Investigation of several biomarkers associated with diabetic nephropathy.

OBJECTIVE: The aim of this study was to facilitate the systematic discovery of diagnostic biomarkers of diabetic nephropathy (DN). METHODS: 3 publicly available independent cohorts were got from Gene Expression Omnibus database. Gene expression array were used to screen for genome-wide relative significance (GWRS) and genome-wide global significance (GWGS). The most significant up- and down-regulated top 100 gene signatures were identified using a fold change based model. Then the protein-protein interaction (PPI) network was constructed, while the hub genes in this PPI network were identified by centrality analysis. Modules detection was performed to explore the functions of the modules. Meanwhile, gene enrichment analysis was performed to illuminate the biological pathways and processes associated with DN. RESULTS: The most significant up- and down-regulated top 100 gene signatures were identified and a PPI network was established. Several hub genes (VEGFA, IL8, MYC, CD14, ALB) were discovered. Several functional modules were revealed. Biological pathways including cytokine-cytokine receptor interaction and p53 signaling pathway, and processes including inflammatory response, response to wounding and enzyme linked receptor protein signaling pathway were identified. CONCLUSION: Our study displayed underlying biomarkers including biological pathways and several hub genes of DN.

Fungal peri-prosthetic joint infection after primary total knee replacement.

We retrospectively reviewed 30 two-stage revision procedures in 28 patients performed for fungal peri-prosthetic joint infection (PJI) after a primary total knee replacement. Patients were followed for at least two years or until the infection recurred. The mean follow-up for patients who remained free of infection was 4.3 years (2.3 to 6.1). Overall, 17 patients were assessed as American Society of Anesthesiologists grade 3 or 4. The surgical protocol included removal of the infected implant, vigorous debridement and insertion of an articulating cement spacer. This was followed by at least six weeks of antimicrobial treatment and delayed reimplantation in all patients. The mean interval between removal of the prosthesis and reimplantation was 9.5 weeks (6 to 24). After reimplantation, patients took antifungal agents orally for a maximum of six months. Two knees became reinfected at one and two months post-operatively, respectively: one of these subsequently required arthrodesis because of uncontrolled infection. Fungal PJIs can be treated successfully by removal of all infected material, appropriate antimicrobial treatment and delayed reimplantation.

A bone replaceable artificial bone substitute: osteoinduction by combining with bone inducing agent.

Bone inducing agent (BIA) isolated from Saos-2 human osteosarcoma cells was added to an artificial bone substitute composed of 980 degrees C-heated carbonate apatite (CAp) and Type I atelocollagen (AtCol) extracted from bovine tail skins (88/12 in wt/wt %), and a CAp-AtCol-BIA substitute was prepared as an osteoinductive bone substitute. Rat calvaria osteoblasts treated by the isolated BIA demonstrated significantly increased alkaline phosphatase (ALP) activity after 3 days (p < 0.05). In vitro cell attachment and proliferation and ALP activity were investigated for the bone substitute combined with BIA. Osteoblasts cultured onto the surface of the CAp-AtCol-BIA substitute demonstrated remarkable morphological changes such as radial spreading, flattening, and projecting filopodia after 5 days. In comparison with the substitute without BIA, osteoblasts grown in the BIA-combined CAp-AtCol substitute expressed significantly increased proliferation and ALP activity, respectively (p < 0.05). Both the substitutes combined with and without BIA were implanted into artificial defects created in rabbit radii. After 4 weeks, the CAp-AtCol-BIA substitute implanted lesion was completely replaced by regenerated host bone in radiological observation whereas the substitute without BIA was partially resorbed. No histologic abnormalities appeared in the substitute either with or without BIA.

A bone replaceable artificial bone substitute: cytotoxicity, cell adhesion, proliferation, and alkaline phosphatase activity.

Cellular toxicity, cell adhesion and proliferation, and alkaline phosphatase (ALP) activity were investigated for an artificial bone substitute composed of heated carbonate apatite (CAp) and Type I atelocollagen (AtCol) extracted from bovine tail skins (88/12 in %wt/wt). To enhance the intramolecular crosslinking between collagen molecules, the CAp-AtCol substitutes were irradiated by ultraviolet rays (wave length 254 nm) at 4 degrees C for 4 h or vacuum dried at 150 degrees C for 2 h. Cytotoxicity tests by a direct contact method and an extract dilution method revealed that the CAp-AtCol substitutes were cytocompatible for balb 3T3 fibroblasts. Osteoblast adhesion studies demonstrated that the substitute disks composed of 980 degrees C-heated CAp and AtCol were significantly more adhesive for osteoblasts than those of 1,200 degrees C-sintered CAp and AtCol (p < 0.05). Proliferation studies showed that the number of osteoblasts grown in the media containing substitutes of 980 degrees C-heated CAp and AtCol was statistically higher than grown in those of 1,200 degrees C-sintered CAp and AtCol after 5 days (p < 0.05). It was found that osteoblasts grown in the substitutes of 980 degrees C-heated CAp and AtCol only expressed similar ALP activity to the controls. These results suggested that the substitutes consisting of 980 degrees C-heated CAp and AtCol show more favorable interactions with osteoblasts than those of 1,200 degrees C-sintered CAp and AtCol.

Behavior of osteoblasts on a type I atelocollagen grafted ozone oxidized poly L-lactic acid membrane.

With oxidizing poly-L-lactic acid (PLLA) surface by ozone, peroxide groups are easily generated on the surface. Those peroxides are broken down by redox-coupling reaction, and provide active species that initiate grafting by reaction with the collagen molecules. The surface density of generated peroxide on a PLLA surface was determined by an iodide method. The maximum concentration of peroxide was about 2.87 x 10(-8) mol/cm2 when ozone oxidation was performed at 60 V for 60 min. After the surface oxidation, type I atelocollagen was grafted onto PLLA surface. All physical measurements on the collagen-grafted surface indicated that the PLLA surface was effectively grafted with type I atelocollagen. Behavior of rat calvaria osteoblasts on type I atelocollagen grafted PLLA (PLLA + COL) surface was observed. Initial attachment of osteoblasts on the surface was significantly enhanced, and it is assumed that the atelocollagen matrix supported the initial attachment and growth of cells. Collagenous protein synthesis of osteoblasts was maintained at relatively low level in the early stage of proliferation due to the primarily existing grafted type I atelocollagen, and then increased in 7 days as the osteoblast differentiated. After 7 days, collagenous protein synthesis in osteoblasts was activated. Alkaline phosphatase (ALPase) activity and mineralization by osteoblasts were promoted on PLLA + COL surface. In comparison with PLLA + COL, non-treated PLLA and tissue culture plate (TCPS) did not show any feature expressed in osteoblasts' maturation up to 9 days in this experiment. The grafted type I atelocollagen provided a favorable matrix for cell migration in relation with collagenase expression. Ozone oxidation might be a favorable method for surface modification of PLLA membranes by collagen grafting, and cell behavior could be modulated by the grafted collagen.

Inhibitor of striate conditionally suppresses cell proliferation in variegated maize.

Since the work done by R.A. Emerson in the 1930s, Inhibitor of striate (Isr) has been recognized as a dose-dependent genetic modifier of variegation in chlorotic leaf striping mutants of maize such as striate2 (sr2). We have shown that Isr specifically inhibits proliferation and differentiation of plastid defective cells in sr2 mutants. Leaf narrowing is due to loss of intermediate veins and ground tissue located at leaf margins, and the few remaining plastid defective cells are of irregular size and aberrant organization. The Isr gene has been cloned by targeted transposon tagging. Isr mRNA is expressed throughout young leaves, but Isr chimeras indicate that the expression of Isr at leaf margins is sufficient to suppress both the lateral expansion of sr2 leaves and the extent of striping. Isr protein appears to encode a chloroplast protein with sequence similarity to a family of bacterial phosphatases involved in carbon catabolite repression or in carbon metabolism. We propose that the action of Isr in nuclear and plastid communication could be triggered by carbon stress.

An overview of cartilage tissue engineering.

Articular cartilage regeneration refers to the formation of new tissue that is indistinguishable from the native articular cartilage with respect to zonal organization, biochemical composition, and mechanical properties. Due to a limited capacity to repair cartilage, scar tissue frequently has a poorly organized structure and lacks the functional characteristics of normal cartilage. The degree of success to date achieved using a purely cell- or biological-based approach has been modest. Potentially the development of a hybrid strategy, whereby, chondrocytes or chondrogenic stem cells are combined with a matrix, making cartilage in vitro, which is then subsequently transplanted, offers a route towards a new successful treatment modality. The success of this approach depends upon the material being biocompatible, processable into a suitable three-dimensional structure and eventually biodegradable without harmful effects. In addition, the material should have a sufficient porosity to facilitate high cell loading and tissue ingrowth, and it should be able to support cell proliferation, differentiation, and function. The cell-polymer-bioreactor system provides a basis for studying the structural and functional properties of the cartilaginous matrix during its development, because tissue concentrations of glycosaminoglycan and collagen can be modulated by altering the conditions of tissue cultivation.

The hemispherical porous acetabular component fixed by press-fit technique and additional screws.

Seventy consecutive primary total hip arthroplasties in which a Harris-Galante porous-coated acetabular component had been used from October, 1986 to December, 1989 were reviewed for 5 to 9 years follow-up. We performed the retrospective, clinical and radiological analysis of the hemispheric, porous-coated, cementless acetabular component using press-fit and screw fixation. No component had detectable migration or any other position change. No acetabular fractures and no screw problems were noted. Initial peripheral gaps were observed in 11 cases (16%). Twelve cases had a postoperative polar gap less than 0.5 mm which were stabilized at 2 years postoperatively. Radiolucent line was present in at least one zone in 53% of cases and occurred most frequently in zones 1 and 3. No continuous radiolucent line greater than 2 mm was seen in any zone and radiolucent lines were stabilized 2-3 years postoperatively. Twelve hips (17.1%) had osteolysis of the acetabulum. No acetabular component was revised because of aseptic loosening. Revision arthroplasties were performed in 9 hips during follow-up. We concluded that the hemispheric porous-coated, acetabular components using press-fit and screw fixation had good results with a 5 to 9-year follow-up.

Molecular analysis of rice plants harboring an Ac/Ds transposable element-mediated gene trapping system.

In rice, limited efforts have been made to identify genes by the use of insertional mutagens, especially heterologous transposons such as the maize Ac/Ds. We constructed Ac and gene trap Ds vectors and introduced them into the rice genome by Agrobacterium-mediated transformation. In this report, rice plants that contained single and simple insertions of T-DNA were analysed in order to evaluate the gene-tagging efficiency. The 3' end of Ds was examined for putative splicing donor sites. As observed in maize, three splice donor sites were identified at the 3' end of the Ds in rice. Nearly 80% of Ds elements were excised from the original T-DNA sites, when Ac cDNA was expressed under a CaMV 35S promoter. Repetitive ratoon culturing was performed to induce new transpositions of Ds in new plants derived from cuttings. About 30% of the plants carried at least one Ds which underwent secondary transposition in the later cultures. Eight per cent of transposed Ds elements expressed GUS in various tissues of rice panicles. With cloned DNA adjacent to Ds, the genomic complexities of the insertion sites were examined by Southern hybridization. Half of the Ds insertion sites showed simple hybridization patterns which could be easily utilized to locate the Ds. Our data demonstrate that the Ac/Ds-mediated gene trap system could prove an excellent tool for the analysis of functions of genes in rice. We discuss genetic strategies that could be employed in a large scale mutagenesis using a heterologous Ac/Ds family in rice.

Effect of polyethylene wear on osteolysis in cementless primary total hip arthroplasty: minimal 5-year follow-up study.

We measured polyethylene wear radiographically in 85 cementless primary total hip arthroplasties. All patients were followed up for at least 5 years (mean, 6.3 years). The mean extent of linear wear was 0.88 mm, and the mean rate of linear wear was 0.14 mm/y. The mean volumetric wear was 539.42 mm3, and the mean rate of volumetric wear was 87.69 mm3/y. Polyethylene wear was correlated inversely with the patients' age only and was not influenced by other clinical factors. Osteolysis developed in 34 femurs and in 11 acetabula. Radiographic loosening was found in 2 cases in the femur and none in the acetabulum. Polyethylene wear was significantly correlated with frequency and size of osteolytic lesions. Therefore, polyethylene wear was considered one of the major causes of osteolysis, but it was not directly associated with prosthesis loosening. To reduce the extent of polyethylene wear, the material characteristics of polyethylene should be improved, or more durable articulating materials should be developed.

Minimizing artifacts caused by metallic implants at MR imaging: experimental and clinical studies.

OBJECTIVE: The purpose of this study was to investigate the effect of metallic implant positioning on MR imaging artifacts, to determine the optimal imaging conditions for minimizing artifacts, and to show the usefulness of artifact-minimizing methods in imaging of the knee. MATERIALS AND METHODS: Using MR images of experimental phantoms (titanium alloy and stainless steel screws), we compared the magnitude of metal-induced artifacts for various pulse sequences, different imaging parameters for the fast spin-echo sequence, and different imaging parameters for several incremental angles between the long axis of the screw and the direction of the main magnetic field. In clinical MR imaging of knees with metallic implants (n = 19), we assessed geometric distortion of anatomic structures to compare the influence of different pulse sequences (n = 19), frequency-encoding directions (n = 7), and knee positions (n = 15). RESULTS: Titanium alloy screws consistently produced smaller artifacts than did stainless steel screws. In experimental MR studies, artifacts were reduced with fast spin-echo sequences, with a screw orientation as closely parallel to the main magnetic field as possible, and, particularly, with smaller voxels that correlated positively with artifact size (R2 = .88, p < .01). In clinical MR studies, fast spin-echo MR imaging obscured articular structures less than did spin-echo imaging (8/19 patients). In particular, the anterior-posterior frequency-encoding direction (3/7 patients) and the flexion position of the knee (5/15 patients) were effective in reducing artifacts. CONCLUSION: MR artifacts can be minimized by optimally positioning in the magnet subjects with metallic implants and by choosing fast spin-echo sequences with an anterior-posterior frequency-encoding direction and the smallest voxel size.

Radiographic parameters of acetabulum for dysplasia in Korean adults.

We analyzed plain radiographs of 591 normal adult hips of various parameters to evaluate the radiological characteristics of the hip joint including the center-edge (CE) angle, acetabular angle, acetabular depth, acetabular roof obliquity and roof angle, and also to verify the rate of acetabular dysplasia. The CE angle was negatively correlated with acetabular angle and acetabular obliquity, but it was positively correlated with acetabular depth and roof angle. The rate of acetabular dysplasia (CE angle < 20 degrees) was 1.8%. We concluded that the CE angle and acetabular angle are more useful parameters for the diagnosis of acetabular dysplasia because there was no significant difference with advancing age and gender, as well as relatively small standard deviations.

Late dissociation of the polyethylene liner from a modular acetabular metal shell after primary total hip arthroplasty--a report of five cases.

Modular designs of hip prostheses have become popular recently. Along with complications inherent in all hip arthroplasty systems, modular systems have the additional potential for dissociation of components. Five male patients underwent total hip arthroplasties, in which all of the acetabular components were Harris-Galante II porous acetabular cups. Many years after the operation, the polyethylene liners were dissociated without any previous trauma or dislocation of the femoral heads, these dissociations and dislodgements were managed with open reduction. This complication can be predicted from clinical symptoms and signs. Roentgenograms must be taken and carefully compared to previous roentgenograms. We postulated two causes for the dissociation. First, the polyethylene liner was not fixed securely within the acetabular metal shell at the time of operation. Second, the locking mechanism of the acetabular metal shell was not strong enough to firmly hold the polyethylene liner within the acetabular metal shell. It does warrant that certain precautions must be taken when implanting modular components. The locking mechanism of the harris-Galante II porous acetabular component is mechanically weak and fails easily, therefore its design must be improved in an attempt to prevent postoperative dissociation of the polyethylene liner.

A new class II rice chitinase, Rcht2, whose induction by fungal elicitor is abolished by protein phosphatase 1 and 2A inhibitor.

Among the four classes of chitinase, a class II chitinase had not yet been reported for rice. We have isolated and characterized a class II acidic chitinase, Rcht2, from rice (Oryza sativa L. cv. Cheongcheongbyeo). The protein consists of a single polypeptide chain of 261 amino acid residues and includes a putative signal sequence of 29 amino acids at its N-terminus. It has a calculated molecular mass of 27,642 Da and an isoelectric point of 5.56. The Rcht2 chitinase lacks the cysteine-rich and hinge domains in the N-terminal region of the protein, which is the criterion for its classification as a class II chitinase. Comparison of the genomic and the cDNA sequence revealed that the coding region of Rcht2 consist of three exons of 301, 112, and 370 bp separated by two introns of 89 and 984 bp. In suspension-cultured rice cells, the transcript level of Rcht2 was dramatically increased by treatment with both glycol chitin and fungal elicitor. The application of protein phosphatase 1 and 2A inhibitors, calyculin A and okadaic acid, effectively abolished the induction of Rcht2 in response to fungal elicitor. In contrast, the activation of Rcht2 transcript was not inhibited by both cycloheximide and protein kinase inhibitors. These results demonstrate that protein dephosphorylation events play a crucial role in the elicitor-mediated induction of Rcht2 in rice cells, while de novo protein synthesis is not required for induction.

Two hevein homologs isolated from the seed of Pharbitis nil L. exhibit potent antifungal activity.

Two antifungal peptides (Pn-AMP1 and Pn-AMP2) have been purified to homogeneity from seeds of Pharbitis nil. The amino acid sequences of Pn-AMP1 (41 amino acid0 residues) and Pn-AMP2 (40 amino acid residues) were identical except that Pn-AMP1 has an additional serine residue at the carboxyl-terminus. The molecular masses of Pn-AMP1 and Pn-AMP2 were confirmed as 4299.7 and 4213.2 Da, respectively. Both the Pn-AMPs were highly basic (pI 12.02) and had characteristics of cysteine/glycine rich chitin-binding domain. Pn-AMPs exhibited potent antifungal activity against both chitin-containing and non-chitin-containing fungi in the cell wall. Concentrations required for 50% inhibition of fungal growth were ranged from 3 to 26 micrograms/ml for Pn-AMP1 and from 0.6 to 75 micrograms/ml for Pn-AMP2. The Pn-AMPs penetrated very rapidly into fungal hyphae and localized at septum and hyphal tips of fungi, which caused burst of hyphal tips. Burst of hyphae resulted in disruption of the fungal membrane and leakage of the cytoplasmic materials. To our knowledge, Pn-AMPs are the first hevein-like proteins that show similar fungicidal effects as thionins do.

Postoperative blood salvage and reinfusion after total joint arthroplasty.

The purpose of this prospective study was to evaluate the safety of salvage and reinfusion of postoperative sanguineous wound drainage using the ConstaVac Blood Conservation System (Stryker, Kalamazoo, MI). A prospective analysis of 135 primary total hip and total knee arthroplasties was carried out. The collection time for reinfusion was limited to 6 hours, and suction pressure was kept to a minimum by using the lowest setting on the device. For all patients, no citrate-phosphate-dextrose anticoagulant was added to the reservoir. To evaluate the effect of reinfusion on hemostasis and the blood coagulation system, antithrombin III, fibrinogen, and D-dimer levels of 40 of 135 patients were measured before surgery and on the first and seventh days after the operation. The mean volume of reinfusion of postoperative drainage was 437 mL for the patients with total hip arthroplasties, 883 mL for those with total knee arthroplasties, and 1,713 mL for those with bilateral total knee arthroplasties. Ninety-nine of 135 patients underwent operations without homologous blood replacement. Transient chills with mild fever were seen in 2 patients during reinfusion. No complications related to air embolism, coagulopathy, renal failure, or sepsis were recognized in any of the patients. This study suggests that postoperative blood salvage and reinfusion appear to be safe and effective in patients undergoing primary total hip and knee arthroplasties.

Prevention of heterotopic bone formation after total hip arthroplasty using 600 rad in single dose in high risk patient.

Nineteen patients received single-dose exposure to 600 rad delivered within 48 hours of total hip arthroplasty (THA) with shielding of the prosthesis region for the prevention of heterotopic ossification. The patients were considered at high risk for developing heterotopic ossification (HO) because of hypertropic osteoarthritis, post-traumatic osteoarthritis or the presence of previously-formed ectopic bone. The average follow-up period was 42 months (range, 37 months-48 months). At a follow-up study, all hips except one were classified as Brooker class 0. The single exception was classified as class I. All patients were asymptomatic at the last follow-up study and no component demonstrated subsidence or radiolucent line indicative of loosening. The authors concluded that 600 rad, single-fraction radiation therapy is cost effective, convenient and safe for the prevention of heterotopic ossification after total hip arthroplasty.

Transforming growth factor-beta 1 responsiveness of human articular chondrocytes in vitro: normal versus osteoarthritis.

The transforming growth factor-beta 1 was known as having the most important influence on chondrocytes among various growth factors, being abundant in articular chondrocytes and osteocytes. We performed in vitro monolayer cultures of human articular chondrocytes from normal and osteoarthritic patients and studied the transforming growth factor-beta 1 responsiveness of those chondrocytes. The cell-growth curve indicated that the primary osteoarthritic chondrocyte culture with transforming growth factor-beta 1 showed a more rapid growth pattern than normal chondrocytes with or without TGF-beta 1 and osteoarthritic chondrocytes without TGF-beta 1. The osteoarthritic group showed a sharp decline in growth pattern with subsequent culture. The shape of osteoarthritic chondrocytes was bigger and more bizarre compared to those of normal chondrocytes. With subsequent culture, this change became prominent. The transforming growth factor-beta 1 increased the [3H]-TdR uptake in each group. The phenotypes of chondrocytes were more clearly expressed in the normal group. The chondrocytes lost their phenotype (production of collagen type II) following subculture in each group. The transforming growth factor-beta 1 could not inhibit or delay the dedifferentiation process (loss of phenotype).

Sequential Tc-99m MDP bone scans after cementless total hip arthroplasty in asymptomatic patients.

PURPOSE: To evaluate the distribution and degree of radioactivity on sequential Tc-99m methylene diphosphonate (Tc-99m MDP) scans in the diagnosis of postoperative loosening in asymptomatic patients who underwent cementless total hip replacement. MATERIAL AND METHODS: Tc-99m MDP scans were obtained every three months where possible, for one year in those patients who underwent total hip replacement using a Harris Galante cementless prosthesis. Eighty-three scans from 45 patients were obtained. Uptake was assessed within the five periprosthetic zones (zone 1: greater trochanter; 2: lateral stem of the prosthesis; 3: tip of the prosthesis; 4: medial stem of the prosthesis; and 5: lesser trochanter) using an arbitrary 5-point grading system. Grade 1 indicated normal uptake and Grade 5 indicated highest uptake. The grade of uptake within each zone was then analyzed against their postoperative ages. RESULTS: The radioactivity regressed to Grade 1 or 2 from Grade 4 or 5 within 12 months after cementless total hip replacement in 87.5% and 93.8% of patients in zones 2 and 4, respectively. Even though the radioactivity within zones 1, 3, and 5 tended to decrease, persistent elevation of activity (over grade 2) lasted up to 12 months after surgery in 31% or 37% of the patients. CONCLUSION: The radioactivity after cementless total hip replacement tended to regress in proportion to the postimplant age. The regression rate varied depending on the periprosthetic zones.

[Effects of alpha 1-adrenergic receptor subtypes activation on proliferation of smooth muscle cells in rabbit aorta].

The effect of alpha 1-Adrenoceptor (alpha 1-AR) and its subtypes, alpha 1A and alpha 1B, activation on the proliferation of cultured vascular smooth muscle cells (VSMC) was studied by [3H]-thymidine (3H-TdR) incorporation and in situ hybridization. Norepinephrine (NE) promotes DNA synthesis of VSMC. The action was induced by activation of alpha 1A-AR subtype and increase of c-fos gene expression. However, the DNA synthesis was inhibited by activation of alpha 1B-AR subtype. There was a synergetic effect on VSMC DNA synthesis between NE and neuropeptide Y (NPY). But only additional effect was found between NE and Ang II. More over, it was found that alpha 1B-AR subtype showed significant constitutive inhibitory effect of VSMC DNA synthesis.