Recombinant human IL-1β was obtained from R&D systems (Minneapolis, MN, USA). Anti-polyhistidine IgG was purchased from Santa Cruz Biotechnologies (Santa Cruz, CA, USA). Nitrate/nitrite colorimetric assay kits were purchased from Cayman Chemical (Ann Arbor, MI, USA). Restriction endonucleases and T4 DNA ligase were purchased from Promega (Madison, WI, USA). Pfu polymerase was obtained from Stratagene (La Jolla, CA, USA). TA-cloning vector was obtained from Invitrogen (Carlsbad, CA, USA). Oligonucleotides were synthesized from Gibco BRL custom primers (Carlsbad, CA, USA). Isopropyl-β-D-thiogalactoside (IPTG) was obtained from Duchefa (Haarlem, the Netherlands). Plasmid pET15b and Escherichia coli strain BL21 (DE3) were from Novagen (Madison, WI, USA). Ni2+-nitrilotriacetic acid Sepharose superflow was purchased from Qiagen (Hilden, Germany). A human Cu, Zn-SOD cDNA fragment was isolated using the PCR technique from the λZap human placenta cDNA library  and a monoclonal antibody against human Cu, Zn-SOD was produced in our laboratory. All other reagents were obtained from Sigma (St. Louis, MO, USA) unless specified otherwise.
Expression and purification of Tat-SOD
The pTat-SOD expression vector was constructed to express the basic domain (amino acids 49 to 57) of HIV-1 Tat as a fusion protein with Cu, Zn-SOD, as described previously . Briefly, two oligonucleotides were synthesized and annealed to generate a double-stranded oligonucleotide encoding nine amino acids from the basic domain of HIV-1 Tat. The sequences were: top strand, 5'-TAGGAAGAAGCGGAGACAGCGACGAAGAC-3'; and bottom strand, 5'-TCGAG-TCTTCGTCGCTGTCTCCGCTTCTTCC-3'. The double-stranded oligonucleotide was directly ligated into the NdeI-XhoI-digested pET15b in frame with the six histidine open-reading frames to generate the HisTat expression plasmid, pHisTat. Next, on the basis of the cDNA sequence of human Cu, Zn-SOD, two oligonucleotides were synthesized. The top strand, 5'-CTCGAGGCGACGAAGGCCGTGTGCGTG-3', contained a XhoI restriction site, and the bottom strand, 5'-GGATCCTTATTG-GGCGATCCCAATTAC-3', contained a BamHI restriction site. The reaction mixture was made up in a 50 μl siliconized reaction tube and heated at 94°C for 5 minutes. The program for PCR consisted of 30 cycles of denaturation at 94°C for 40 seconds, annealing at 54°C for 1 minute, and elongation at 70°C for 3 minutes, and the final extension at 72°C for 10 minutes. The PCR products were purified by preparative agarose gel electrophoresis. The purified products were ligated into a TA-cloning vector and then transformed into a competent cell. The bacterial cells (E. coli BL21) transformed with pTat-SOD were harvested and disrupted by sonication in 5 ml binding buffer (5 mM imidazole, 0.5 M NaCl, 20 mM Tris-HCl, pH 7.9) containing 6 M urea. After centrifugation, supernatant was immediately loaded onto a 2.5 ml Ni2+-nitrilotriacetic acid Sepharose column (Qiagen, Valencia, CA, USA). After the column was washed with 10 volumes of a binding buffer and 6 volumes of a washing buffer (60 mM imidazole, 0.5 M NaCl, 20 mM Tris-HCl, pH 7.9), the fusion protein was eluted with an elution buffer (1 M imidazole, 0.5 M NaCl, 20 mM Tris-HCl, pH 7.9). The fusion protein containing fractions were combined and the salts were removed using PD10 column chromatography. To enhance the transduction efficacy of Tat-SOD, copper ion recovery of overexpressed Tat-SOD was conducted as described previously . We purified six histidine residue-tagged SODs without an HIV-1 Tat PTD by using a SOD expression vector (pSOD) as a control SOD protein. pTat-green fluorescent protein (GFP) was also constructed to express the basic domain of HIV-1 Tat as a fusion with GFP as was described previously  and used as a control Tat protein.
Cartilage samples were obtained from the femoral condyle and tibial plateau of OA patients at the time of joint replacement surgery. All cartilage samples were procured after obtaining oral informed consent from the patients and institutional approval. Chondrocytes from articular cartilage were cultured in monolayer as described previously . After about seven days, confluent chondrocytes were split once, plated and these first passage adherent chondrocytes were used in subsequent experiments. For cartilage explant culture, full-thickness cartilage slices were obtained above the subchondral bone from a relatively lesion-free area of the femoral condyle of OA patients. Each slice was cut further and a piece of approximately 2 mm width by 5 mm length by full thickness was cultured in a 48-well culture plate in 200 μl per well of the same medium in which monolayer chondrocytes were cultured. Explants were incubated in the medium for three days before protein transduction to allow them to stabilize in in vitro conditions.
Transduction of Tat-SOD into chondrocytes
For the transduction of Tat-SOD protein into the monolayer cultured chondrocytes, cells were seeded at 5 × 105/well in six well plates. Then, the culture medium was replaced with 1 ml of fresh serum free DMEM containing various concentrations of fusion protein. For the transduction of Tat-SOD into the cartilage explant culture, the culture medium was replaced with 200 μl of fresh serum free DMEM containing various concentrations of fusion proteins. The transduction procedures were the same for control SOD and Tat-GFP proteins.
Western blot analysis
Monolayer cultured chondrocytes were extensively washed after protein transduction, and trypsinized. Cellular proteins were extracted in lysis buffer containing 50 mM sodium acetate, pH 5.8, 10% v/v SDS, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, and 1 μg/ml aprotinin at 4°C. Western blotting was performed as described previously , using a rabbit anti-polyhistidine IgG (Santa Cruz Biotechnologies; dilution 1:500), the epitope of which is specific for the polyhistidine domain of Tat-SOD and control SOD. For explant-cultured chondrocytes, tissues were milled in liquid nitrogen after protein transduction and extensive washing. Protein was extracted in 4 M guanidine hydrochloride buffer containing 50 mM sodium acetate, pH 5.8, 1 mM phenylmethylsulfonyl fluoride, and 1 μg/ml aprotinin at 4°C and dialyzed. Electrophoresis and western blotting procedures were the same as those for monolayer chondrocytes.
SOD enzyme assay
Proteins extracted from the monolayer chondrocytes were used for the analysis of dismutase activity of SOD. The inhibition of ferricytochrome c reduction by the xanthine/xanthine oxidase reaction was monitored, according to a method reported previously . The standard assay was performed in 3 ml of 50 mM potassium phosphate buffer at pH 7.8 containing 0.1 mM EDTA in a cuvette at 25°C. The reaction mixture contained 10 μM ferricytochrome c, 50 μM xanthine and sufficient xanthine oxidase to produce a rate of reduction of ferricytochrome c at 550 nm of 0.025 absorbance unit per minute. Under these defined conditions, the amount of superoxide dismutase required to inhibit the rate of reduction of ferricytochrome c by 50% is defined as 1 unit of activity.
Fluorescence analysis and immunohistochemistry
For direct detection of transduced SOD protein in monolayer cultured chondrocytes, purified Tat-SOD and control SOD were labeled with FITC using an EZ-Label FITC protein labeling kit according to the manufacturer's instructions (Pierce, Rockford, IL, USA). Chondrocytes were seeded on glass coverslips and treated with 3 μM Tat-SOD or control SOD proteins. After incubation for 1 hour at 37°C, the cells were washed extensively with PBS. The distribution of fluorescence in non-fixed cells was analyzed with a Carl Zeiss Axiophot fluorescence microscope (Oberkochen, Germany). Immunohistochemical studies were carried out to identify transduced Tat-SOD in cartilage explant cultures. The cartilage explants were fixed with 4% paraformaldehyde after protein transduction and extensive washing. After fixation, explants were stored in 30% sucrose in PBS, and then cut in a cryotome. After removal of non-specific immunoreactivity using 0.3% Triton X-100 and 10% normal rabbit serum in PBS, immunohistochemistry was performed using a mouse monoclonal anti-human Cu, Zn-SOD IgG (dilution 1:500). The presence and distribution of Cu, Zn-SOD were determined by a confocal scanning fluorescent microscope (LSM510, Zeiss).
The final products of NO in vivo are nitrite and nitrate, the sum of which can be used as an index of total NO production. Chondrocytes were transduced with Tat-SOD protein, control SOD protein or Tat-GFP protein. After transduction, cells were extensively washed, and fresh serum free DMEM with or without IL-1 (1 ng/ml) was replenished. Culture media were harvested after 24 hours, and then analyzed using a nitrate/nitrite colorimetric assay kit, as recommended by the manufacturer. Briefly, nitrate was converted to nitrite using nitrate reductase, and then the Griess reagents were added to form a deep-purple azo compound. Absorbance was measured at 540 nm using a plate reader to determine nitrite concentrations. The detection limit of the assay was 1 μM.
After transduction of monolayer chondrocytes, fresh serum free DMEM with or without IL-1 (1 ng/ml) was replenished. Total RNA was isolated from chondrocytes after 4 hours using the RNeasy Mini Kit (Qiagen, Valencia, CA, USA). Total RNA (200 ng) was reverse transcribed using the SuperScript™ First-strand synthesis system for reverse transcriptase (RT)-PCR (Invitrogen, Gaithersburg, MD, USA) with oligo(dT)20 primers. PCR amplification of cDNA aliquots was performed with the following sense and antisense primers (5'→3'): iNOS sense, GTG AGG ATC AAA AAC TGG GG; iNOS antisense, ACC TGC AGG TTG GAC CAC; glyceraldehyde 3-phosphate dehydrogenase (GAPDH) sense, CGA TGC TGG GCG TGA GTA C; and GAPDH antisense, CGT TCA GCT CAG GGA TGA CC. Reactions were processed through 25 cycles of 30 seconds of denaturation at 94°C, 1 minute of annealing at 55°C for GAPDH and 30 cycles of 45 seconds of denaturation at 95°C, 30 seconds of annealing at 55°C for iNOS, followed by 1 minute of elongation at 72°C. PCR conditions were chosen to be non-saturating in all cases. PCR products were run on a 1.5% agarose gel, stained with ethidium bromide and visualized using a UVP transilluminator(Ultraviolet Product, Upland, CA). The band densities were quantified using the National Institues of Health,(NIH) image program (Bethesda, MD).
Data are expressed as means ± standard deviations. Differences between treatment groups were tested by using the Mann-Whitney U test (GraphPad Prism, version 3, GraphPad Software, San Diego, CA, USA). Significance was established at the 95% confidence level (p < 0.05).