Compensatory structural and functional adaptation after radical nephrectomy for renal cell carcinoma according to preoperative stage of chronic kidney disease. Choi DK, Jung SB, Park BH, Jeong BC, Seo SI, Jeon SS, Lee HM, Choi HY, Jeon HG.J Urol. 2015 Oct;194(4):910-5. [Epub 2015 Apr 28]. doi: 10.1016/j.juro.2015.04.093.

PURPOSE: We investigated structural hypertrophy and functional hyperfiltration as compensatory adaptations after radical nephrectomy in patients with renal cell carcinoma according to the preoperative chronic kidney disease stage. MATERIALS AND METHODS: We retrospectively identified 543 patients who underwent radical nephrectomy for renal cell carcinoma between 1997 and 2012. Patients were classified according to preoperative glomerular filtration rate as no chronic kidney disease-glomerular filtration rate 90ml/min/1.73m2 or greater (230, 42.4%), chronic kidney disease stage II-glomerular filtration rate 60 to less than 90ml/min/1.73m2 (227, 41.8%), and chronic kidney disease stage III-glomerular filtration rate 30 to less than 60ml/min/1.73m2 (86, 15.8%). Computerized tomography performed within 2 months before surgery and 1 year after surgery was used to assess functional renal volume for measuring the degree of hypertrophy of the remnant kidney, and the preoperative and postoperative glomerular filtration rate per unit volume of functional renal volume was used to calculate the degree of hyperfiltration. RESULTS: Among all patients (mean age = 56.0y) mean preoperative glomerular filtration rate, functional renal volume, and glomerular filtration rate/functional renal volume were 83.2ml/min/1.73m2, 340.6cm3, and 0.25ml/min/1.73m2/cm3, respectively. The percent reduction in glomerular filtration rate was statistically significant according to chronic kidney disease stage (no chronic kidney disease 31.2% vs. stage II 26.5% vs. stage III 12.8%, P<0.001). However, the degree of hypertrophic functional renal volume in the remnant kidney was not statistically significant (no chronic kidney disease 18.5% vs. stage II 17.3% vs. stage III 16.5%, P = 0.250). The change in glomerular filtration rate/functional renal volume was statistically significant (no chronic kidney disease 18.5% vs. stage II 20.1% vs. stage III 45.9%, P<0.001). Factors that increased glomerular filtration rate/functional renal volume above the mean value were body mass index (P = 0.012), diabetes mellitus (P = 0.023), hypertension (P = 0.015), and chronic kidney disease stage (P<0.001). CONCLUSIONS: Patients with a lower preoperative glomerular filtration rate had a smaller reduction in postoperative renal function than those with a higher preoperative glomerular filtration rate due to greater degrees of functional hyperfiltration.

COUP-TFII Stimulates Dentin Sialophosphoprotein Expression and Mineralization in Odontoblasts.

Chicken ovalbumin upstream promoter transcription factor 2 (COUP-TFII), an orphan nuclear receptor belonging to the steroid-thyroid hormone receptor superfamily, plays an important role in cell fate determination of various tissues. However, the specific role of COUP-TFII in tooth development has not yet been elucidated. In the present study, we aimed to explore the role of COUP-TFII in dentin sialophosphoprotein (DSPP) expression and matrix mineralization in odontoblast-lineage cells. In primary human dental pulp cells (HDPCs) and murine dental papilla-derived cells (MDPC-23) cultured in a mineralizing medium, the expression of COUP-TFII was induced along with the increased odontoblast-specific dentin matrix protein-1 (DMP-1) and DSPP expression. Endogenous expression of COUP-TFII in maxillary second molar germs of rats showed an increasing tendency as development of the tooth progressed. Also, COUP-TFII protein was detected in greater quantity in the odontoblastic layer of second molar germs than in that of third molar germs of rats. Overexpression of COUP-TFII using an adenoviral system upregulated the expression of odontoblast-specific genes with increased alkaline phosphatase activity and matrix mineralization in odontoblast-lineage cells. In contrast, knockdown of COUP-TFII using small interfering RNA decreased the expression of odontoblast-specific genes, which reduced matrix mineralization. Mechanistic studies revealed that COUP-TFII increased DSPP transcription by direct binding on the DSPP promoter. In addition, COUP-TFII physically interacted with the homeodomain transcription factor Msx2 and antagonistically regulated the Msx2 effect on DSPP promoter activity. Taken together, these results suggest that COUP-TFII has a stimulatory role in DSPP expression and matrix mineralization in odontoblast-lineage cells.

MicroRNA-194 reciprocally stimulates osteogenesis and inhibits adipogenesis via regulating COUP-TFII expression.

Osteoblasts and adipocytes are differentiated from common mesenchymal stem cells (MSCs) in processes which are tightly controlled by various growth factors, signaling molecules, transcriptional factors and microRNAs. Recently, chicken ovalbumin upstream promoter-transcription factor II (COUP-TFII) was identified as a critical regulator of MSC fate. In the present study, we aimed to identify some microRNAs (miR), which target COUP-TFII, and to determine the effects on MSCs fate. During osteoblastic or adipocytic differentiation from MSCs lineage cells, miR-194 expression was found to be reversal. In the cultures of mesenchymal C3H10T1/2 and primary bone marrow stromal cells, osteogenic stimuli increased miR-194 expression with accompanying decreases in COUP-TFII expression, whereas adipogenic stimuli reduced miR-194 expression with accompanying increases in COUP-TFII expression. A luciferase assay with COUP-TFII 3'-untranslated region (UTR) reporter plasmid, including the miR-194 binding sequences, showed that the introduction of miR-194 reduced the luciferase activity. However, it did not affect the activity of mutated COUP-TFII 3'-UTR reporter. Enforced expression of miR-194 significantly enhanced osteoblast differentiation, but inhibited adipocyte differentiation by decreasing COUP-TFII mRNA and protein levels. In contrast, inhibition of the endogenous miR-194 reduced matrix mineralization in the MSCs cultures, promoting the formation of lipid droplets by rescuing COUP-TFII expression. Furthermore, overexpression of COUP-TFII reversed the effects of miR-194 on the cell fates. Taken together, our results showed that miR-194 acts as a critical regulator of COUP-TFII, and can determinate the fate of MSCs to differentiate into osteoblasts and adipocytes. This suggests that miR-194 and COUP-TFII may be good target molecules for controlling bone and metabolic diseases.

Clinical applications of platelet-rich plasma in patellar tendinopathy.

Platelet-rich plasma (PRP), a blood derivative with high concentrations of platelets, has been found to have high levels of autologous growth factors (GFs), such as transforming growth factor-ß (TGF-ß), platelet-derived growth factor (PDGF), fibroblastic growth factor (FGF), vascular endothelial growth factor (VEGF), and epidermal growth factor (EGF). These GFs and other biological active proteins of PRP can promote tissue healing through the regulation of fibrosis and angiogenesis. Moreover, PRP is considered to be safe due to its autologous nature and long-term usage without any reported major complications. Therefore, PRP therapy could be an option in treating overused tendon damage such as chronic tendinopathy. Here, we present a systematic review highlighting the clinical effectiveness of PRP injection therapy in patellar tendinopathy, which is a major cause of athletes to retire from their respective careers.

Transcriptional factor ATF6 is involved in odontoblastic differentiation.

ATF6 is an endoplasmic reticulum (ER) membrane-bound transcription factor that regulates various cellular functions. The purpose of this study was to investigate the role of ATF6 in odontoblast differentiation. Rat tooth germs were isolated, changes in gene expression were evaluated over time, and localization of ATF6 was determined by immunohistochemistry. Human dental pulp cells (HDPCs) were cultured with 50 µg/mL ascorbic acid and 5 mmol/L ß-glycerophosphate or 100 ng/mL bone morphogenetic protein 2 to induce differentiation. Translocation of ATF6 was observed by immunofluorescence and confocal microscopy. Overexpression of ATF6 was performed with an adenoviral vector. Matrix mineralization was evaluated by alizarin red staining. Immunoreactivity to anti-ATF6 was observed in the odontoblastic layer of the molar tooth germ, and expressions of ATF6, dentin sialophosphoprotein (DSPP) and dentin matrix protein 1 (DMP1) increased gradually during tooth germ development. When HDPCs were cultured in differentiation media, ATF6, DSPP, and DMP1 expression increased with the expression of unfolded protein response (UPR) markers, BiP and CHOP. Immunofluorescence results showed that ATF6 protein moved from cytoplasm to nucleus when cells were exposed to differentiation media. Notably, overexpression of ATF6 increased DSPP and DMP1 expression, alkaline phosphatase (ALP) activity, and matrix mineralization in HDPC cultures. Inhibition of ATF6 decreased ALP activity and mineralization. These results suggest that ER membrane-bound transcriptional factor ATF6 may be involved in odontoblastic differentiation.

Erythrocyte sedimentation rate and anaemia are independent predictors of survival in patients with clear cell renal cell carcinoma.

BACKGROUND: The 1997 international consensus conference on renal cell cancer (RCC) prognosis suggested erythrocyte sedimentation rate (ESR), alkaline phosphatase (ALP), and anaemia as prognostic biomarkers, but most studies reviewed were limited by small sample sizes. METHODS: The Cox proportional hazards model was used to evaluate whether ESR, ALP, haemoglobin (Hb), and haematocrit (Hct) could predict survival outcomes in 1307 patients with clear cell RCC (ccRCC) who underwent nephrectomy during 1994-2008. RESULTS: During a median follow-up of 43 months, we found that the patients with preoperative high levels of ESR, had a 2.10-fold (95% confidence interval (CI): 1.21-3.67) greater risk of dying from RCC compared with patients with low levels (normal range). Patients with preoperative anaemia, assessed by Hb and Hct, had a 3.11-fold (95% CI: 1.17-8.25) and 6.20-fold (95% CI: 2.30-16.72) greater risk of dying from other illnesses, respectively, compared with patients without anaemia. ALP levels were not associated with ccRCC patients' survival. These associations for ESR and anaemia were more pronounced in patients with body mass index (BMI) <25 compared with patients with BMI ≥ 25 kg m(-2). CONCLUSION: Preoperative high ESR, but not ALP, was a significant predictor for cancer-specific survival among ccRCC patients. Anaemia increases the risk of death from other illness.

Radiotolerance of phosphatases of a Serratia sp.: potential for the use of this organism in the biomineralization of wastes containing radionuclides.

Aqueous wastes from nuclear fuel reprocessing present special problems of radiotoxicity of the active species. Cells of Serratia sp. were found previously to accumulate high levels of hydrogen uranyl phosphate (HUP) via the activity of a phosphatase enzyme. Uranium is of relatively low radiotoxicity whereas radionuclide fission products such as (90)Sr and (137)Cs are highly radiotoxic. These radionuclides can be co-crystallized, held within the bio-HUP "host" lattice on the bacterial cells and thereby removed from contaminated solution, depending on continued phosphatase activity. Radiostability tests using a commercial (60)Co γ-source showed that while cell viability and activity of purified phosphatase were lost within a few hours on irradiation, whole-cell phosphatase retained 80% of the initial activity, even after loss of cell culturability, which was increased to 100% by the incorporation of mercaptoethanol as an example radioprotectant, beyond an accumulated dose of >1.3 MGy. Using this co-crystallization approach (without mercaptoethanol) (137)Cs(+) and (85)Sr(2+) were removed from a simulated waste selectively against a 33-fold excess of Na(+).

Lactone form 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors (statins) stimulate the osteoblastic differentiation of mouse periodontal ligament cells via the ERK pathway.

BACKGROUND AND OBJECTIVE: Recent studies reported that the lactone forms of 3-hydroxy- 3-methylglutaryl-coenzyme A reductase inhibitors, which are also known as statins, have a bone stimulatory effect. However, there are few reports on the effect of statins on periodontal ligament cells. This study examined the statin-induced osteoblastic differentiation of mouse periodontal ligament cells as well as its mechanism. MATERIAL AND METHODS: Mouse periodontal ligament cells were cultured with lovastatin or simvastatin, and their viability was measured. The levels of alkaline phosphatase (ALP), osteocalcin, bone sialoprotein and bone morphogenetic protein-2 mRNA expression were evaluated by RT-PCR. The osteoblastic differentiation was characterized by the ALP activity and Alizarin Red-S staining for calcium deposition. The activity of the osteocalcin gene (OG2) and synthetic osteoblast-specific elements (6× OSE) promoter with statins was also measured using a luciferase assay. For the signal mechanism of statins, the ERK1/2 MAPK activity was determined by western blot analysis. RESULTS: A statin treatment at concentrations < 1 µM did not affect the cell viability. Lovastatin or simvastatin at 0.1 µM increased the levels of ALP, osteocalcin, bone sialoprotein and bone morphogenetic protein-2 mRNA in mouse periodontal ligament cells. In addition, the ALP activity, mineralized nodule formation and OG2 and OSE promoter activity were higher in the lovastatin- or simvastatin-treated cells than the control cells. Western blot analysis confirmed that the statins stimulated the phosphorylation of ERK1/2. CONCLUSION: Lovastatin and simvastatin may stimulate the osteoblastic differentiation of periodontal ligament cells via the ERK1/2 pathway. This suggests that the statins may be useful for regenerating periodontal hard tissue.

Association of the bla(CMY-10) gene with a novel complex class 1 integron carrying an ISCR1 element in clinical isolates from Korea.

The bla(CMY-10) gene responsible for ß-lactam resistance was located on a new complex class 1 integron within a conjugative plasmid. The sul1-type class 1 integron, containing an aadA2a gene cassette, was identified upstream of bla(CMY-10). A unique gene array (yqgF-yqgE-gshB-orf97--orf105) was identified downstream of bla(CMY-10.).

Cloning, expression, and characterization of a methionyl aminopeptidase from a hyperthermophilic archaeon Thermococcus sp. NA1.

Genomic analysis of a hyperthermophilic archaeon Thermococcus sp. NA1 revealed the presence of an 885-bp open reading frame encoding a protein of 295 amino acids with a calculated molecular mass of 32,981 Da. Analysis of the deduced amino acid sequence showed that amino acid residues important for catalytic activity and the metal binding ligands conserved in all of methionyl aminopeptidases (MetAP) were also conserved and belonged to type IIa MetAP. The protein, designated TNA1_MetAP (Thermococcus sp. NA1 MetAP), was cloned and expressed in Escherichia coli. The recombinant enzyme was a Mn(2+)-, Ni(2+)-, Fe(2+)-, or Co(2+)-dependent metallopeptidase. Optimal MetAP activity against L: -methionine p-nitroanilide (Met-pNA) (K (m) = 0.68 mM) occurred at pH 7.0 and 80 to 90 degrees C. The MetAP was very unstable compared to Pyrococcus furiosus MetAP, which was completely inactivated by heating at 80 degrees C for 5 min. It seemed likely that the cysteine residue (Cys53) played a critical role in regulating the thermostability of TNA1_MetAP.

Investigation of extended-spectrum beta-lactamases produced by clinical isolates of Klebsiella pneumoniae and Escherichia coli in Korea.

AIMS: Isolates obtained from various regions in Korea in 2002 were identified and their susceptibility to extended-spectrum cephalosporins, monobactams and/or cephamycins was studied along with any production of extended-spectrum beta-lactamases (ESBLs). METHODS AND RESULTS: Bacteria identified by the conventional techniques and Vitek GNI card were Klebsiella pneumoniae and Escherichia coli. Using disk diffusion and double-disk synergy tests, we found that 39.2% of strains produced ESBLs. About 52% of isolates transferred resistance to ceftazidime by conjugation. Banding patterns of PCR amplification with the designed primers showed that 837- and 259-bp fragments specific to bla(TEM) genes were amplified in 63.3% of strains. 929- and 231-bp fragments (bla(SHV)), 847- and 520-bp fragments (bla(CMY)), 597- and 858-bp fragments (bla(CTX-M)) were amplified in 61.5, 17.3 and 7.7% of strains respectively. About 51.9% of strains contained more than two types of beta-lactamase genes. Especially, one strain contained bla(TEM), bla(CMY) and bla(CTX-M) genes. SIGNIFICANCE: Resistance mechanisms to beta-lactams, comprising mostly ESBL production, lead to the resistance against even recently developed beta-lactams in enterobacteria, which is now a serious threat to antibiotic therapy. The high prevalence of bla(CMY) genes and multidrug-resistant genes may also make therapeutic failure and lack of eradiation of these strains by extended-spectrum cephalosporins or cephamycins.

Molecular identification of Oxalobacter formigenes with the polymerase chain reaction in fresh or frozen fecal samples.

OBJECTIVE: To develop a simple and rapid polymerase chain reaction (PCR) method for detecting Oxalobacter formigenes (which degrades oxalate in the gut) in fecal specimens from healthy volunteers and patients with urolithiasis, and to determine whether O. formigenes can be detected in frozen or fresh fecal samples. MATERIALS AND METHODS: Whole bacterial DNA was isolated directly from fresh and frozen fecal samples obtained from 30 healthy volunteers free from urolithiasis and from fresh fecal samples obtained from 38 patients with urolithiasis. Genus-specific oligonucleotide sequences were designed, corresponding to homologous regions residing in the oxc gene that encodes for oxalyl-coenzyme A decarboxylase. A PCR-based assay was used on both fresh and frozen fecal samples, and the nucleotide sequences analysed to confirm oxc. RESULTS: A PCR product of 416 bp encoding the oxc gene was detected in 23 (77%) of 30 healthy volunteers free from urolithiasis and in 14 (37%) of 38 patients with urolithiasis. In healthy volunteers, the results of PCR for the fresh and the frozen samples were identical in each subject. The nucleotide sequence analysis showed that the sequence of the amplified product was compatible with that of oxc. CONCLUSION: O. formigenes can be identified easily and efficiently using this PCR-based detection system. The colonization rate of O. formigenes in patients with urolithiasis was significantly lower than that in healthy volunteers known to be free from urolithiasis. Furthermore, as the PCR-based assay results in the frozen fecal samples were identical to those from fresh samples in each subject, immediate processing of fecal samples may not be necessary to detect O. formigenes in the clinical setting.

Purification and characterization of acid-type phosphatases from a heavy-metal-accumulating Citrobacter sp.

An acid phosphatase from a heavy-metal-accumulating strain of a Citrobacter sp. was resolved into two forms on the basis of their nonbinding (phosphatase I) or binding (phosphatase II) behaviour on the cation-exchange resin SP-Sephadex C50. Both holoenzymes had a molecular mass of 103-108 kDa as determined by Superose Q-6 column chromatography in the presence of 150 mM KCl and a subunit molecular mass of 27 kDa as determined by SDS-PAGE; the enzyme was tetrameric. Both enzymes had a pI approximately 9.0 and were immunologically cross-reactive. There were minor differences in amino acid composition and in peptide maps following tryptic digest. The pH optimum for phosphatases I and II was 5.5 and 6.25, respectively; phosphatase II alone retained activity at pH values up to 9.0. Phosphatase I was more resistant to mechanical shear, gamma-irradiation, high temperature, and toxins (F- and formaldehyde). Glycerol increased the thermostability of both enzymes, particularly the more thermosensitive phosphatase II. Phosphatase II had a lower Km and a lower Vmax for glycerol 2-phosphate hydrolysis. The production of enzyme isoforms is a phenomenon similar to that described previously for the alkaline phosphatase of Escherichia coli, where the isoforms relate to precursive and final processed forms of the enzyme. Acid phosphatase is physiologically distinct, with a role that is still obscure but that may relate to cellular stress responses.

Enzymically accelerated biomineralization of heavy metals: application to the removal of americium and plutonium from aqueous flows.

A biological process for the removal of heavy metals from the aqueous flows is described. Metals are precipitated on the surface of immobilized cells of a Citrobacter sp. as cell-bound metal phosphates. This uses phosphate liberated by the activity of a cell-bound phosphatase. Some radionuclides (e.g. 241americium) form metal phosphates readily; efficient removal of 241Am on a continuous basis is demonstrated. At low phosphatase activities, the efficiency of uranium removal correlates with enzyme activity. High phosphatase activities are not realised as an increase in metal removal, suggesting that chemical events become rate-limiting. Studies have suggested that maximal metal uptake occurs only after nucleation and the formation of precipitation foci. A model is presented to illustrate how nucleation and crystallization processes could enhance the removal of plutonium and neptunium from dilute solutions.