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In order to protect implanted glucose sensors from biofouling, novel hydrogels (146–217% water by mass) were developed based on a copolymer of hydroxyethyl methacrylate (HEMA) and 2,3-dihydroxypropyl methacrylate (DHPMA). The porosity and mechanical properties of the hydrogels were improved using N-vinyl-2-pyrrolidinone (VP) and ethyleneglycol dimethacrylate (EGDMA). The results of SEM and DSC FT-IT analyses showed that the hydrogel (VP30) produced from a monomeric mixture of 34.5% HEMA, 34.5% DHPMA, 30% VP and 1% EDGMA (mol%) had an excellent pore structure, high water content at swelling equilibrium (Weq = 166% by mass) and acceptable mechanical properties. Two kinds of VP30-coated sensors, Pt/GOx/VP30 and Pt/GOx/epoxy-polyurethane (EPU)/VP30 sensors were examined in glucose solutions during a period of 4 weeks. The Pt/GOx/VP30 sensors produced large response currents but the response linearity was poor. Therefore, further studies were focused on the Pt/GOx/EPU/VP30 sensors. With a diffusion-limiting epoxy-polyurethane membrane, the linearity was improved (2–30 mM) and the response time was within 5 min. Eight Pt/GOx/EPU/VP30 sensors were subcutaneously implanted in rats and tested once per week over 4 weeks. All of the implanted sensors kept functioning for at least 21 days and 3 out of 8 sensors still functioned at day 28. Histology revealed that the fibrous capsules surrounding hydrogel-coated sensors were thinner than those surrounding Pt/GOx/EPU sensors after 28 days of implantation.

Fibroblast growth factor-18 (FGF-18) has been shown to regulate the growth plate chondrocyte proliferation, hypertrophy and cartilage vascularization necessary for endochondral ossification. The heparan sulfate proteoglycan perlecan is also critical for growth plate chondrocyte proliferation. FGF-18 null mice exhibit a skeletal dwarfism similar to that of perlecan null mice. Growth plate perlecan contains chondroitin sulfate (CS) and heparan sulfate (HS) chains and FGF-18 is known to bind to heparin and to heparan sulfate from some sources. We used cationic filtration and immunoprecipitation assays to investigate the binding of FGF-18 to perlecan purified from the growth plate and to recombinant perlecan domains expressed in COS-7 cells. FGF-18 bound to perlecan with a K(d) of 145 nM. Near saturation, approximately 103 molecules of FGF-18 bound per molecule of perlecan. At the lower concentrations used, FGF-18 bound with a K(d) of 27.8 nM. This binding was not significantly altered by chondroitinase nor heparitinase digestion of perlecan, but was substantially and significantly reduced by reduction and alkylation of the perlecan core protein. This indicates that the perlecan core protein (and not the CS nor HS chains) is involved in FGF-18 binding. FGF-18 bound equally to full-length perlecan purified from the growth plate and to recombinant domains I-III and III of perlecan. These data indicate that low affinity binding sites for FGF-18 are present in cysteine-rich regions of domain III of perlecan. FGF-18 stimulated 3H-thymidine incorporation in growth plate chondrocyte cultures derived from the lower and upper proliferating zones by 9- and 14-fold, respectively. The addition of perlecan reversed this increased incorporation in the lower proliferating chondrocytes by 74% and in the upper proliferating cells by 37%. These results suggest that perlecan can bind FGF-18 and alter the mitogenic effect of FGF-18 on growth plate chondrocytes.

We report here the isolation and sulphation isomer analyses of trisaccharides GalNAcS(beta1,4)GlcA(beta1,3)GalNAcS (in which S indicates sulphate) derived from the non-reducing termini of aggrecan chondroitin sulphate. Rat chondrosarcoma and human aggrecans were digested for 1 h at 37 degrees C with 30 micro-units of endo-chondroitinase ABC per microgram of chondroitin sulphate, and trisaccharides were isolated from the digests by ToyoPearl HW40S gel-filtration chromatography. Four trisaccharide species were identified; their sulphation isomer compositions, as determined by digestion with chondroitinase ACII and fluorescence-based ion-exchange HPLC, were GalNAc4Sbeta1,4GlcAbeta1,3GalNAc4S, GalNAc4Sbeta1,4GlcAbeta1,3GalNAc6S, GalNAc4,6Sbeta1,4GlcAbeta1, 3GalNAc4S and GalNAc4,6Sbeta1,4GlcAbeta1,3GalNAc6S. The abundances of such sequences in chondroitin sulphate on aggrecan from normal (foetal to 72 years of age) and from osteoarthritic human knee cartilages were also established. The results showed that non-reducing terminal GalNAc4S or GalNAc4,6S can be linked to either a 4-sulphated or a 6-sulphated disaccharide, suggesting that the sulphation of the last disaccharide might not have a direct effect on the specificity of chondroitin sulphate terminal GalNAc sulphotransferases. Furthermore, for each aggrecan preparation examined, the 4S-to-6S ratio of all chain interior disaccharides was equivalent to that in the last repeating disaccharides at the non-reducing terminus, suggesting that neither chondroitin 4-sulphotransferase nor chondroitin 6-sulphotransferase shows preferential activity near the chain terminus.

Chondroitin lyase products of aggrecan and small proteoglycans from normal and osteoarthritic cartilages were analyzed for chain internal Δdisaccharides and terminal mono- or disaccharides. Chondroitin and dermatan sulfate chains from arthritic cartilages were of essentially normal size and internal sulfation but had significantly altered sulfation of the terminal residues. Whereas in normal cartilage, ∼60% of terminal GalNAc4S was 4,6-disulfated, it was reduced to ∼30% in osteoarthritic cartilage. This is most likely due to a lower terminal GalNAc4,6S-disulfotransferase activity and reveals that metabolic changes in osteoarthritis can affect this distinct sulfation step during chondroitin and dermatan sulfate synthesis. GlcAβ1,3GalNAc6S-, the mimotope for antibody 3B3(−), was present on ∼8 and ∼10% of chains from normal and osteoarthritic cartilages, respectively. 3B3(−) assayed by immunodot blot was within the normal range for most osteoarthritic samples, with only 5 of 24 displaying elevated reactivity. This resulted not from a higher content of mimotope, but possibly from other structural changes in the proteoglycan that increase mimotope reactivity. In summary, chemical determination of sulfation isomers at the non-reducing termini of chondroitin and dermatan sulfate provides a reliable assay for monitoring proteoglycan metabolism not only during normal growth of cartilage but also during remodeling of cartilage in osteoarthritis.

Clinical reports of ethanol-lock use for the prevention of catheter-related bloodstream infections have been marked by the occurrence of serious catheter occlusions, particularly among children with mediports. We hypothesized that precipitate forms when ethanol mixes with heparin at the concentrations relevant for vascular access devices, but that the use of a combination of two alcohols, ethanol and isopropanol, would diminish heparin-related precipitation, while retaining anti-bacterial and anti-fungal effects.Heparin (0-100units/mL) was incubated in ethanol-water solutions (30%-70% vol/vol) or in an aqueous solution containing equal parts (35% and 35% vol/vol) of isopropanol and ethanol. Precipitation at temperatures from 4 to 40°C was measured in nephelometric turbidity units using a benchtop turbidimeter. Growth of Escherichia coli, Staphylococcus aureus, and Candida albicans colonies were measured following exposure to solutions of ethanol or isopropanol-ethanol. Groupwise comparisons were performed using analysis of variance with Bonferroni-corrected, post-hoc T-testing.Seventy percent ethanol and heparin exhibit dose-dependent precipitation that is pronounced and significant at the concentrations typically used in mediports (p0.05). Precipitate is significantly reduced by use of a combined 35% isopropanol-35% ethanol solution rather than 70% ethanol (p0.05), while maintaining the solution's anti-bacterial and anti-fungal properties. On the other hand, although ethanol solutions under 70% form less precipitate with heparin, such concentrations are also less effective at bacterial colony inhibition than solutions of either 70% ethanol or 35% isopropanol-35% ethanol (p0.05).A combined 35% isopropanol-35% ethanol locking solution inhibits bacterial and fungal growth similarly to 70% ethanol, but results in less precipitate than 70% ethanol when exposed to heparin. Further study of a combined isopropanol-ethanol locking solution for the prevention of catheter-related bloodstream infections should focus on the determination as to whether such a locking solution may reduce the rate of precipitation-related catheter occlusion, and whether it may be administered with low systemic toxicity.

Prader-Willi syndrome (PWS) is characterized by hypotonia, obesity, hypogonadism, small hands and feet, and mental deficiency. Obesity and hypogonadism are also frequently associated with slipped capital femoral epiphysis (SCFE), suggesting that children with PWS might be at increased risk of developing SCFE. Members of the Prader-Willi Syndrome Association (U.S.A.) were surveyed regarding the history of orthopaedic problems in general and of SCFE in particular. A total of 565 (63%) responses were received. The prevalence of orthopaedic conditions included 47% with flat feet, 41% with scoliosis, 19% with knock knees, 10% with hip dysplasia, 9% with osteoporosis, 7% with patellofemoral instability, 3% with bowlegs, 2% each with clubfeet, nursemaid's elbow, or leg-length inequality, and one patient (0.2%) with SCFE. The results of the survey indicate that SCFE is uncommon in patients with PWS, but the prevalence of hip dysplasia is increased approximately 10-fold compared with the general population.

We have previously shown that lymphocytes from idiopathic minimal-lesion nephrotic patients produce a lymphokine (supernatant factor) that increases the 35sulfate uptake in the glomerular b

The cytolytic Clostridium perfringens delta toxin lyses selectively cells which express ganglioside GM2. In this study, we investigated whether delta toxin can be used to characterize GM2 on tumor cell membranes and as an antitumor agent. The sensitivity to lysis by delta toxin of various murine and human malignant cell lines and also normal tissues was quantified using a 51Cr-release assay. The cytotoxicity titers were correlated with the 125I-labeled toxin binding capacity of sensitive and insensitive cells. Seven of eight human melanomas tested were lysed by the toxin and, of these, four were very sensitive (cytotoxicity titers below 12 ng of toxin). All neuroblastomas, gliomas and the retinoblastoma tested were lysed with 3–18 ng of toxin. Three of six carcinomas and one of two sarcomas were also very sensitive (cytotoxicity titers 0.6–15 ng) whereas leukemias and lymphoma cells were insensitive. Normal human tissues were insensitive (erythrocytes, skin fibroblasts) or poorly sensitive (brain, lung, spleen). The in vivo antitumor activity of delta toxin was tested in tumor-bearing mice. Daily intra-tumor injections of 0.5–1 mg of toxin for 4–5 days in carcinoma Me180- and melanoma A375-bearing nude mice, and neuroblastoma C1300-bearing A/J mice significantly inhibited tumor growth for 12–36 days. Intravenous administration of 100 ng of toxin per day for 5 days in Me180- bearing nude mice and C1300-bearing A/J mice gave significant inhibition of tumor growth only during the treatment period, and 10 injections of the same dose of toxin had no significant effect on SK-MEL28, a tumor lacking GM2.

The preparation and properties of poly(vinyl benzoate) nanoparticle suspensions as molecular carriers are described for the first time. These nanoparticles can be formed by nanoprecipitation of commercial poly(vinyl benzoate) in water using Pluronic F68 as surfactant, to create spherical nanostructures measuring 200-250nm in diameter. These nanoparticles are stable in phosphate buffer and blood serum, and only slowly degrade in the presence of esterases. Pluronic F68 stabilizes the nanoparticle and also protects it from enzymatic degradation. Up to 1.6% by weight of a lipid-soluble molecule such as coumarin-6 can be introduced into the nanoparticle during nanoprecipitation, compared to a water-soluble compound (5(6)-carboxyfluorescein) which gave almost no loading. Kinetics experiments in phosphate buffer indicate that 78% of the coumarin-6 was encapsulated within the polymer matrix of the nanoparticle, and the residual 22% of coumarin-6 was surface-bound and quickly released. The nanoparticles are non-toxic in vitro towards human epithelial cells (IC(50)>1000μg/mL) and primary bovine aortic endothelial cells (IC(50)>500μg/mL), and non-bactericidal against a selection of representative test microbes (MIC >250μg/mL). These properties suggest that the poly(vinyl benzoate) nanoparticles may be suitable carriers for molecular delivery of lipophilic small molecules such as pharmaceutical and imaging agents.

We have developed a new 3D porous and biostable collagen scaffold for implantable glucose sensors. The scaffolds were fabricated around the sensors and crosslinked using nordihydroguaiaretic acid (NDGA) or glutaraldehyde (GA) to enhance physical and biological stability. The effect of the scaffolds on sensor function and biocompatibility was examined during long-term (≥28 days) in vitro and in vivo experiments and compared with control bare sensors. To evaluate the effect of the sensor length on micromotion and sensor function, we also fabricated short and long sensors. 3D porous scaffold application around glucose sensors did not significantly affect the long-term in vitro sensitivity of the sensors. The scaffolds, crosslinked by either NDGA or GA, remained stable around the sensors during the 4 week in vitro study. In the long-term in vivo study, the sensitivity of the short sensors was higher than the sensitivity of long sensors presumably because of less micromotion in the subcutis of the rats. The sensors with NDGA-crosslinked scaffolds had a higher sensitivity than the sensors with GA-crosslinked scaffolds. Histological examination showed that NDGA-crosslinked scaffolds retained their physical structure with reduced inflammation when compared with the GA-crosslinked scaffolds. Therefore, the application of NDGA-crosslinked collagen scaffolds might be a good method for enhancing the function and lifetime of implantable biosensors by minimizing the in vivo foreign body response. © 2009 Wiley Periodicals, Inc. J Biomed Mater Res, 2010

We have developed a new dexamethasone (Dex)-loaded poly(lactic-co-glycolic acid) microspheres/porous collagen scaffold composite for implantable glucose sensors. The scaffolds were fabricated around the sensing element of the sensors and crosslinked using nordihydroguaiaretic acid (NDGA). The microspheres containing Dex were incorporated into the NDGA-crosslinked collagen scaffold by dipping in microsphere suspension in either water or Pluronic. The loading efficiencies of Dex in the microspheres and the scaffold were determined using high performance liquid chromatography. The microspheres/scaffold composite fabricated using microspheres in the hydrogel solution had a better loading efficiency than when microspheres were in water suspension. The composite fabricated using the hydrogel also showed a slower and more sustained drug release than the standard microspheres in vitro during a 4 week study and did not significantly affect the function of the sensors in vitro. The sensors with the composite that were still functional retained above 50% of their original sensitivity at 2 weeks. Histology showed that the inflammatory response to the Dex-loaded composite was much lower than for the control scaffold at 2 and 4 weeks after implantation. The Dex-loaded composite system might be useful to reduce inflammation to implanted glucose sensors and therefore extend their function and lifetime.

The long-term performance stability of minimally invasive glucose biosensors was evaluated in vitro and in vivo.Coil-type glucose biosensors were constructed using an epoxy-polyurethane membrane. Seven sensors were continuously polarized for 12 weeks in a 5 mM glucose-phosphate-buffered saline (PBS) solution, and the sensor sensitivities were tested weekly. Glucose biosensors (n = 44) were also subcutaneously implanted in rats, and the in vivo sensitivities were determined for up to 4 weeks. Histological analysis was performed on the tissue surrounding the sensors.During a period of 12 weeks, the normalized sensitivity (S/S(0)) of the sensors tested in vitro first increased from 1.10 +/- 0.13 (week 1) to 2.30 +/- 0.90 at week 6 and then decreased to 1.07 +/- 0.24 at week 12 (n = 7). After 6 weeks, the sensors showed a much more significant response to acetaminophen. With continuous polarization in 5 mM glucose-PBS, the sensor functioned for at least 3 months, or about a half of the observed lifetime of sensors stored in the solution with occasional sensitivity measurements (e.g., tested twice each month). For the 15 implanted sensors that lasted for at least 28 days, the average sensitivities values were 4.4 +/- 2.0 (S(0), in vitro), 3.5 +/- 1.3 (day 7, in vivo), 3.3 +/- 1.1 (day 14), 3.6 +/- 1.4 (day 21), and 2.9 +/- 2.2 nA/mM (day 28). Histological analysis showed that the implanted sensors were covered by a 200-800-mu-thick fibrous capsule after 1 week. Blood vessels were found in the fibrous tissue from day 7 through day 34. In addition, the background current that was observed during in vivo sensor testing could be successfully eliminated by using an enzyme-free sensor.This study confirms that coil-type glucose biosensors based on an epoxy-polyurethane membrane can perform stably in vitro for months and in vivo for weeks.

Perlecan is a heparan sulfate proteoglycan present in the growth plate and essential for endochondral ossification. We evaluated the synthesis and structure of perlecan in the different zones of the growth plate. The growth plates from fetal bovine ribs were isolated and sequentially sliced into 1-mm sections containing the hypertrophic zone, lower proliferative zone, upper proliferative zone, intermediate zone, and resting zone, respectively. The slices were then either incubated in culture medium with 35SO4 to measure total sulfated proteoglycan synthesis and perlecan synthesis, extracted for perlecan core protein analysis by Western blot, or extracted for perlecan isolation and subsequent characterization of glycosaminoglycan size and disaccharide composition. 35SO4 incorporation into perlecan was three–fourfold higher in the proliferating/hypertrophic zone than the resting zone. Western blot showed perlecan content was greatest in the lower and upper proliferating zones and that a perlecan fragment lacking portions of the N- and C-terminal domains containing heparan sulfate was also present in all zones. Purified perlecan from the hypertrophic/lower proliferative zone had larger chondroitin sulfate chains and a different composition of CS and HS disaccharides than the perlecan isolated from the resting zone. These results indicate perlecan deposition is increased and is turned over during proliferation to be replaced by a perlecan with a different sulfation pattern. © 2006 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 24:1317–1326, 2006

FGF-2 is a regulator of chondrocyte proliferation in the developing growth plate and has been shown to bind to perlecan, a heparan sulfate proteoglycan. We evaluated the effect of perlecan isolated from the growth plate on the binding of FGF-2 to its low and high affinity receptors on resting and proliferating chondrocytes. Chondrocytes were isolated by pronase/collagenase digestion of 1 mm thick slices from the resting and proliferating zones of fetal bovine ribs and were plated in serum-free DMEM. Chondrocytes maintained their zone-specific level of DNA and matrix synthesis over a two-day culture period. The collagen, aggrecan, and perlecan components of the matrix produced were associated with the cell layer and were secreted into the medium. Most of the perlecan made by the chondrocytes was secreted into the medium. Western blots showed medium perlecan to contain two high molecular weight core proteins and overlay assays showed only the large core protein bound FGF-2. Cell layer perlecan contained only the smaller core protein. Immunoprecipitation assays of media showed that the medium perlecan bound (125)I-FGF-2, that the bound FGF-2 was eluted from perlecan by 2 M NaCl at pH 7.4, and that this binding was eliminated by prior digestion with heparatinase. This indicates that the perlecan secreted into the medium is a low affinity receptor for FGF-2. (125)I-FGF-2 also bound to the chondrocytes in cell culture. Competition studies showed exogenous FGF-2 reduced (125)I-FGF-2 binding to high affinity receptor but not the low affinity receptor in the cell layer. Exogenous perlecan, however, reduced (125)I-FGF-2 binding to both the low and the high affinity receptors in the cell layer by approximately 60%. The results suggest that perlecan made by growth plate chondrocytes is a low affinity receptor for FGF-2 and acts to sequester FGF-2 away from the high affinity receptor.

Heparan sulfate proteoglycans are thought to mediate the action of growth factors. The heparan sulfate-containing proteoglycans in extracts of the bovine fetal rib growth plate were detected using the monoclonal antibody 3G10, which recognizes a neoepitope generated by heparitinase digestion (David, G., Bai, X. M., Van der Schueren, B., Cassiman, J. J., and Van den Berghe, H. (1992) J. Cell Biol. 119, 961–975). The heparan sulfate proteoglycans that react with this antibody were identified using antisera to known proteoglycans; purified using CsCl density gradient centrifugation, molecular sieve, and ion exchange chromatography; and then characterized. The major heparan sulfate proteoglycans in the growth plate had core proteins of 200 kDa and larger and were identified as perlecan and aggrecan. These two heparan sulfate proteoglycans could be effectively separated from each other by CsCl density gradient centrifugation alone. Perlecan contained 25% heparan sulfate and 75% chondroitin sulfate. The heparan sulfate chains on growth plate perlecan were considerably smaller than the chondroitin sulfate chains, and the heparan sulfate disaccharide content was different than that found for heparan sulfate from either kidney, tumor tissue, or growth plate aggrecan. Aggrecan contained only 0.1% heparan sulfate, which was localized to the CS-1 domain of aggrecan. These results indicate that perlecan and aggrecan would be the principal candidate proteoglycans involved in the action of heparan sulfate-binding proteins in the developing growth plate.

Many tissues contain glycoproteins and proteoglycans, which are substituted with N-or O-linked keratan sulfate, a glycosaminoglycan in which the lactosamine (-galbeta1,4glcNAc-) disaccharide backbone is variably modified by sulfation, fucosylation, and sialylation. We report here a rapid, sensitive, and quantitative procedure for obtaining a complete disaccharide compositional analyses for keratan sulfates after FACE separation of products generated by hydrolysis of the glycosaminoglycans with B. fragillis keratanase II and E. freundii endo-beta-galactosidase. Seven digestion end products are separable in a single electrophoretic step using Monosaccharide composition gels. These are: the unsulfated disaccharide, glcNAcbeta1,3gal, the fucosylated trisaccharide, galbeta1,2[fucalpha1,3]glcNAc6S, the mono- and disulfated disaccharides, galbeta1,4glcNAc6S or gal6Sbeta1,4glcNAc6S from the chain interior, and the sialylated mono- and disulfated trisaccharides neuAalpha2,3galbeta1,4glcNAc6S or neuAalpha2,3gal6Sbeta1,4glcNAc6S from the nonreducing terminus. FACE analyses also revealed the presence of a contaminant beta-galactosidase activity in keratanase II enzyme preparations which cleaves the disaccharide, galbeta1,4glcNAc6S to its constituent monosaccharides, gal and glcNAc6S. It was particularly prominent at enzyme concentrations > 2 mU per nmole substrate glcNH(2) or after prolonged digestion times (> 12 h), and was not inhibitable by thiogalactosides or N-acetyl-lactosamine. As these monosaccharide products would not be detectable using the commonly described analytical methods for KS hydrolase products, such as (1)H-NMR and HPLC analyses, our data illustrate that the FACE procedure represents an improved approach for accurate compositional microanalyses of corneal and skeletal keratan sulfates, especially applicable to experimentation involving small amounts (1-2 microg) of this glycosaminoglycan.

The content and fine structure of keratan and chondroitin/dermatan sulfate in normal human corneas and corneas affected by macular corneal dystrophies (MCD) types I and II were examined by fluorophore-assisted carbohydrate electrophoresis. Normal tissues (n = 11) contained 15 microg of keratan sulfate and 8 microg of chondroitin/dermatan sulfate per mg dry weight. Keratan sulfates consisted of approximately 4% unsulfated, 42% monosulfated, and 54% disulfated disaccharides with number of average chain lengths of approximately 14 disaccharides. Chondroitin/dermatan sulfates were significantly longer, approximately 40 disaccharides per chain, and consisted of approximately 64% unsulfated, 28% 4-sulfated, and 8% 6-sulfated disaccharides. The fine structural parameters were altered in all diseased tissues. Keratan sulfate chain size was reduced to 3-4 disaccharides; chain sulfation was absent in MCD type I corneas and cartilages, and sulfation of both GlcNAc and Gal was significantly reduced in MCD type II. Chondroitin/dermatan sulfate chain sizes were also decreased in all diseased corneas to approximately 15 disaccharides, and the contents of 4- and 6-sulfated disaccharides were proportionally increased. Tissue concentrations (nanomole of chains per mg dry weight) of all glycosaminoglycan types were affected in the disease types. Keratan sulfate chain concentrations were reduced by approximately 24 and approximately 75% in type I corneas and cartilages, respectively, and by approximately 50% in type II corneas. Conversely, chondroitin/dermatan sulfate chain concentrations were increased by 60-70% in types I and II corneas. Such changes imply a modified tissue content of individual proteoglycans and/or an altered efficiency of chain substitution on the core proteins. Together with the finding that hyaluronan, not normally present in healthy adult corneas, was also detected in both disease subtypes, the data support the conclusion that a wide range of keratocyte-specific proteoglycan and glycosaminoglycan remodeling processes are activated during degeneration of the stromal matrix in the macular corneal dystrophies.

We have previously shown that the in vivo infusion of interleukin-8 (IL8) in rats causes albuminuria and an increased catabolism of the heparan sulfate (HS) glycosaminoglycans (GAG). The purpose of this study was to characterize further the in vivo effect of IL8 on rat HSGAG. The left renal artery of male rats was continuously infused with IL8 (750 ng/ml, 4 rats) or 1% bovine serum albumin (3 rats) using an osmotic pump. On the 5th day of infusion, 35sulfate (1.0 mCi/100 g) was given intraperitoneally and the rat was killed 8 h later. Glomerular HSGAG was isolated and its size and charge was determined by liquid chromatography. The HSGAG 35sulfate uptake was higher in IL8-treated rats than controls (387+/-138 vs. 246+/-15 cpm/1,000 glomeruli, mean +/- SD, P0.05). No differences in HSGAG size were observed between IL8-treated and control animals. However, after ion exchange chromatography, there was a shift to the left in the elution profile of IL8-treated rats, demonstrating the presence of chains with decreased negative charge. IL8 infusion into rats results in a decreased total negative charge of the HSGAG. Because IL8 has been found in the serum of some patients with minimal lesion nephrotic syndrome, this finding may explain, at least in part, the reported reduced glomerular charge barrier observed in these patients.

Introduction Heparin sulfate (HS) has long been implicated in the bone deformity hereditary multiple exostoses (HME), and it is now clear that HME is associated with mutations in the HS biosynthetic genes EXT1 and EXT2. Interestingly, HME is also associated with an increased risk of chondro- and osteo-sarcomas. Methods and results Preliminary analysis of GAG samples purified from fibroblasts of both HME and isolated non-HME exostoses patients reveal a dramatic shift in the ratio of CS : HS, with the HME and isolated cases having a much higher proportion of CS relative to normal controls. This is true in the case of both shed and cell surface material but is far more extreme in the latter, with the HS reducing from approximately 45% in the controls to less than 10% in HME patients. Initial analysis also reveals shortened chain length within these samples; indeed they often have two populations of chains present. Simple analysis of the total disaccharide composition of these samples demonstrates no significant differences against controls. However, detailed analysis of the subpopulations of chains (as determined by chain length) within these samples as well as cartilaginous samples from exostoses patients may provide further insight into the changes that occur within the biosynthetic pathway following disrupted EXT function. We are also carrying out immunocytochemistry with a variety of HS-specific antibodies with the aim to further investigate normal HS structure and localization. This is being carried out on human primary chondrocytes isolated from normal patients and also adult mesenchymal stem cells as they undergo differentiation into chondrocytes. HS has been identified in both these cell types, and it is hoped that the manipulation of these cells through RNAi of different enzymes of the HS biosynthetic pathway will provide a suitable model for studying what changes may occur in cellular HS structures over the initial differentiation process in the growth plate. Discussion Together, these investigations should provide a good model to allow us to determine the role of HS in chondrocyte differentiation and maturation in both normal and diseased states.

We report a new class of peptide mimetics, α-AApeptides, that display broad-spectrum activity against both Gram-negative and Gram-positive bacteria and fungi. With non-hemolytic activity, resistance to protease hydrolysis, and easy sequence programmability, α-AApeptides may emerge as a novel class of antibiotics.

There is considerable interest in using DNA nanowires or nanotubes in a wide variety of bioelectronic applications and microcircuitry. Various methods have been developed to construct DNA nanostructures. Here, we report a novel method to construct semiconducting DNA nanowires by applying a suitable DC bias to a gold plating solution containing double-stranded DNA. The self-assembled nanowires fabricated by this method contain attached gold nanoparticles. Further, we report that the dimensions of the nanowires can be easily manipulated by altering the applied DC bias. We also confirmed the semiconducting nature of the DNA nanowires by studying their resistance-temperature behavior from 25 to 65 degrees C in a microelectrode system. These studies describe a simple process by which gold-decorated, semiconducting DNA nanowires could be created and may lead to a breakthrough in the field of self-assembly of nanometer-scale circuits. The self-assembled structures do have some similarity with tube-like structures but in the present work we are using the term 'DNA nanowires' to define the structures.

The present studies were conducted to determine whether luteinizing hormone (LH), a hormone which increases intracellular cAMP, also increases "second messengers" derived from inositol phospholipid hydrolysis in isolated bovine luteal cells. In luteal cells prelabeled with 32PO4, LH provoked increases in labeling of phosphatidic acid, phosphatidylinositol, and polyphosphatidylinositol (PIP). No reductions in 32P-prelabeled PIP and PIP2 were observed in LH-treated cells. In luteal cells prelabeled with myo-[2-3H]inositol, LH provoked rapid (10-30 s) and sustained (up to 60 min) increases in the levels of inositol mono-, bis-, and trisphosphates (IP, IP2, and IP3, respectively. IP3 was formed more rapidly than IP2 or IP following LH treatment. In addition, LH increased (50%) levels of [3H]inositol phospholipids in 30-min incubations. LiCl (10 mM) enhanced inositol phosphate accumulation in response to LH. Maximal increases in IP3 occurred at 1-10 micrograms/ml of LH. Similar temporal and dose-response relationships were observed for LH-stimulated IP3 and cAMP accumulation. However, exogenous cAMP (8-bromo-cAMP, 5 mM) and forskolin (10 microM) had no effect on inositol phosphate synthesis. The initial (1 min) effects of LH on IP3 and cAMP were independent of extracellular calcium concentrations, whereas the sustained (5 min) effect of LH on IP3, but not cAMP, was dependent on a source of extracellular calcium. LH-stimulated progesterone synthesis was also dependent on the presence of extracellular calcium. LH induced rapid and concentration-dependent increases in [Ca2+]i as measured by Quin 2 fluorescence. The LH-induced increases in [Ca2+]i were maximal within 30 s (approximately 2-fold) and remained elevated for at least 10 min. In Ca2+-free media containing 2 mM [ethylenebis(oxyethylenenitrilo)]tetraacetic acid, LH was still able to increase [Ca2+]i, but the increase was slightly less in magnitude and of shorter duration (2-4 min). These findings demonstrate that LH can rapidly raise levels of IP3 and [Ca2+]i, as well as, cAMP in bovine luteal cells. These findings suggest that at least two second messenger systems exist to mediate the action of LH in the corpus luteum.

ANF did not prevent the formation of [3H] inositol trisphosphate in response to All but inhibited aldosterone secretion in calf adrenal glomerulosa cells. 8-bromo cGMP did not affect either inositol phosphate formation or aldosterone secretion. Changes in cytosolic Ca++ concentration induced by All, as measured by Quin 2 fluorescence, were also unaffected by ANF. No difference in adrenal cell protein phosphorylation with All or All+ANF was observed. The results suggest that ANF may inhibit aldosterone secretion through a non-guanyl cyclase linked receptor system not involving the formation of phosphoinositide-derived second messengers. Interference with protein kinase C activity cannot be ruled out.

The present studies were conducted to determine the effects of gonadotropins (LH and hCG) and prostaglandin F2a (PGF2a) on the production of "second messengers" and progesterone synthesis in purified preparations of bovine small luteal cells. Corpora lutea were removed from heifers during the luteal phase of the normal estrous cycle. Small luteal cells were isolated by unit-gravity sedimentation and were 95-99% pure. LH provoked rapid and sustained increases in the levels of [3H]inositol mono-, bis-, and trisphosphates (IP, IP2, IP3, respectively), cAMP and progesterone in small luteal cells. LiCl (10 mM) enhanced inositol phosphate accumulation in response to LH but had no effect on LH-stimulated cAMP or progesterone accumulation. Time course studies revealed that LH-induced increases in IP3 and cAMP occurred simultaneously and preceded the increases in progesterone secretion. Similar dose-response relationships were observed for inositol phosphate and cAMP accumulation with maximal increases observed with 1-10 micrograms/ml of LH. Progesterone accumulation was maximal at 1-10 ng/ml of LH. LH (1 microgram/ml) and hCG (20 IU/ml) provoked similar increases in inositol phosphate, cAMP and progesterone accumulation in small luteal cells. 8-Bromo-cAMP (2.5 mM) and forskolin (1 microM) increased progesterone synthesis but did not increase inositol phosphate accumulation in 30 min incubations. PGF2a (1 microM) was more effective than LH (1 microgram/ml) at stimulating increases in inositol phosphate accumulation (4.4-fold vs 2.2-fold increase for PGF2a and LH, respectively). The combined effects of LH and PGF2a on accumulation of inositol phosphates were slightly greater than the effects of PGF2a alone. In 30 min incubations, PGF2a had no effect on cAMP accumulation and provoked small increases in progesterone secretion. Additionally, PGF2a treatment had no significant effect on LH-induced cAMP or progesterone accumulation in 30 min incubations of small luteal cells. These findings provide the first evidence that gonadotropins stimulate the cAMP and IP3-diacylglycerol transmembrane signalling systems in bovine small luteal cells. PGF2a stimulated phospholipase C activity in small cells but did not reduce LH-stimulated cAMP or progesterone accumulation. These results also demonstrate that induction of functional luteolysis in vitro requires more than the activation of the phospholipase C-IP3/calcium and -diacylglycerol/protein kinase C transmembrane signalling system.

Rat granulosa cells isolated from mature Graafian follicles were incubated with luteinizing hormone under various conditions in order to follow the synthesis and degradation of phospholipids. During acute incubations, luteinizing hormone provoked rapid and concentration-dependent increases in the incorporation of 32PO4 into phosphatidic acid, phosphatidylinositol, and the polyphosphoinositides. Similarly, luteinizing hormone provoked increases in labeling of phosphatidylinositol and the polyphosphoinositides when granulosa cells were incubated with myo-[2-3H]inositol. When granulosa cells were prelabeled with 32PO4 in order to label phosphatidylinositol to constant specific radioactivity (4 h), luteinizing hormone treatment significantly increased 32P-phosphatidylinositol levels (23%). Comparable increases (27%) in the cellular concentrations of phosphatidylinositol were observed in response to luteinizing hormone. In pulse-chase experiments employing 32PO4 - or [3H]inositol-prelabeled cells, luteinizing hormone did not alter phospholipid degradation. In addition, luteinizing hormone did not stimulate degradation of polyphosphoinositides. These results demonstrate that: (a) luteinizing hormone has selective effects on phospholipid metabolism in rat granulosa cells which involve phosphatidic acid, phosphatidylinositol, and the polyphosphoinositides, (b) luteinizing hormone increases net levels of phosphatidylinositol and presumably phosphatidic acid and the polyphosphoinositides, and (c) luteinizing hormone does not increase phospholipid degradation. Our findings suggest that luteinizing hormone provokes increases in de novo synthesis of phosphatidylinositol in rat granulosa cells. These changes in phospholipid metabolism may be important for steroidogenesis and other enzymatic processes during treatment with luteinizing hormone.

The present studies were conducted to determine whether prostaglandin F2 alpha (PGF2 alpha) stimulates the production of "second messengers" derived from inositol phospholipid hydrolysis and increases intracellular free Ca2+ ([Ca2+]i) in isolated bovine luteal cells. PGF2 alpha provoked rapid (10 sec) and sustained (up to 60 min) increases in the levels of inositol mono-, bis-, and trisphosphates (InsP, InsP2, and InsP3, respectively). InsP3 was formed more rapidly than InsP2 or InsP after PGF2 alpha treatment. In addition, PGF2 alpha increased inositol phospholipid turnover, as evidenced by increased 32PO4 incorporation into phosphatidic acid and phosphatidylinositol. LiCl (1-20 mM) enhanced inositol phosphate accumulation in response to PGF2 alpha. Maximal increases in InsP3 occurred at 1 microM PGF2 alpha, with half-maximal stimulation occurring at 36 nM. The acute effects of PGF2 alpha on InsP3 levels were independent of reductions in extracellular calcium. Prostaglandins E1 and E2 also stimulated increases in inositol phosphate levels, albeit to a lesser extent. PGF2 alpha also induced rapid and concentration-dependent increases in [Ca2+]i as measured by quin-2 fluorescence. The PGF2 alpha-induced increases in [Ca2+]i were maximal within 30 sec (approximately 2- to 3-fold), and [Ca2+]i remained elevated for 8-10 min. The PGF2 alpha-induced increases in [Ca2+]i were also independent of extracellular calcium. These findings demonstrate that the action of PGF2 alpha is coupled to the phospholipase C-InsP3 and diacylglycerol second messenger system in the corpus luteum.

Fetal rat retina was excised from donor rats at 15 days of gestation and transplanted to the occipital cortex of neonatal host rats in combination with and adjacent to: 1) the appropriate portion of the superior colliculus to serve as a specific target tissue in an attempt to stimulate an outgrowth of optic fibers from the isolated retinal transplant; 2) a sample of the medial thalamus to provide a alternate tissue not normally recipient to the retinofugal projection, thereby serving as a control to further test for the target specificity of the axonal outgrowth from the retinal transplant. After 5–30 days of development samples of transplant and surrounding host cortex were removed and subjected to light and electron microscopic study.

The effects of human chorionic gonadotropin (hCG) and prostaglandin F2 alpha (PGF2 alpha) on the adenylate cyclase-cAMP and inositol phospholipid-phospholipase C-inositol trisphosphate and diacylglycerol transmembrane signalling systems were evaluated in cultured human granulosa-luteal cells. Granulosa-luteal cells obtained from patients undergoing in vitro fertilization were cultured for 72 h prior to addition of hormones. During the last 24 h of culture granulosa-luteal cells were incubated with [3H]inositol. Neither hCG nor gonadotropin-releasing hormone (GnRH) stimulated the inositol phospholipid-phospholipase C signalling system. PGF2 alpha stimulated increases in inositol mono-, bis-, and trisphosphate accumulation in 30 min incubations. NaF (20 mM) mimicked the stimulatory effect of PGF2 alpha on inositol phosphate accumulation suggesting the involvement of a guanine nucleotide regulatory protein in the activation of phospholipase C. In contrast, hCG but not PGF2 alpha or NaF stimulated cAMP accumulation in 30 min incubations. Simultaneous treatment with hCG and PGF2 alpha did not alter the stimulatory effect of PGF2 alpha on inositol phosphate accumulation but reduced (37%) the stimulatory effect of hCG on cAMP accumulation. The protein kinase C activator, 12-O-tetradecanoylphorbol 13-acetate (TPA) inhibited the stimulatory effects of hCG (76%) and PGF2 alpha (62%) on cAMP and inositol phosphate accumulation, respectively. Thus, cultures of human granulosa-luteal cells possess multiple transmembrane signalling systems which may be modulated by the activation of protein kinase C.

Human chorionic gonadotropin, hCG, a hormone which increases intracellular cAMP, provoked rapid (30 s) and sustained (up to 30 min) increases in the levels of inositol mono-, bis- and trisphosphates (IP, IP 2 and IP 3 , respectively) in bovine luteal cells. LiCl (10 mM) enhanced inositol phosphate accumulation in response to hCG. Concentration-dependent increases in inositol phosphates, cAMP and progesterone accumulation were observed in hCG-treated luteal cells. hCG also induced rapid and concentration-dependent increases in cytosolic free Ca 2+ as measured by quin 2 fluorescence. These findings demonstrate that hCG stimulates the phospholipase C-IP 3 and diacylglycerol ‘second messenger’ system in the bovine corpus luteum.

Luteinizing hormone (LH) stimulates the formation of adenosine 3',5'-cyclic monophosphate (cAMP) and inositol trisphosphate (IP3) in rat granulosa cells. This report describes the effects of protein kinase C activators on second messenger generation in isolated rat granulosa cells. The protein kinase C activator 12-O-tetradecanoylphorbol 13-acetate (TPA) completely inhibited LH-stimulated inositol phosphate accumulation. The inhibitory effects of TPA were rapid (5-15 min) and concentration dependent with 50 nM TPA producing maximally inhibitory effects. However 30-min incubations with 10-100 nM TPA had no effect on LH-stimulated cAMP or progesterone levels. The inhibitory effect of TPA could not be overcome by high concentrations of LH. TPA also inhibited gonadotropin-releasing hormone-stimulated phospholipase C activity, although to a much lesser extent. Increased inositol phosphate degradation and reduced inositol phospholipid synthesis were unlikely explanations for the effects of TPA. The results indicate that phorbol esters modulate the inositol phospholipid-phospholipase C transmembrane signaling system in rat granulosa cells. The results suggest that phorbol esters may alter the coupling of the hormone receptor complex to phospholipase C.

Since the intracellular messengers of various proopiomelanocortin-derived peptides remain ambiguous at best, we have investigated the possible involvement of phosphoinositide metabolism in aldosterone secretion evoked by alpha-MSH, beta-LPH, as well as ACTH in rat and calf adrenal glomerulosa cells. We have also examined the cAMP responses in the adrenal glomerulosa cells to alpha-MSH comparing it with those of ACTH. Our results showed that neither alpha-MSH, beta-LPH, nor ACTH increased inositol triphosphate (IP3) or other inositol phosphates in adrenal glomerulosa cells while increasing aldosterone secretion from the same cells. Angiotensin II, known to cause hydrolysis of the phosphoinositides, increased IP3 in these adrenal cells in a dose-dependent manner. Both ACTH and alpha-MSH raised the cAMP levels in the calf adrenal glomerulosa cells, although the magnitude of the increase of cAMP in response to ACTH was greater. These findings suggest that IP3 as a mediator of alpha-MSH- and beta-LPH-induced aldosterone secretion is not likely and other mediator(s) may be involved.

Maintenance of the corpus luteum (CL) is dependent on a balance between a number of luteotropic and luteolytic factors (recently reviewed in references 1 and 2). Prostaglandins (PG) are important paracrine and endocrine factors which control CL function. Prostaglandins of the E and I series stimulate steroidogenesis in bovine luteal tissues (2–4), presumably via their ability to activate adenylate cyclase (4,5). Thus, their actions are analogous to the stimulatory actions of luteinizing hormone (LH) on adenylate cyclase and progesterone synthesis. Although PGF2α has a luteolytic effect in vivo, PGF2α also stimulates progesterone synthesis in incubations of cells (2) or slices (3,6) of bovine luteal tissue in vitro. This luteotropic action of PGF2α, however, does not involve the activation of adenylate cyclase (4) or an increase in cAMP (4,7) in bovine luteal tissue. Instead, PGF22α stimulates the inositol phospholipid-phospholipase C transmembrane signalling system (7–10). This system is coupled to increases in intracellular calcium and protein kinase C activation (8,10,11). Recent studies also show that LH activates the inositol phospholipid-phospholipase C signalling system in bovine luteal cells (12,13). The present studies were performed to examine whether the luteotropic prostaglandins of the E and I series also activate this transmembrane signalling system.

The sensitivity of rat corpora lutea to luteolytic agents increases with luteal age. We examined the effect of prostaglandin F2 alpha (PGF2 alpha) and [D-Ala6,Des-Gly10]GnRH ethylamide (GnRHa) on inositol phospholipid metabolism in day 2 and day 7 corpora lutea from PMSG-treated rats. Isolated corpora lutea were incubated with 32PO4 or [3H]inositol and were treated with LH, PGF2 alpha, or GnRHa. Phospholipids were purified by TLC, and the water-soluble products of phospholipase-C activity (inositol phosphates) were isolated by ion exchange chromatography. In day 2 corpora lutea, PGF2 alpha, (10 microM) and GnRHa (100 ng/ml) significantly increased 32PO4 incorporation into phosphatidic acid (PA) and phosphatidylinositol (PI), but not into other fractions. LH provoked slight increases in PA. Results were similar with 30 min of prelabeling or simultaneous addition of 32PO4 and stimulants. In other experiments, PGF2 alpha and GnRHa provoked rapid increases (1-5 min) in the accumulation of inositol mono-, bis-, and trisphosphates. LH did not significantly increase inositol phosphate accumulation, but stimulated cAMP accumulation in 2-day-old corpora lutea. Inositol phospholipid metabolism was increased in day 7 corpora lutea compared to that in day 2 corpora lutea. This increase was associated with increased incorporation of 32PO4 into PA and PI and increased accumulation of [3H]inositol phosphates. In day 7 corpora lutea, which are very sensitive to the luteolytic effect of PGF2 alpha, the PG-induced increase in PA labeling was small and inconsistent, whereas PI labeling was unaffected in 30-min incubations. GnRHa was without effect in such corpora lutea. LH, PGF2 alpha, or GnRHa did not increase inositol phosphate accumulation in 7-day-old corpora lutea. These studies demonstrate that the transformation of young (day 2) to mature (day 7) corpora lutea is associated with an increase in luteal inositol phospholipid metabolism. The results also show that PGF2 alpha and GnRHa stimulate phospholipase-C activity in young corpora lutea, but are ineffective in mature corpora lutea, and suggest that an increase in inositol phospholipid metabolism by itself is not sufficient to explain the acute luteolytic action of PGF2 alpha and GnRH in vitro. However, phospholipase-C-derived second messengers may be involved in the action of hormones that control luteal function.

The inositol phospholipid-phospholipase C transmembrane signalling system provides second messengers that regulate cellular differentiation as well as metabolic and secretory activities (reviewed in references 1,2). These cellular events are presumably mediated by increases in inositol 1,4,5-trisphosphate and diacylglycerol, the two immediate products of phosphatidylinositol 4,5-bisphosphate hydrolysis. Both products contribute to the cellular activity by regulating intracellular calcium mobilization and the activities of protein kinases.

This report describes the rapid effects of GnRH and an agonist [D-Ala6, des-Gly10] GnRH ethylamide (GnRHa) on polyphosphoinositide metabolism in rat granulosa cells. As indicated by the depletion of cellular levels of 32P-prelabeled triphosphoinositide (TPI) and diphosphoinositide (DPI), GnRHa rapidly stimulated the hydrolysis of TPI and DPI. The effect of GnRHa was maximal at the earliest time point examined (30 sec) and preceded GnRHa-induced increases in labeling of phosphatidylinositol. A specific GnRH antagonist had no effect on TPI or DPI levels, but prevented the polyphosphoinositide depletion induced by GnRH. LH did not stimulate depletion of 32P-polyphosphoinositides. The rapid and specific effects of GnRH on polyphosphoinositide depletion may represent an early and possibly initiating event in the action of GnRH.

The following studies were conducted to determine whether luteinizing hormone (LH), a hormone which increases cellular levels of cyclic AMP, also provokes increases in ‘second messengers’ derived from inositol lipid metabolism (i.e. inositol phosphates and diacylglycerol). Rat granulosa cells isolated from mature Graafian follicles were prelabelled for 3 h with myo-[2-3H]inositol. LH provoked rapid (5 min) and sustained (up to 60 min) increases in the levels of inositol mono-, bis, and trisphosphates (IP, IP2 and IP3, respectively). Time course studies revealed that IP3 was formed more rapidly than IP2 and IP following LH treatment. The response to LH was concentration-dependent with maximal increases at LH concentrations of 1 microgram/ml. LiCl (2-40 mM) enhanced the LH-provoked accumulation of all [3H]inositol phosphates, presumably by inhibiting the action of inositol phosphate phosphatases. The effectiveness of LH, however, was dependent on the concentration of lithium employed; maximal increases in IP were observed at 10 mM-LiCl, whereas maximal increases in IP2 and IP3 were observed at 20 mM- and 40 mM-LiCl, respectively. The stimulatory effects of LH on inositol phosphate and progesterone accumulation were also compared with changes in cyclic nucleotide levels. LH rapidly increased levels of inositol phosphates, progesterone and cyclic AMP, but transiently reduced levels of cyclic GMP. These results demonstrate that LH increases both cyclic AMP and inositol trisphosphate (and presumably diacylglycerol) in rat granulosa cells. Our findings suggest that two messenger systems exist to mediate the action of LH in granulosa cells.

The present studies were conducted to determine whether the large or small bovine luteal cell was the site for the stimulatory effect of prostaglandin F2 alpha (PGF) on phospholipase C-catalyzed inositol phospholipid hydrolysis. Corpora lutea were removed from heifers during the luteal phase of the normal estrous cycle. Small luteal cells were isolated by unit-gravity sedimentation and large luteal cells were isolated by flow cytometry using a Becton Dickson FACS 440 cell sorter. PGF provoked rapid (5-30 s) and sustained (up to 30 min) increases in the levels of inositol mono-, bis-, and trisphosphates (IP, IP2, IP3, respectively) in small luteal cells. IP3 was formed more rapidly than IP2 or IP following PGF treatment. The PGF-stimulated increase in IP3 was accompanied by a transient reduction in the levels of 3H-labeled phosphatidylinositol 4,5-bisphosphate. LiCl (10 mM) enhanced inositol phosphate accumulation in response to PGF. Maximal increases in inositol phosphate accumulation were observed with 1-10 microM PGF and half-maximal increases were observed with 60 nM PGF. PGF (1-10 microM) had no effect on cAMP levels but stimulated small increases in progesterone accumulation in 30 min incubations of small luteal cells. PGF also increased the accumulation of inositol phosphates in large luteal cells. The increases were apparent within 5 min of incubation (the earliest time examined) and further increases were observed in incubations lasting 30 min. PGF had no significant effect on cAMP or progesterone in 30 min incubations of large cells.(ABSTRACT TRUNCATED AT 250 WORDS)