Sjögren's syndrome (SS) and to compare US with parotid magnetic resonance (MR) imaging and MR sialography.Methods. US examination of parotid, submandibular and sublingual glands was performed on 27 patients with primary SS, 27 healthy controls and 27 symptomatic controls without SS. The results were compared with parotid MR imaging and MR sialography and the clinical features of the patients.Results. Salivary gland abnormalities, parenchymal inhomogeneity or adipose degeneration, were visualized in 21 (78%) SS patients, in one healthy control and in two symptomatic controls by US. Eighteen (67%) patients had changes in the parotid and submandibular glands and 8 (30%) changes in the sublingual glands. In the comparison, MR sialography was found to be the most sensitive method (96%), followed by MR imaging (81%) and US (78%), in detecting glandular changes. The specificity of US was 94%. The US and MR results were related to anti-Ro/SSA positivity but not to saliva secretion. The focus scores were related only to parotid MR imaging findings.Conclusions. US, MR imaging and MR sialography with modern technology have reached such a good accuracy in visualizing glandular structural changes that they are promising alternatives to the conventional invasive examinations in the diagnostics of SS.

MMP‐8 (collagenase‐2) is the most effective collagenase to initiate type I collagen degradation. Since initiation of lysis of the surrounding collagen matrix is an essential prerequisite for carcinoma cells to spread, this study investigated the expression of MMP‐8 in squamous cell carcinoma (SCC) of the head and neck in vivoand in vitro. Most of the recently established head and neck carcinoma cell lines (22/25), corresponding tumour (5/7) and dermal (2/2) fibroblasts, commercial tongue carcinoma (HSC‐3 and SCC‐25), and transformed keratinocyte cell lines of the tongue (IHGK) and skin (HaCaT) expressed MMP‐8 mRNA analysed by the PCR method. Western blotting revealed a latent 50 kD band in concentrated culture media of carcinoma cells and corresponding tumour and dermal fibroblasts. The expression of immunoreactive MMP‐8 protein was reduced 30% by transforming growth factor beta‐1 (TGF‐β1) at 1 ng/ml concentration and 60% at 10 ng/ml concentration, but up‐regulated 2‐ and 2.5‐fold after 10 nMand 100 nMphorbol 12‐myristate 13 acetate (PMA), respectively. Immunohistological staining localized MMP‐8 protein in a few malignant invading tumour cell islands, certain fibroblasts, polymorphonuclear neutrophils (PMNs), and plasma cells. In situhybridization revealed a faint sporadic signal in carcinoma cells of all eight tissue sections analysed. It is concluded that tissue from head and neck carcinomas can express MMP‐8 both in vivoand in vitro. Since the amount of MMP‐8 in carcinoma and stromal cells is rather low, MMP‐8 may have a potential role, with other collagenases, in the proteolysis of connective tissue associated with the spreading of invasive carcinoma. Copyright © 2002 John Wiley & Sons, Ltd.

The purpose of this study was to determine whether other cellular sources than neutrophils can express matrix metalloproteinase (MMP)-8 protein and mRNA in bronchiectatic (BE) lung. The molecular forms of MMP-8 in the BE bronchoalveolar lavage fluid (BALF) and healthy control BALF were analysed by western immunoblotting. MMP-8 expression was demonstrated by immunohistochemistry and in situ hybridization in BE lung tissue and by immunohistochemistry in control lung tissue. In the BE BALF, different MMP-8 species were detected: 70-80 kD MMP-8 apparently of polymorphonuclear leukocyte (PMN) origin and also 40-60 kD MMP-8 from non-PMN cellular sources, such as bronchial epithelial cells, glandular cells or monocytes/macrophages. Both of these MMP-8 species were elevated and converted to a significant extent to activated forms in BE BALF compared with healthy control BALF. The levels of high molecular weight (>80 kD) MMP-8 complexes, evidently representing MMP-8 trapped by endogenous MMP inhibitors and/or MMP-8 dimers, were significantly elevated in BE BALF compared with healthy control BALF. In BE lung tissue, the MMP-8 protein and mRNA expression was found in bronchial ciliated epithelial cells, glandular cells, neutrophils, and monocytes/macrophages infiltrating the bronchial epithelial area. Minimal MMP-8 expression was observed in neutrophils, monocytes/macrophages, and epithelial cells in control lung tissues. In this study, new potential cellular sources have been demonstrated for MMP-8 in the inflamed lung. MMP-8 from multiple cellular sources, including inflamed lung epithelium, was activated to a significant extent in the BE BALF, indicating a major role for MMP-8 in the destruction of lung and bronchial tissues. Copyright © 2001 John Wiley & Sons, Ltd.

Matrix metalloproteinases (MMPs) collectively degrade extracellular matrix and basement membrane proteins in chronic inflammation and bone-destructive lesions. This study examined the ability of immunoglobulin-producing plasma cells, typically present in sites of chronic inflammation, to express collagenases (MMP-8 and -13) in vivo and in vitro. Phorbol-12-myristate-13-acetate, interleukin-6, and tumour necrosis factor-α and heparin with the tumour promoter or cytokines potently enhanced (up to nine-fold) MMP-8 and -13 expression by the RPMI 8226 myeloma cell line, as evidenced by western blotting and semi-quantitative reverse transcriptase-polymerase chain reaction. Immunohistochemical analysis and in situ hybridization revealed that plasma cells expressed MMP-8 and -13 focally in periapical granulomas, odontogenic cysts, and malignant plasmacytomas. MMP-8 and MMP-13 from plasma cells can participate in bone organic matrix destruction at sites of chronic inflammation and neoplastic growth. Since MMP-13 was more frequently expressed than MMP-8 in plasma cells of strongly recurring keratocysts and malignant plasmacytomas, it is concluded that plasma cell MMP-13 has a particularly important role in benign and malignant bone-destructive lesions. Copyright © 2001 John Wiley & Sons, Ltd.

For entire Dirichlet series of the form $$F\left( z \right) = \sum\nolimits_{n = 0}^{ + \infty } {a_n e^{z{\lambda }_n } ,0 \leqslant {\lambda }_n \uparrow + \infty ,\;n \to + \infty }$$ , we establish conditions under which the relation $$F\left( {{\sigma } + iy} \right) = \left( {1 + o\left( 1 \right)} \right)a_{{\nu }\left( {\sigma } \right)} e^{\left( {{\sigma + }iy} \right){\lambda }_{{\nu }\left( {\sigma } \right)} }$$ holds uniformly in $$y \in \mathbb{R}\;{as}\;{\sigma } \to + \infty$$ outside a certain set Efor which $$DE = \mathop {\lim \sup }\limits_{{\sigma } \to + \infty } h\left( {\sigma } \right)\;{meas}\;\left( {E \cap \left[ {{\sigma ,} + \infty } \right)} \right) = 0$$ where h(σ) is a positive continuous function increasing to +∞ on [0, +∞).

OBJECTIVES: Integrin α9 subunit is a member of β1 integrin family and binds tenascin (TN). It is expressed by stratified squamous epithelium and may be associated with cell differentiation and growth. We studied if the expression of α9 integrin and TN is altered in leukoplakia, lichen planus, and squamous cell carcinoma (SCC).

Abstract: Objective: To study the effect of antihypertensive medications on autonomic nervous system in patients with hypertension and sleep apnea syndrome using frequency domain measures of heart rate and blood pressure variabilities. Methods: The β-receptor blocking agent atenolol (50 mg), the calcium antagonist isradipine SRO (2.5 mg), the diuretic hydrochlorothiazide (25 mg) and the ACE inhibitor spirapril (6 mg) once daily were given in a double-blind crossover schedule for 8 weeks. Cardiovascular autonomic control was assessed using frequency domain measures of heart rate variability during the spontaneous and controlled breathing tests. During orthostatic maneuver and cold pressor test the blood pressure variability analysis also was performed. Results: In general, the responses of heart rate and blood pressure variabilities were abnormal in the patients with arterial hypertension and sleep apnea syndrome compared to reference data. Of the four drugs, only atenolol effected heart rate and blood pressure variabilities as it shifted the autonomic regulation to the vagal direction. Other antihypertensive drugs did not change any parameter of heart rate or blood pressure variabilities. Conclusion: The short-term treatment with atenolol in patients with arterial hypertension and sleep apnea syndrome is associated with normalization of autonomic nervous control judged by heart rate and blood pressure variability. Thus, β-receptor blockade may have adjunctive beneficial effects beyond blood pressure reduction in these patients. However, the long-term effects of blood pressure reduction on autonomic nervous control remain to be studied.

We investigated the expression of proteoglycans (PGs) and hyaluronan (HA) during healing of human mucosal wounds. Biopsy specimens of experimental wounds were taken 1, 3, and 7 days after wounding. Frozen sections were used for immunolocalization of CD44, syndecan-1, basement membrane-associated heparan sulfate proteoglycan (BM-HSPG), decorin, and biglycan. HA was localized in paraffin sections with a specific HA-binding probe. Epithelium showed first signs of migration on Day 1, more progressive migration on Day 3, and epithelial sheets confronted on Day 7. CD44 surrounded migrating keratinocytes at all stages of wound healing. In epithelium, CD44 and HA remarkably localized to the same region. Expression of syndecan-1 was switched from the suprabasal cell layer of unwounded epithelium to the basal cell layer of the migrating wound epithelium. BM-HSPG was absent under migrating keratinocytes. It started to reappear at the basement membrane zone on Day 7. The area under the wound epithelium containing newly synthesized collagen fibers first became positive for decorin on Day 7, whereas staining of biglycan was negative. Granulation tissue was also strongly positive for CD44 and hyaluronan. Our results indicate that migrating keratinocytes express both CD44 and syndecan-1 but not BM-HSPG. During differentiation of keratinocytes, expression of CD44 preceded that of syndecan-1. The results suggest that different HSPGs have multiple functions in keratinocyte migration and differentiation during reepithelialization.

We have sequenced the Streptomyces plicatus beta-N-acetylhexosaminidase (SpHex) gene and identified the encoded protein as a member of family 20 glycosyl hydrolases. This family includes human beta-N-acetylhexosaminidases whose deficiency results in various forms of GM2 gangliosidosis. Based upon the x-ray structure of Serratia marcescens chitobiase (SmChb), we generated a three-dimensional model of SpHex by comparative molecular modeling. The overall structure of the enzyme is very similar to homology modeling-derived structures of human beta-N-acetylhexosaminidases, with differences being confined mainly to loop regions. From previous studies of the human enzymes, sequence alignments of family 20 enzymes, and analysis of the SmChb x-ray structure, we selected and mutated putative SpHex active site residues. Arg162 --> His mutation increased Km 40-fold and reduced Vmax 5-fold, providing the first biochemical evidence for this conserved Arg residue (Arg178 in human beta-N-acetylhexosaminidase A (HexA) and Arg349 in SmChb) as a substrate-binding residue in a family 20 enzyme, a finding consistent with our three-dimensional model of SpHex. Glu314 --> Gln reduced Vmax 296-fold, reduced Km 7-fold, and altered the pH profile, consistent with it being the catalytic acid residue as suggested by our model and other studies. Asp246 --> Asn reduced Vmax 2-fold and increased Km only 1.2-fold, suggesting that Asp246 may play a lesser role in the catalytic mechanism of this enzyme. Taken together with the x-ray structure of SmChb, these studies suggest a common catalytic mechanism for family 20 glycosyl hydrolases.

Increased production of proteinases, such as matrix metalloproteinases (MMPs), is a characteristic feature of malignant tumors. Some human cancers and cell lines derived from them also express trypsinogen, but the function of the extrapancreatic trypsin has remained unclear. In this study we cloned and sequenced trypsinogen-2 cDNA from human COLO 205 colon carcinoma cells and characterized the ability of the enzyme to activate latent human type IV procollagenases (proMMP-2 and proMMP-9). As shown by cloning and N-terminal amino acid sequencing, the amino acid sequence of tumor-associated trypsin-2 is identical to that of pancreatic trypsin-2. We found that both pancreatic trypsin-2 and tumor cell-derived trypsin-2 are efficient activators of proMMP-9 and are capable of activating proMMP-9 at a molar ratio of 1:1000, the lowest reported so far. Human trypsin-2 was a more efficient activator than widely used bovine trypsin and converted the 92-kDa proMMP-9 to a single 77-kDa product that was not fragmented further. The single peptide bond cleaved by trypsin-2 in proMMP-9 was Arg87-Phe88. The generation of the 77-kDa species coincided with the increase in specific activity of MMP-9. In contrast, trypsin-2 only partially activated proMMP-2. Trypsin-2 cleaved the Arg99-Lys100 peptide bond of proMMP-2 generating 62-65-kDa MMP-2 species. Trypsin-2-induced proMMP-2 and -9 conversions were inhibited by tumor-associated trypsin inhibitor added either prior to or during activation indicating that proMMPs were not activated autocatalytically. Trypsin-2 also activated proMMPs associated with tissue inhibitor of matrix metalloproteinases, the complexes of which are thought to be the major MMP forms in vivo. The ability of human tumor cell-derived trypsin-2 to activate latent MMPs suggests a role for trypsin-2 in initiating the proteinase cascade that mediates tumor invasion and metastasis formation.

Two benign mutations, C739T(R247W) and C745T(R249W), in the alpha-subunit of beta-hexosaminidase A (Hex A) have been found in all but one of the currently identified Hex A-pseudodeficient subjects. To confirm the relationship of the benign mutations and Hex A pseudodeficiency and to determine how the benign mutations reduce Hex A activity, we transiently expressed each of the benign mutations, and other mutations associated with infantile, juvenile, and adult onset forms of GM2 gangliosidosis, as Hex S (alphaalpha) and Hex A (alphabeta) in COS-7 cells. The benign mutations decreased the expressed Hex A and Hex S activity toward the synthetic substrate 4-methylumbelliferyl-6-sulfo-beta-N-acetylglucosaminide (4-MUGS) by 60-80%, indicating that they are the primary cause of Hex A pseudodeficiency. Western blot analysis showed that the benign mutations decreased the enzymatic activity by reducing the alpha-subunit protein level. No change in heat sensitivity, catalytic activity, or the substrate specificity to the synthetic substrates, 4-methylumbelliferyl-beta-N-acetylglucosaminide or 4-methylumbelliferyl-6-sulfo-beta-N-acetylglucosaminide, was detected. The effects of the benign mutations on Hex A were further analyzed in fibroblasts, and during transient expression, using pulse-chase metabolic labeling. These studies showed that the benign mutations reduced the alpha-subunit protein by affecting its stability in vivo, not by affecting the processing of the alpha-subunit, i.e. phosphorylation, targeting, or secretion. Our studies also demonstrated that these benign mutations could be readily differentiated from disease-causing mutations using a transient expression system.

Neutrophil collagenase (matrix metalloproteinase-8 or MMP-8) is regarded as being synthesized exclusively by polymorphonuclear neutrophils (PMN). However, in vivo MMP-8 expression was observed in mononuclear fibroblast-like cells in the rheumatoid synovial membrane. In addition, we detected MMP-8 mRNA expression in cultured rheumatoid synovial fibroblasts and human endothelial cells. Up-regulation of MMP-8 was observed after treatment of the cells with either tumor necrosis factor-alpha (10 ng/ml) or phorbol 12-myristate 13-acetate (10 nM). Western analysis showed a similar regulation at the protein level. The size of secreted MMP-8 was 50 kDa, which is about 30 kDa smaller than MMP-8 from PMN. Conditioned media from rheumatoid synovial fibroblasts contained both type I and II collagen degrading activity. However, degradation of type II collagen, but not that of type I collagen, was completely inhibited by 50 microM doxycycline, suggesting specific MMP-8 activity. In addition, doxycycline down-regulated MMP-8 induction, at both the mRNA and protein levels. Thus MMP-8 exerts markedly wider expression in human cells than had been thought previously, implying that PMN are not the only source of cartilage degrading activity at arthritic sites. The inhibition of both MMP-8 activity and synthesis by doxycycline provides an incentive for further studies on the clinical effects of doxycycline in the treatment of rheumatoid arthritis.

Abstract. Objective and Design: To determine whether an inhibitor of matrix metalloproteinases (MMPs), administered to human subjects in a dental school research clinic, can reduce bone-type collagen degradation fragments in oral inflammatory exudates containing excessive levels of collagenase.¶Materials and Subjects: Gingival crevicular fluid (GCF) was collected from 18 subjects with adult periodontitis whose clinical findings (gingival inflammation, pocket depth, and bone loss on radiographs) predicted excessive MMP activity in their periodontal pockets.¶Treatment: One month before the baseline appointment, plaque and calculus were removed from the teeth by supra- and subgingival scaling. After collection of GCF from 8-12 pocket sites per subject and recording of clinical indices, 12 of the 18 subjects were treated with doxycycline at a low dosage (20 mg b.i.d.) known via an extensive literature to suppress mammalian MMP activity by a non-antimicrobial mechanism. The remaining 6 subjects were followed without drug treatment.¶Methods: At the baseline, 1 and 2-month appointments, GCF samples were analyzed for ICTP (carboxyterminal peptide, a pyridinoline-containing fragment of Type I collagen) and osteocalcin by radioimmunoassay, as well as collagenolytic enzyme activity and MMP species (Western blot). Statistical analyses were determined by ANOVA.¶Results: GCF ICTP and functional collagenase activity (but not osteocalcin levels) were significantly reduced (p < 0.05) in the doxycycline-treated subjects at both 1 and 2 month evaluations; there was no such change in the non-treated subjects. Western blots revealed that neutrophil-type collagenase (MMP-8) was the predominant MMP; MMP-13, which has been associated with pathologic collagenolysis including bone resorption, was detected in human GCF for the first time and was more substantially reduced than MMP-8. ¶Conclusion: This is the first demonstration in human subjects of the simultaneous reduction of excessive MMP activity with concomitant reduction in levels of collagen degradation fragments. The findings are potentially applicable to a wide variety of human diseases characterized by excessive collagenase activity.

Although matrix metalloproteinases (MMPs) are among the potential key mediators of cancer invasion, their involvement in premalignant lesions and conditions is not clarified. Therefore, we studied, using in situ hybridization, immunohistochemistry and zymography the expression and distribution of MMP-1 and -2, and their tissue inhibitors (TIMPs -1, -2 and -3) in oral squamous cell carcinomas (SCC) and lymph node metastases as well as in oral lichen planus, epithelial dysplasias and normal buccal mucosa. In oral SCC and lymph node metastasis, MMP-1 mRNA was detected in fibroblastic cells of tumoral stroma. In two out of ten carcinomas studied, the peripheral cells of neoplastic islands were also positive. MMP-2 mRNA expression was noted in fibroblasts surrounding the carcinoma cells, and no signal in carcinoma cells was detected. A clear TIMP-3 mRNA expression was seen in stromal cells surrounding the neoplastic islands in all SCCs and lymph node metastases studied. TIMP-1 mRNA was detected in some stromal cells surrounding the neoplastic islands, whereas the mRNA expression for TIMP-2 was negligible. On the other hand, expression of MMPs and TIMPs was consistently low in oral epithelial dysplasias, lichen planus and normal mucosa. In certain epithelial dysplasias and lichen planus, MMP-1 and -2 mRNA expressions were detected in few fibroblasts under the basement membrane zone, but normal mucosa was completely negative. In SCC and lymph node metastasis, a detectable immunostaining for MMP-1 in stromal cells and in some carcinoma cells was observed. MMP-2 immunoreactivity was detected in the peripheral cell layer in neoplastic islands and in some fibroblast-like cells of tumoral stroma. Immunostaining for TIMP-3 was detected in stromal cells surrounding the neoplastic islands. A weak positive staining for TIMP-1 was located in tumoral stroma, whereas the immunostaining for TIMP-2 was negative. Using zymography, elevated levels of MMP-2 and MMP-9 were observed in carcinoma samples in comparison with lichen planus or normal oral mucosa. Our results indicate that the studied MMPs and TIMPs are clearly up-regulated during invasion in oral SCC. However, there was also a clear, although weak, up-regulation of the expression of the MMPs but not TIMPs in some of the lichen planus and dysplastic lesions.

Nickel-titanium alloy (Nitinol) is a metallic biomaterial that has a unique thermal shape memory, superelasticity, and high damping properties. Nitinol is potentially very useful in orthopedic surgery, for example. At present, there are not enough confirmative biocompatibility data available on Nitinol. The aim of our study was to clarify the primary cytotoxicity and corrosion rate of Nitinol in human cell cultures. Comparisons were made with stainless steel (Stst), titanium (Ti), composite material (C), and control cultures with no test discs. Human osteoblasts (OB) and fibroblasts (FB) were incubated for 10 days with test discs of equal size, 6 × 7 mm. The cultures were photographed and the cells counted. Samples from culture media were collected on days 2, 4, 6, and 8, and the analysis of metals in the media was done using flameless atomic absorption spectrophotometry. The proliferation of FB was 108% (Nitinol), 134% (Ti) (p< 0.02), 107% (Stst), and 48% (C)(p< 0.0001) compared to the control cultures. The proliferation of OB was 101% (Nitinol), 100% (Ti), 105% (Stst), and 54% (C) (p< 0.025) compared to the controls. Initially, Nitinol released more nickel (129–87 µg/L) into the cell culture media than Stst (7 µg/L), but after 2 days the concentrations were about equal (23–5 µg/L versus 11–1 µg/L). The titanium concentrations from both Nitinol and Ti samples were all <20 µg/L. We conclude that Nitinol has good in vitrobiocompatibility with human osteoblasts and fibroblasts. Despite the higher initial nickel dissolution, Nitinol induced no toxic effects, decrease in cell proliferation, or inhibition on the growth of cells in contact with the metal surface. © 1997 John Wiley & Sons, Inc. J Biomed Mater Res, 35, 451–457, 1997.

A sulfated glycoprotein was isolated from the culture media of Drosophila Kc cells and named papilin. Affinity purified antibodies against this protein localized it primarily to the basement membranes of embryos. The antibodies cross-reacted with another material which was not sulfated and appeared to be the core protein of papilin, which is proteoglycan-like. After reduction, papilin electrophoresed in sodium dodecyl sulfate-polyacrylamide gel electrophoresis as a broad band of about 900,000 apparent molecular weight and the core protein as a narrow band of approximately 400,000. The core protein was formed by some cell lines and by other cells on incubation with 1 mM 4-methylumbelliferyl xyloside, which inhibited formation of the proteoglycan-like form. The buoyant density of papilin in CsCl/4 M guanidine hydrochloride is 1.4 g/ml, that of the core protein is much less. Papilin forms oligomers linked by disulfide bridges, as shown by sodium dodecyl sulfate-agarose gel electrophoresis and electron microscopy. The protomer is a 225 +/- 15-nm thread which is disulfide-linked into a loop with fine, protruding thread ends. Oligomers form clover-leaf-like structures. The protein contains 22% combined serine and threonine residues and 25% combined aspartic and glutamic residues. 10 g of polypeptide has attached 6.4 g of glucosamine, 3.1 g of galactosamine, 6.1 g of uronic acid, and 2.7 g of neutral sugars. There are about 80 O-linked carbohydrate chains/core protein molecule. Sulfate is attached to these chains. The O-linkage is through an unidentified neutral sugar. Papilin is largely resistant to common glycosidases and several proteases. The degree of sulfation varies with the sulfate concentration of the incubation medium. This proteoglycan-like glycoprotein differs substantially from corresponding proteoglycans found in vertebrate basement membranes, in contrast to Drosophila basement membrane laminin and collagen IV which have been conserved evolutionarily.

The effect of dexamethasone on the synthesis and degradation of type IV collagen was studied in human fibrosarcoma cells, HT-1080. A dexamethasone concentration as low as 0.1 microM markedly increased collagen synthesis in HT-1080 cells labelled with [14C]proline. The increase in type IV collagen synthesis was not specific, since total protein synthesis was also increased. Further studies indicated that part of the increase was due to an increase in the specific radioactivity of the intracellular proline pool, after dexamethasone treatment. In fact, with dexamethasone concentrations of 0.1-10 microM the relative collagen synthesis was decreased, indicating that synthesis of other protein was increased more than that of type IV collagen. This was also confirmed by measuring the relative amount of type IV collagen RNA by using recombinant plasmid cDNA specific for the human procollagen pro alpha l (IV) RNA. The results indicated that relative collagen synthesis and the relative amount of type IV collagen messenger RNA was decreased similarly, indicating that dexamethasone affected type IV collagen synthesis at the pre-translational level. The dexamethasone-induced effect on total protein and collagen synthesis was maximal after 12-24 h. Dexamethasone induced a marked accumulation of collagen into the cell layer, leading to diminished deposition of soluble collagen into the medium. Since bacterial-collagenase treatment of the cell layer drastically decreased the collagen content of the dexamethasone-treated cells, this indicates that dexamethasone caused an accumulation of collagen into the extracellular matrix of the cell layer. In contrast, the amount of fibronectin was markedly increased in the medium. Dexamethasone decreased the type IV collagen-degrading activity in HT-1080 cells. The HT-1080 cells contained glucocorticoid receptors, as demonstrated by two different methods: by a whole-cell binding assay and by using a cytosol-gel-filtration method. The number of specific binding sites was similar to that in human skin fibroblasts. In conclusion, glucocorticoids affect the metabolism of type IV collagen and fibronectin in HT-1080 cells, and, since these cells contain specific glucocorticoid receptors, the effects are apparently receptor-mediated.

The secretion of a type IV collagen-specific proteinase is stimulated in cultured human skin fibroblasts by the phorbol ester tumor promoter 12-O-tetradecanoyl phorbol 13-acetate (TPA) and during cell proliferation. Exposure of the cells at the late log phase of growth to 10(-9) to 10(-6) M TPA resulted in the secretion of type IV collagenase activity to the medium, this effect being reversible. Incubation of intact type IV procollagen with TPA-induced fibroblast medium protein produced six peptides, four of which corresponded in size to the fragments produced by a type IV collagen-specific collagenase (Fessler, L., Duncan, K., Fessler, J., Salo, T., and Tryggvason (1984) J. Biol. Chem. 259, 9783-9789). The TPA-induced type IV collagen-degrading enzyme could be activated by trypsin, was inhibited by EDTA, but was not affected by soybean trypsin inhibitor, N-ethylmaleimide, aprotinin, or cysteine. Therefore, in human skin fibroblasts, TPA can induce a type IV collagen-specific, metal-dependent collagenase as was previously described in some invasive tumor cells. Furthermore, another metalloprotease is apparently secreted under the same conditions of TPA exposure. The production of metal-dependent, type IV collagen-degrading activity was also studied at different stages of cellular proliferation. In early log phase, a significant amount of enzyme activity was observed in the control cell medium; this activity disappeared during both late log and stationary growth phases. This activity could be markedly increased by the addition of 10(-8) M TPA to the culture medium. The production of matrix-degrading proteinases is therefore likely to be associated with rapid cell proliferation in both transformed and untransformed cells.

Proteoglycans (PGs) are extracellular and cell surface-associated macromolecules that regulate cell adhesion, cell growth, matrix formation, and bind growth factors. In this work we studied the distribution of core proteins of four PGs (decorin, biglycan, a large molecular weight PG, and CD44) in human gingiva and periodontal ligament by immunohistochemical staining of frozen tissue sections with specific antibodies. Decorin, a major PG of this tissue, was localized on collagen fiber bundles in the gingival and periodontal connective tissues. Staining for decorin was most intense at the subepithelial region. Biglycan was a minor PG component of the human periodontium, showing some accumulation in connective tissue under the oral epithelium. At the immunohistochemical level, biglycan appeared to form fine filament-like structures on extracellular matrix fibers. Localization of large molecular weight PG differed from that of decorin and biglycan. It was concentrated in deep connective tissue areas of the gingiva and in the periodontal ligament, and was only weakly present at the subepithelial region. CD44 was mainly concentrated in cell-cell contact areas of basal and spinous layers of oral epithelium. In the connective tissue of gingiva and periodontal ligament, CD44 was localized on fibroblast cell surfaces. Connective tissue area under the junctional epithelium contained relatively small amounts of PGs. The results indicate that different parts of human periodontium contain a typical variety of PGs, suggesting a specific function for each PG species in the location at which they accumulate.

The specific mammalian collagenase isolated from cultures of metastatic mouse PMT sarcoma cells cleaves murine procollagen IV into two segments, of approximate mass ratio 3:1. These fragments were separated by velocity sedimentation, visualized by electron microscopy, and analyzed. The longer COOH-terminal procollagen segment has a 270-nm collagenous portion with a knob at one end. This knob consists of the three previously identified, noncollagenous carboxyl propeptides, of approximately 30,000 daltons each. These carboxyl propeptides are chain-specific, and the three chains of each segment have the same amino to carboxyl orientation. The collagenase cuts through all three chains at one site, and the three-component chains of both the longer COOH-terminal procollagen segment and the shorter NH2-terminal procollagen segment are linked by interchain disulfide bridges. The enzyme cuts off the same COOH-terminal procollagen segment from procollagen IV monomers and tetramers, and the flexibility of this segment is similar to that of interstitial collagen helices. The amino ends of the NH2-terminal procollagen segments derived from tetramers remain joined as the 32-nm long “7 S collagen” junctional complex, and the remaining 89-nm long projecting threads are significantly more flexible than the COOH-terminal procollagen segment. The electrophoretic mobilities of the enzyme cleavage products are consistent with a heterotrimeric composition of this procollagen IV.

A type IV collagen-degrading enzyme activity secreted by a highly metastatic mouse tumor was purified by concanavalin A- and type IV collagen-agarose affinity chromatographies followed by gel filtration on Bio-Gel A-0.5 m. The apparent molecular weight of the enzyme was 160,000 but about 70,000 when Triton X-100 was added to the column buffer. The purified enzyme protein was resolved on sodium dodecyl sulfate-polyacrylamide gel electrophoresis into two polypeptide chains of about 68,000 and 62,000 daltons. The enzyme activity could be increased by preincubation with trypsin and it is possible that the two chains represent latent and active enzyme forms. The enzyme activity was not reduced in the presence of dithiothreitol, it had a pH optimum of 7.6 and was inhibited by EDTA but not N-ethylmaleimide, phenylmethylsulfonyl fluoride, or Trasylol. The inhibition with EDTA was reversible. The pro-alpha 1(IV) and pro-alpha 2(IV) chains of the type IV procollagen substrate were both degraded at a similar rate to form two pairs of degradation fragments corresponding in molecular weights to about 70 and 30% of the original size chains. The presence of Triton X-100 increased slightly the activity of the enzyme and diminished the reduction of its activity upon freezing, indicating that the enzyme is a hydrophobic protein.

Interstitial collagenase present in human jaw cyst extract and purified human fibroblast-type collagenase (MMP-1) were both efficiently inhibited in vitroby clodronate, an osteoactive, antiresorptive bisphosphonate. The IC50of clodronate to inhibit MMP-1 is 150 μM. These findings suggest an extended and hitherto undescribed properties for clodronate/biphosphonates in prevention and treatment of tissue degradation in both bone and soft tissue destructive diseases.

The aim of this work was to determine whether human polymorphonuclear neutrophilic interstitial collagenase (matrix metalloproteinase 8 [MMP-8]) levels are reduced during long-term doxycycline treatment in humans with reactive arthritis. Serum MMP-8 levels were reduced (mean +/- standard error of the mean, 678.9 +/- 185.6 versus 491.2 +/- 144.8 ng of MMP-8 per ml), but not statistically significantly. However, the reduction of salivary MMP-8 levels was statistically significant (3,729 +/- 1,905.3 versus 1,866 +/- 780.0 ng of MMP-8 per ml, P < 0.05). This study demonstrated that a 2-month regimen of doxycycline can reduce MMP-8 levels in serum and especially in body fluids (i.e., saliva) containing inflammatory exudates and thus may contribute to reduced tissue destruction.

A fully differential 80 MHz fourth-order bandpass ΔΣ modulator, meant for a 100 MHz GSM/WCDMA multimode IF receiver, is presented. The modulator is based on a double-delay single opamp SC-resonator structure which is well suited for low supply voltages. Furthermore, the centre frequency of the topology is insensitive to different component variances. The measured peak SNR is 78 dB and 43.3 dB for 270 kHz (GSM) and 3.84 MHz (WCDMA) bandwidths, respectively.