Study of ciprofloxacin biodegradation by a Thermus sp. isolated from pharmaceutical sludge.

Ciprofloxacin (CIP) is an antibiotic drug frequently detected in manure compost and is difficult to decompose at high temperatures, resulting in a potential threat to the environment. Microbial degradation is an effective and environmentally friendly method to degrade CIP. In this study, a thermophilic bacterium that can degrade CIP was isolated from sludge sampled from an antibiotics pharmaceutical factory. This strain is closely related to Thermus thermophilus based on 16S rRNA gene sequence analysis and is designated C419. The optimal temperature and pH values for CIP degradation are 70°C and 6.5, respectively, and an appropriate sodium acetate concentration promotes CIP degradation. Seven major biodegradation metabolites were identified by an ultra-performance liquid chromatography tandem mass spectrometry analysis. In addition, strain C419 degraded other fluoroquinolones, including ofloxacin, norfloxacin and enrofloxacin. The supernatant from the C419 culture grown in fluoroquinolone-containing media showed attenuated antibacterial activity. These results indicate that strain C419 might be a new auxiliary bacterial resource for the biodegradation of fluoroquinolone residue in thermal environments.

Biodegradation of sulfamethazine by an isolated thermophile-Geobacillus sp. S-07.

Sulfamethazine (SM2) is an antimicrobial drug that is frequently detected in manure compost, is difficult to degrade at high temperatures and is potentially threatening to the environment. In this study, a thermophilic bacterium was isolated from the activated sludge of an antibiotics pharmaceutical factory; this bacterium has the ability to degrade SM2 at 70 °C, which is higher than the traditional manure composting temperature. The strain S-07 is closely related to Geobacillus thermoleovorans based on its 16S rRNA gene sequence. The optimal conditions for the degradation of SM2 are 70 °C, pH 6.0, 50 rpm rotation speed and 50 mL of culture volume. More than 95% of the SM2 contained in media was removed via co-metabolism within 24 h, which was a much higher percentage than that of the type strain of G. thermoleovorans. The supernatant from the S-07 culture grown in SM2-containing media showed slightly attenuated antibacterial activity. In addition, strain S-07 was able to degrade other sulfonamides, including sulfadiazine, sulfamethoxazole and sulfamerazine. These results imply that strain S-07 might be a new auxiliary bacterial resource for the biodegradation of sulfonamide residue in manure composting.

[Effects of different reconstruction methods in treatment of avulsion of ureteral mucosa: experiment with dogs].

OBJECTIVE: To investigate the curative effects of different reconstruction methods in the treatment of avulsion of ureteral mucosa. METHODS: Twenty adult dogs were randomly divided into 4 equal groups: Group III (control group in which the ureter was incised and a ureteral stent was placed therein), Group II (the ureter was incised and a ureteral mucosal avulsion about 5 cm in length was created and the avulsed mucosa was replaced to the original position and a ureteral stent was placed in addition), Group III (a piece of bladder mucosa was incised and grafted to the avulsed ureter mucosa and a ureteral stent was placed in addition), and Group IV (a tubularized peritoneal free graft and a ureteral stent were placed in the avulsed ureter). Ten weeks later intravenous pyelography (IVP) was conducted to observe the images of ureter. Then the dogs were killed and pathological examination of the reconstructed ureter was conducted. RESULTS: IVP showed atresia and stenosis in all control group dogs. No obvious stenosis was observed in Groups II and IV. Pathological examination showed that the newly grown mucosa was similar to the normal ureter structure in Groups II and IV. In Group III one dog died, slight distension of ureter superior to the reconstructed part was found in 2 dogs, and no obvious stenosis was observed in 3 dogs. CONCLUSION: Orthotopic replacement and tabularized peritoneal free grafting with placement of ureter stent are effective in treatment of avulsion of ureter mucosa. Bladder mucosa can be used as selectable material.

[Combined treatment applied to advanced cancer of abdominal cryptorchidism (report of 12 cases)].

OBJECTIVE: To discuss the treatment of advanced cancer of abdominal cryptorchidism. METHODS: The combined method, including preoperation chemotherapy + surgery + postoperation radiotherapy and chemotherapy, was used to treat 12 cases of the advanced cancer of abdominal cryptorchidism and the effects were evaluated. RESULTS: The patients recovered smoothly without complications of operation. The side effect of chemotherapy and radiotherapy was very slight. Eleven out of 12 cases were followed up. All 11 cases survived and had no recurrence. CONCLUSION: The results of combined method to treat advanced cancer of abdominal cryptorchidism is very perfect.

[Expression and implication of cyclooxygenase-2 mRNA in prostate cancer].

OBJECTIVE: To investigate the relationship between cyclooxygenase-2 (COX-2) mRNA expression and the biological behaviors of prostate carcinoma (PCa). METHODS: The expression of COX-2 mRNA was detected by RT-PCR method in 32 samples of PCa and the COX-2/GAPDH value was determined. Seven normal prostate tissues were served as control. RESULTS: The expression of COX-2 mRNA in normal tissue of 7 control cases was all negative. There was statistical correlation between the COX-2/GAPDH and the Gleason scores of PCa. There also showed statistical correlation between the COX-2/GAPDH and the stages of PCa. CONCLUSION: COX-2 mRNA play an important role in occurrence and progression of the PCa. COX-2 is a tumor marker which may be the possible prognostic factor of PCa.

[Expression of different genes in transitional zone and peripheral zone of human normal prostate].

OBJECTIVE: To detect the expression of different genes in the transitional zone and peripheral zone of human normal prostate. METHODS: Seventeen specimens of normal prostate gland were obtained from dead kidney donors and patients undergoing total cystectomy. The transitional zone and peripheral zone were isolated. Twenty specimens of benign prostate hypertrophy (BPH) were obtained from patients undergoing prostatic enucleation. The periurethral significantly hypertrophic nodules were isolated. BioDoor Chip-40S containing 6700 clones of targeting gene cDNA was used to detect the gene expression. The peripheral zone RNA samples were labeled with cy3, and the transitional zone RNA samples labeled with Cy5. The genes with the ratio average (RA) < 0.5 or > 2 were identified as differentially expressed genes, and those with a RA < 0.2 or > 4 were identified as the most significantly differentially expressed genes. RT-PCR was used to investigate semi-quantitatively the expression of epidermal growth factor (EGF) mRNA in the 17 specimens of transitional zone and peripheral zone from the 17 normal prostate glands and the 20 specimens of periurethral zone from BPH. RESULTS: A total of 640 genes differentially expressed in transitional zone and peripheral zone were screened out (comprising 269 unknown genes and 371 known genes), of which 294 genes were expressed in the transitional zone and 346 in the peripheral zone. Fourteen genes were identified as markedly differentially expressed, among which the EGF gene was highly expressed in the central zone, and the OSF-1 (osteoblast stimulating factor-1) gene was highly expressed in the peripheral zone. RT-PCR proved that in comparison with the expression of the internal marker beta-actin gene, the expression levels of EGF in the normal transitional zone and in the periurethral zone from BPH were markedly higher than that in the peripheral zone. CONCLUSION: There are differently expressed genes between the peripheral zone and central zone of normal human prostate, which may be the background of different biological behavior between these zones. EGF may markedly contribute to the growth of central zone of normal prostate.

Association between clinical characteristics and expression abundance of RTKN gene in human bladder carcinoma tissues from Chinese patients.

PURPOSE: Bladder carcinoma is the most common urological malignancy in China. Gene mutation may be one of causes of carcinogenesis in the cancer. We therefore investigated the mRNA expression of RTKN gene in clinic malignant bladder carcinoma and explored the relationship between the novel gene and the cancer. METHODS: Total RNA was extracted from 33 surgically resected specimens of bladder carcinoma and 19 specimens of tumor-free bladder tissues. After the optimal reverse-transcription polymerase chain reaction condition was established, the mRNA expression levels of the RTKN gene in the lesions and tumor-free bladder tissues were examined semiquantitatively, and the relationships between expression levels of RTKN and clinical pathological features were analyzed. RESULTS: The expression of RTKN gene mRNA in 33 human bladder carcinoma tissues was significantly higher than that in 19 human tumor-free bladder tissues (0.937+/-0.103 vs. 0.350+/-0.082). The average ratio of RTKN expression in neoplasms to that in tumor-free bladder tissues was 0.350+/-0.164. Based on this ratio the 33 patients were divided into three groups: a down-regulated expression group (n=2), an up-regulated expression group (n=22), and an unchanged group (n=9). Although the chi(2) test demonstrated a statistically nonsignificant differences in RTKN expression between tumor stages Ta, T(1), and T(2) overall in the 33 human bladder carcinoma, the t test showed that there were statistically significant differences between solitary and multiple tumors, between the paired group aged younger or older than 70 years in 27 de novo bladder carcinoma patients, and between the groups with tumor larger or smaller than 2.25 cm(3). CONCLUSIONS: These results suggest that the RTKN gene is involved in bladder carcinogenesis and progression in bladder carcinoma, indicating that RTKN gene could be a molecular target in cancer therapy.

Anergic cells induced by the blockade of CD40-CD154 and CD28-B7 costimulatory pathways act as potent immunoregulatory cells in vitro and vivo.

BACKGROUND: This study was to evaluate whether anergic cells induced by the blockade of CD40-CD154 and CD28-B7 costimulatory pathways can act as potent immunoregulatory cells in vitro and prolong cardiac allograft survival after adoptive transfer. METHODS: Anergic cells were induced in vitro by the addition of anti-CD154 and anti-CD80 monoclonal antibodies (mAbs) to primary MLR (mixed lymphocyte reaction) consisting of BALB/c as responder and C3H as stimulator. Anergic cells were added to a newly formed MLR in assessing the regulatory capacity and antigen specificity of anergic cells. The ability of anergic cells to respond to antigen and/or exogenous recombinant mouse interleukin-2 (rmIL-2) was tested. For in vivo studies, anergic cells were intravenously injected into 3.0-Gy gamma-irradiated BALB/c mice immediately after heterotopic abdominal cardiac transplantation. To prolong allograft survival, recipient mice injected with anergic cells received rapamycin therapy [1 mg.day(-1).kg(-1)]. RESULTS: Anergic cells strongly suppressed the proliferation of naicaron;ve BALB/c splenocytes against the original (C3H) stimulator in a dose-dependent manner, but they failed to suppress the proliferation of naicaron;ve BALB/c splenocytes against the third-party (C57BL/6J) stimulator. The anergic state was reversed by both original (C3H) stimulator and additional exogenous IL-2. In in vivo studies, untreated irradiated BALB/c mice rejected C3H cardiac allografts with a mean survival time of (8.6 +/- 1.1) days, whereas those injected with the anergic cells rejected the allografts with a mean survival time of (11.8 +/- 1.9) days, which was slightly longer than that of the untreated mice. The protocol based on anergic cells injection plus rapamycin therapy could prolong allograft survival significantly [(29.6 +/- 4.4) days]. CONCLUSIONS: Anergic cells induced by the blockade of CD40-CD154 and CD28-B7 costimulatory pathways can act as potent immunoregulatory cells in vitro, and prolong cardiac allograft survival after adoptive transfer in the presence of rapamycin therapy. This procedure might be clinically useful for prolonging allograft survival if optimal protocols are developed.

Marked prolongation of murine cardiac allograft survival using recipient immature dendritic cells loaded with donor-derived apoptotic cells.

We investigated whether recipient dendritic cells (DCs), pretreated with nuclear factor-kappaB oligodeoxyribonucleotide decoy (NF-kappaB ODN decoy) and loaded with ultraviolet B-irradiated donor apoptotic splenocytes (Apo-SCs), were able to induce murine cardiac allograft tolerance. Heterotopic vascularized heart transplantation was performed from BALB/c to C57BL/6 mice, and recipients (C57BL/6) were given one injection of recipient DCs pretreated with NF-kappaB ODN decoy and loaded with donor (BALB/c) apoptotic SCs (decoy Apo-SCs DCs) through the portal vein at 7 days, before heart transplantation in the absence of immunosuppression. The cardiac allograft survival time and the expressive levels of intragraft cytokine genes [interleukin (IL)-2, IL-10, and interferon-gamma] were evaluated. Our results indicated that injection of decoy Apo-SCs DCs significantly prolonged vascularized heart allograft survival and led to skewing of intragraft cytokine expression towards T helper 2 (IL-10). The mechanisms can be useful for therapy of allograft rejection with minimization of systemic immunosuppression.

[Expression of androgen receptor isoforms in normal human prostate].

OBJECTIVE: To investigate the expression of androgen receptor (AR) isoforms in normal human prostate. METHODS: Fourteen normal prostatic specimens from donors, aged 25 on average (21-28 yr), were analyzed by high resolution isoelectric focusing (IEF). The expression of AR isoforms was demonstrated in all 14 normal human prostatic tissues. RESULTS: Four types of AR isoforms were detected with isoelectric point value at 6.5, 6.0, 5.8 and 5.3 in 14 prostatic specimens. Binding of 3H-dihydrotestosterone (DHT) to these four AR isoforms was inhibited by the addition of 100-fold excess of DHT and testosterone. No effect of progesterone, estradiol and diethylstilbestrol on tritiated hormone binding was observed. CONCLUSIONS: There are four AR isoforms in normal human prostate. The expression of AR isoforms is different from one another.

[Some controversial conditions in the management of chronic prostatitis/chronic pelvic pain syndrome].

Chronic prostatitis (CP)/chronic pelvic pain syndrome (CPPS) is a common problem of medically controversial condition that causes considerable morbidity and impact on life. Although there are many competing causes proposed, the etiology and pathogenesis of CP/CPPS remain unclear. The causative factors underlying the CPPS are not fully understood. The optimal management of CP/CPPS is still unknown. The guideline of diagnosis and management of CP/CPPS based on evidence base medicine is not yet established. Many problems are still not resolved, such as the significance of leukocytes and the role of inflammation in CP/CPPS, the significance of bacteria presence and the role of infection in CP/CPPS, the correlation between leukocytes/bacteria and severity of symptoms, how to divide the subgroups of CP/CPPS, the role of antimicrobial therapy in the treatment of men with CP/CPPS, why patients with category IIIb complain of symptoms, while those with category IV complain of none. Although CP/CPPS is now achieving greater recognition, well-designed studies with large sample size should be performed.

[The treatment of benign prostatic hyperplasia by means of transurethral holmium laser enucleation].

OBJECTIVES: To evaluate the clinical effect of the holmium laser enucleation and morcellation of prostate (HoLEP) in the treatment of benign prostatic hyperplasia(BPH). METHODS: In the treatment of 35 BPH patients, 100 watt high-powered holmium laser set was used transurethrally and a reciprocating blade tissue morcellator was introduced via a nephroscope to enucleate and morcellate the prostatic tissue. RESULTS: Operations in all 35 cases were successful. The average operation time was 60 +/- 23.2 (range 30-180) min. The removed prostatic tissue weighed 31 +/- 9 (range 10-56) g on average. The average catheter time was 1.5 d (from 20 h to 4 d). No blood transfusion was performed in all cases. Histopathological analysis confirmed the diagnosis of BPH in all cases. In the 3-month follow up of 32 cases, IPSS dropped from 24 +/- 6.2 to 5.6 +/- 3.6; the peak urinary flow rate(Qmax) went up from 8.5 +/- 3.9 ml/s to 22.0 +/- 7.2 ml/s; the residual volume of urine dropped from 138 +/- 125 ml to 21 +/- 15 ml. No serious complications were found. CONCLUSIONS: HoLEP is effective in treating BPH. It can completely enucleate the hyperplastic tissue with little bleeding in operation. The treatment has the advantages of short catheter time and significant clinical improvements.

[Expression of fractalkine and its receptor in acute cardiac allografts rejection].

OBJECTIVE: To investigate the expression of fractalkine (FKN) and its receptor CX3CR1 in cardiac allografts and the effect of Cyclosporin A (CsA). METHODS: Three groups of rats underwent heterotopic cardiac transplantation, 45 cases in each group and 5 cases in control group: SD to SD regarded as isograft group (group A), Wistar to SD divided into CsA untreated allograft group (group B) and CsA treated allograft group (group C), normal SD rats as control group. The FKN mRNA expression was detected by one-step RT-PCR method and the expression of FKN and CX3CR1 protein was detected by standard ABC immunohistochemical technique. RESULTS: The expression of FKN mRNA and protein was weak in both isografts and normal heart specimens. The changes of FKN mRNA expression were correlated with the process of acute allograft rejection. The peak of FKN mRNA expression (0.8 +/- 0.26) appeared on the seventh day after transplantation, which could be down-regulated by CsA significantly (t = 2.390, P < 0.05). FKN protein was located in endothelia cells and its receptor CX3CR1 was located in infiltrating mononuclear cells in allografts. CONCLUSIONS: Upregulation of FKN and its receptor was significantly correlated with the trafficking of mononuclear cells which play an important role in acute allograft rejection. It may be one of the important mechanisms of CsA to intervene the acute rejection by inhibiting the activation of the FKN-CX3CR1 pathway.

[Androgen receptor isoforms in LNCaP cell and human prostate].

OBJECTIVES: To investigate the androgen receptor (AR) isoforms expression in human benign and malignant prostatic tissues and LNCaP cells. METHODS: Using high resolution isoelectric focusing (IEF), the different expression of AR isoforms were demosntrated in human benign and malignant prostatic tissues and LNCaP cells. RESULTS: Data were obtained from 41 AR-positive BPH, three prostatic cancer specimens, and LNCaP cells. From these materials, three types of AR isoforms were detected with pI values at 6.5, 6.0 and 5.3. In the case of BPH tissues, 15 (36.5%) specimens expressed all the three types of isoforms at pI 6.5, 6.0 and 5.3, and 10 (24.4%) samples contained isoforms at pI 6.5 and 5.3, five (12.2%) samples indicated isoforms at pI 6.5 and 6.0, four (9.8%) showed the isoforms at pI 6.0 and 5.3. Of all the 41 specimens, two (4.9%) and two (4.9%) as well as three (7.3%) denoted the isoforme at pI 6.5, 6.0 and 5.3 respectively. As for three prostatic cancer specimens, one sample showed all the three types of AR isoforms at pI 6.5, 6.0, 5.3, but another specimen expressed at pI 6.5 and 6.0, and only one failed to indicate any types of isoforms. LNCaP cells expressed all three types of AR isoforms at pI 6.5, 6.0 and 5.3. Binding of 3H-dihydrotestosterone to these three types of isoforms was inhibited by the addition of 100-fold excess of DHT and testosterone. No effect of progesterone, oestradiol and diethylstilboestrol on tritiated hormone binding was observed. CONCLUSIONS: The expression of AR isoforms is different among various patients and different between BPH and LNCaP cells, though no clear explanation could be induced for this. These results suggest the possibility of explaining effective hormonal therapy to prostatic disease in the future.

[Expression of NF-kappa B in human bladder cancer and its clinical significance].

BACKGROUND & OBJECTIVE: Studies on solid cancer(such as breast cancer, hepatocellular cancer, pancreatic cancer) indicated that the abnormal expression of nuclear transcription factor Kappa B (NF-kappa B) regulates angiogenesis and cyclin-related genes. This study was designed to investigate the expression differences of NF-kappa B and its regulated genes between human primary transitional cell carcinoma(TCCs) of bladder and non-cancer bladder mucosa and its clinical significance. METHODS: Forty-three frozen sections including 30 bladder cancer and 13 non-cancer bladder mucosa were subjected to immunohistochemistry and nucleus staining for determining levels of NF-kappa B family and I kappa B alpha; Five paired cancer and non-cancer specimens were subjected to Western blot for analysis p65, an important subtype of NF-kappa B; Thirteen paired specimens were subjected to RT-PCR for determination mRNA levels of p50, p52, p65, c-Rel, RelB, I kappa B alpha, CyclinD1, IL-8. RESULTS: Expressions of p50, p52, p65, c-Rel, RelB, I kappa B alpha, CyclinD1, IL-8 mRNAs in bladder cancer were higher than that in non-cancer bladder mucosa (P < 0.01, P < 0.05, P < 0.01, P < 0.01, P < 0.05, P < 0.05, P < 0.05 and P < 0.05, respectively). Nucelus stainings of p50, p52, p65, c-Rel, RelB were also stronger in bladder cancer(P < 0.01, P < 0.01, P < 0.01, P < 0.01 and P < 0.01, respectively). Moreover, p65 was expressed more in cancer tissue than that in non-cancer mucosa evidenced by Western blot. In bladder cancer, the ranking score differences of p52, p65, c-Rel protein between lymphatic metastasis group and non-lymphatic metastasis group were statistically significant (P < 0.01, P < 0.01, P < 0.05, respectively). CONCLUSIONS: Compared to noncancer bladder mucosa, expressions of NF-kappa B family and its regulated genes in bladder cancer are markedly higher. NF-kappa B may be related to lymphatic metastasis.

Characteristic pattern of human prostatic growth with age.

AIM: To study the characteristic pattern of the age-related growth of the human prostate gland. METHODS: The volume (weight) of the prostate in 1,601 males, aged from newborn to 92 years, was determined by B-ultrasonography. RESULTS: Prostatic volume determination by B-ultrasonography in 1601 males (1301 normal subjects and 300 BPH patients) pointed out that the age-stratified growth of human prostate could be categorized into 4 life stages: (1) the first slow growing phase (from newborn to 9 years): the prostate grows slowly at a rate of 0.14 g per year; (2) the first rapid growing phase (from 10 to 30 years): the prostate grows at a rate of 0.84 g per year; (3) the second slow growing phase (from 30 to 50 years), the prostate grows at a rate of 0.21 g per year; (4) the second rapid growing phase (from 50 to 90 years): the prostate grows at one of the following rates: in one group the growth rate is of 0.50 g per year and in the other 1.20 g per year, leading to benign prostatic hyperplasia (BPH). CONCLUSION: The volumes of the prostate are different in different age groups and it grows with age at different rates in four life phases. The prostate growth in phases can be expressed by the following equation: Y=19.36+1.36X'-0.58X'(2+0.33X'3), where Y = prostate volume, X = age (up to 70 years), X'=(X-35.5)/10.

[Expression of cyclooxygenase-2 in human transitional cell bladder carcinomas].

BACKGROUND & OBJECTIVES: Cyclooxygenase(COX) is a rate-limiting enzyme in prostaglandin synthesis. Recent study suggested that COX-2 was associated with carcinogenesis. This study was designed to investigate the role of COX in the development and progression of bladder cancer through examining the expression of COX-1 and COX-2 in human bladder cancer tissue, normal bladder mucosa, and cystitis tissue. METHODS: Reverse transcriptase-polymerase chain reaction(RT-PCR) and immunohistochemistry(Envision method) were applied to detect the mRNA and protein expression of COX-1 and COX-2 in bladder transitional cell carcinoma(BTCC), tissue adjacent to cancer, normal bladder mucosa, and chronic cystitis tissue. Furthermore, the relationship between COX expression level and cancer pathologic parameters was analyzed. RESULTS: Using RT-PCR, all 15 frozen specimens from bladder cancer were shown to express COX-2 mRNA, while only 1 of 5 specimens from grossly normal mucosa adjacent to cancer expressed COX-2 mRNA, with significant difference. But COX-1 mRNA was expressed constitutionally in all of the fresh cancer tissue samples. The immunohistochemical result was analogous to RT-PCR result. COX-2 protein was mainly localized at cytoplasm of malignant cell (positive rate was 50%) but was not expressed in normal bladder mucosa (n = 4) and chronic cystitis tissues (n = 5). In contrast, COX-1 protein was mainly localized at smooth muscle cell of normal tissue and cystitis tissue(positive rate was 100%) but was not expressed in cancer tissue. In 40 paraffin embedded specimens of BTCC, the intensity of COX-2 expression was associated with tumor grade and stage. The level of COX-2 expression was higher in grade III tumor, than in grade I, II tumors, and was higher in invasive tumor (T2-T4) than in superficial tumor(Ta-T1). CONCLUSIONS: The expression of COX-2 mRNA and protein was enhanced in human BTCC, and was associated with the malignant degree of the tumor, indicating that COX-2 may play a key role in the development and progression of BTCC.