A new feather-degrading bacterium was isolated from a local feather waste site and identified as Bacillus subtilis based on morphological, physiochemical, and phylogenetic characteristics. Screening for mutants with elevated keratinolytic activity using N-methyl-N'-nitro-N-nitrosoguanidine mutagenesis resulted in a mutant strain KD-N2 producing keratinolytic activity about 2.5 times that of the wild-type strain. The mutant strain produced inducible keratinase in different substrates of feathers, hair, wool and silk under submerged cultivation. Scanning electron microscopy studies showed the degradation of feathers, hair and silk by the keratinase. The optimal conditions for keratinase production include initial pH of 7.5, inoculum size of 2% (v/v), age of inoculum of 16 h, and cultivation at 23 degrees C. The maximum keratinolytic activity of KD-N2 was achieved after 30 h. Essential amino acids like threonine, valine, methionine as well as ammonia were produced when feathers were used as substrates. Strain KD-N2, therefore, shows great promise of finding potential applications in keratin hydrolysis and keratinase production.
Amino Acids ; biosynthesis ; Bacillus subtilis ; genetics ; metabolism ; Culture Media ; Hydrogen-Ion Concentration ; Keratins ; metabolism ; Mutation ; Peptide Hydrolases ; biosynthesis

OBJECTIVETo determine the effect of tea polyphenol (TP) on the proliferation of human periodontal ligament fibroblasts (HPDLFs).

METHODSHPDLFs were primary cultured from tissue explants, and the cells of the 5th to 8th passages were used after immunohistochemical identification (with SABC method) of keratin and vimentin expressions. The cells were divided into 5 groups and treated with TP at 1, 0.5, 0.25, 0.125, and 0.0625 mg/ml, respectively, with another group without TP treatment as the blank control group. Cell counting and MTT colorimetric assay were performed to assess the cell proliferation, and flow cytometry was employed to determine the DNA content of the HPDLFs.

RESULTSDifferent concentrations of TP all significantly increased the proliferation and DNA synthesis of the HPDLFs (P<0.05), and TP treatment at 0.5 mg/ml for 6 h produced the optimal effect.

CONCLUSIONTP has obviously effect in promoting the proliferation of HPDLFs.

Cell Proliferation ; drug effects ; Cells, Cultured ; DNA ; biosynthesis ; Fibroblasts ; cytology ; drug effects ; metabolism ; Flavonoids ; pharmacology ; Flow Cytometry ; Humans ; Immunohistochemistry ; Keratins ; biosynthesis ; Periodontal Ligament ; cytology ; Phenols ; pharmacology ; Polyphenols ; Tea ; chemistry ; Vimentin ; biosynthesis

OBJECTIVETo observe the effect of bombesin noncytoskeleton form and intracellular free calcium ([Ca2+]i) concentration in PC-3 prostate cancer cell line.

METHODSImmunofluorescent histochemistry (IH) combined with laser scanning confocal microscopy (LSCM) was used to examine the expression of cytokeratin (CK) in PC-3 cells treated with definite concentrations of BBS and observe its effect on cytoskeleton form. Fluo-3/AM fluorescence technique and LSCM were adopted to measure the [Ca2+]i concentration after different concentrations (10(-9), 10(-7) and 10(-5) mol/L) of BBS were added in PC-3 cells.

RESULTSBBS (10(-5) mol/L) stimulated the expression of CK in PC-3 cells and the formation of lamellipodium, and increased the [Ca2+]i concentration, with concentration dependence.

CONCLUSIONDefinite concentrations of BBS could obviously enhance the [Ca2+] i concentration, CK expression and cytoskeleton morphology of PC-3 cells. The results provide a basis for further studies on the role of BBS in tumour researches as well as in intracellular signal transmission.

Bombesin ; pharmacology ; Calcium ; analysis ; Cytoskeleton ; drug effects ; metabolism ; Fluoroimmunoassay ; Humans ; Keratins ; biosynthesis ; Male ; Microscopy, Confocal ; Prostatic Neoplasms ; metabolism ; Serum Albumin, Bovine ; Tumor Cells, Cultured

OBJECTIVETo investigate the value of detection of AMACR (P504S), P63 and 34betaE12 cocktail in the early diagnosis of prostate cancer (PCa).

METHODSThe expressions of AMACR, P63 and 34betaE12 were examined in the biopsy specimens of 42 cases of prostate cancer, 12 cases of high-grade prostatic intraepithelial neoplasia (HGPIN) and 30 cases of benign prostatic hyperplasia (BPH) using the Maxvision single-step immunohistochemical method with triple-antibody cocktail (AMACR/P63/34betaE12) staining and double-color chromogens in single paraffin sections .

RESULTSThe expressions of AMACR, P63 and 34betaE12 were significantly different between PCa and BPH (P < 0.01). The staining of PCa was positive for AMACR and negative for P63 and 34betaE12, and the positivity rate of AMACR was 100%. BPH was strongly expressed for P63 and 34betaE12, but negatively for AMACR. The expression of AMACR was significantly different between HGPIN and BPH (P < 0.01), but not between HGPIN and PCa (P > 0.05), and the positivity rate of AMACR in HGPIN was 91.67%. However, the expressions of P63 and 34betaE12 were significantly different between HGPIN and PCa (P < 0.01), but not between HGPIN and BPH (P > 0.05), and the positivity rate of AMACR in HGPIN was 100%. The level of AMACR expression was not correlated with PCa Gleason score (P > 0.05).

CONCLUSIONAMACR is a sensitive and specific marker for PCa. P63 and 34betaE12 cocktail staining can increase the sensitivity and specificity of the basal cell detection. The immunohistochemical analysis with triple-antibody cocktail (AMACR/P63/34betaE12) staining and double-color chromogens can improve diagnostic accuracy and has an important applied value for the early diagnosis of prostate cancer.

Aged ; Carcinoma, Basal Cell ; diagnosis ; metabolism ; Early Diagnosis ; Humans ; Immunohistochemistry ; Keratins ; biosynthesis ; Male ; Membrane Proteins ; biosynthesis ; Middle Aged ; Prostatic Hyperplasia ; diagnosis ; metabolism ; Prostatic Neoplasms ; diagnosis ; metabolism ; Racemases and Epimerases ; biosynthesis

OBJECTIVETo culture interleukin-1beta (IL-1beta)-activated Schwann cells (SCs) with human hair keratins (HHKs) for artificial nerve bridge construction.

METHODSSCs purified by primary culture with or without IL-1beta activation were cultured with HHKs decorated by extracellular matrix (ECM), and the artificial nerve bridge was implanted into the defect of rat sciatic nerve. The morphology of the SCs cultured with HHKs was monitored by inverted microscope, scanning electron microscope and evaluated by immunocytochemical staining, and the expression of nerve growth factor (NGF) in the sciatic nerve was observed by in situ hybridization.

RESULTSActivated SCs showed better ability to adhere to the HHKs and grew well. The HHKs component in the artificial nerve bridge underwent degradation in the sciatic nerve defect after 3 to 4 weeks, and IL-1beta activation resulted in enhanced NGF expression in the SCs.

CONCLUSIONThe constructed artificial nerve bridge by three-dimensional culture of IL-1beta-activiated SCs with HHKs decorated by ECM promotes the repair of sciatic nerve defects and accelerates sciatic nerve regeneration.

Animals ; Animals, Newborn ; Axons ; physiology ; Cell Culture Techniques ; Cell Movement ; physiology ; Cells, Cultured ; Hair ; chemistry ; Humans ; Interleukin-1beta ; pharmacology ; Keratins ; pharmacology ; Microscopy, Electron, Scanning ; Nerve Growth Factor ; biosynthesis ; Nerve Regeneration ; drug effects ; Rats ; Rats, Sprague-Dawley ; Schwann Cells ; drug effects ; metabolism ; ultrastructure ; Sciatic Nerve ; injuries ; physiopathology ; surgery ; Tissue Engineering ; methods

This study is aimed at establishing a sensitive approach to detect disseminated tumor cells in peripheral blood and evaluate its clinical significance. A total of 198 blood samples including 168 from colorectal carcinoma (CRC) patients and 30 from healthy volunteers were examined by quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR) to evaluate the expression of carcinoembryonic antigen (CEA), cytokeratin 20 (CK20) and cytokeratin 19 (CK19) mRNA. CEA mRNA was detected in 35.8% of patients and 3.3% of controls, CK20 mRNA in 28.3% of patients and 6.7% of controls, and CK19 mRNA in 41.9% of patients and 3.3% of controls. CEA and CK20 mRNA positive ratio increased with the advancing Dukes stages, but there was no significant difference in positive ratio between any two stages (P>0.05). Also, relatively high positive ratio of CEA, CK20 and CK19 mRNA expression was observed in some CRC patients with earlier Dukes stages. A higher positive ratio was obtained when two or three detection markers were combined compared to a single marker. Our study indicates that quantitative real-time RT-PCR detection for CEA, CK20 and CK19 mRNA in peripheral blood is a valuable tool for monitoring early stage dissemination of CRC cells in blood circulation.
Adult ; Aged ; Aged, 80 and over ; Biomarkers, Tumor ; biosynthesis ; blood ; genetics ; Carcinoembryonic Antigen ; biosynthesis ; blood ; genetics ; Carcinoma ; blood ; genetics ; Colorectal Neoplasms ; blood ; genetics ; Female ; Humans ; Keratin-20 ; Keratins ; biosynthesis ; blood ; genetics ; Male ; Middle Aged ; RNA, Messenger ; blood ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Sensitivity and Specificity

Metaplastic breast carcinoma is very rare, and metaplastic carcinoma with chondroid differentiation is even rarer. Here, we report a case of metaplastic carcinoma with extensive chondroid differentiation mimicking chondrosarcoma that was challenging to diagnose. The tumor was characterized by an abundant chondromyxoid matrix. The definitive area of classic invasive ductal carcinoma was minimal. The peripheral portion of the tumor showed increased cellularity with pleomorphism and definitive invasive growth. Tumor cells in the chondrosarcomatous areas were diffusely immunoreactive for S-100 protein, patchy positive for cytokeratin, but negative for epithelial membrane antigen (EMA). Tumor cells in carcinomatous areas were diffusely positive for cytokeratin, S-100 protein, and patchy positive for EMA. In both areas, tumor cells were negative for smooth muscle actin (SMA) and CD34, while oncoprotein p53 was overexpressed. When pathologists encounter breast tumors with chondroid differentiation, careful sampling and immunohistochemistry for cytokeratin and SMA are most helpful to differentiate metaplastic carcinoma from malignant phyllodes tumor and malignant adenomyoepithelioma.
S100 Proteins/chemistry ; Neoplasm Metastasis ; Muscle, Smooth/pathology ; Middle Aged ; Metaplasia ; Keratins/metabolism ; Immunohistochemistry ; Humans ; Female ; Cell Differentiation ; Carcinoma/*complications/metabolism/pathology ; CA-15-3 Antigen/metabolism ; Breast Neoplasms/complications/metabolism/*pathology ; Antigens, CD34/biosynthesis ; Actins/metabolism

OBJECTIVETo improve the level of clinical diagnosis and differential diagnosis of benign and malignant prostate lesions.

METHODSOne hundred and nine cases of prostate cancer and prostate hyperplasia were evaluated by the expression of high molecular weight cytokeratin (CK34BE12), prostate specific antigen (PSA) and protein P53 gene using the immunohistochemical technique.

RESULTSThe basal-cells in all of the benign lesions were stained with the CK34BE12 and PSA, while it had not immunoreactivity with P53. In contrast, the prostate carcinoma were not stained or partly stained with the CK34BE12 and PSA, but P53 show significant immunoreactivity with the tissue.

CONCLUSIONBased on the routine histological studies with the expression of CK34BE12 and PSA together, they can indicate the existence of basal-cell distinctly and show indirectly whether the basal-cell is integrated. Combining the expression of P53 to determine the existence of cancer gene, it can help to distinguish benign and malignant prostate lesions.

Diagnosis, Differential ; Humans ; Immunohistochemistry ; Keratins ; biosynthesis ; Male ; Prostate-Specific Antigen ; biosynthesis ; Prostatic Neoplasms ; diagnosis ; metabolism ; pathology ; Staining and Labeling ; Tumor Suppressor Protein p53 ; biosynthesis

OBJECTIVETo study immunohistochemical expression of cytokeratin19 (CK19), galectin-3 (Gal-3) and HBME-1 in thyroid lesions and to assess their usefulness as markers in the differential diagnoses of thyroid nodular lesions.

METHODSImmunohistochemical staining was performed on formalin-fixed paraffin-embedded tissue of 21 cases of nodular goiters, 14 cases of toxic goiters, 15 cases of follicular adenomas (FA), 13 cases of follicular carcinomas (FC), 13 cases of follicular variant papillary carcinomas (FVPC) and 48 cases of classic papillary carcinomas (CPC).

RESULTSAll three markers were expressed in the cytoplasm with no or weak expression in benign lesions and diffuse and strong in malignant cases. Positive expressions of CK19, Gal-3 and HBME-1 were present in 11of 21, two of 21, four of 21 in nodular goiters, seven of 14, one of 14, one of 14 in toxic goiters, nine of 15, two of 15, two of 15 in FA, 10 of 13, eight of 13, seven of 13 in FC, 13 of 13, 11 of 13, 12 of 13 in FVPC, and 48 of 48, 45 of 48, 46 of 48 in CPC. The expression rates of the three markers between benign lesions (nodular goiters, toxic goiters and FA) and malignant lesions (FA, FVPC and CPC) were statistically significant. Among the three follicular lesions (FA, FC and FVPC), the differences were statistically significant as well. Nine, seven and six cases were negative for all three markers in nodular goiters, toxic goiters and FA, respectively. Only one case in FC was negative for all three markers, no case was all negative in FVPC and CPC; the rate of one case with two or more positive marker expression in nodular goiters, toxic goiters, FA, FC, FVPC and PC was 14.2% (3/21), 21.43% (3/14), 20.0% (3/15), 69.2% (9/13), 92.3% (12/13), 100.0% (48/48), the differences between benign lesions and malignant lesions and between FA, FC and FVPC were also statistically significant.

CONCLUSIONSImmunohistochemical stains of CK19, Gal-3 and HBME-1, especially when used in combination, can be an important adjunct to the histopathological diagnoses of thyroid lesions.

Adenocarcinoma, Follicular ; chemistry ; diagnosis ; pathology ; Adenoma ; chemistry ; diagnosis ; pathology ; Biomarkers, Tumor ; biosynthesis ; genetics ; Carcinoma, Papillary, Follicular ; pathology ; Diagnosis, Differential ; Galectin 3 ; biosynthesis ; genetics ; Goiter, Nodular ; metabolism ; pathology ; Humans ; Immunohistochemistry ; Keratins ; biosynthesis ; genetics ; Thyroid Neoplasms ; chemistry ; diagnosis ; pathology ; Thyroid Nodule ; chemistry ; diagnosis ; pathology

OBJECTIVETo investigate the expression of CEA mRNA and CK(19) mRNA in peripheral blood cells of patients with lung cancer and evaluate its clinical significance.

METHODSPeripheral blood nucleated cells of 50 patients with lung cancer were studied by RT-PCR to detect the expression of CEA mRNA and CK(19) mRNA.

RESULTSThe combined positive rate of CEA mRNA and CK(19) mRNA in patients with lung cancer (82.0%) was significantly higher than that in patients with benign lung diseases (28.0%) and healthy volunteers (8.1%) (P < 0.001). The expression rate had no relation to the clinical staging or histological type. Compared with single detection, combined detection increased the detection rate but did not decrease the specificity.

CONCLUSIONCombined detection of CEA mRNA and CK(19) mRNA expression in peripheral blood nucleated cells increase the sensitivity of detecting hematogenous dissemination of cancer cells. Long-term survival analysis and more specimens would be helpful for evaluating its clinical significance.

Adenocarcinoma ; blood ; pathology ; Adult ; Aged ; Carcinoembryonic Antigen ; biosynthesis ; blood ; genetics ; Carcinoma, Small Cell ; blood ; pathology ; Carcinoma, Squamous Cell ; blood ; pathology ; Female ; Humans ; Keratins ; biosynthesis ; blood ; genetics ; Lung Neoplasms ; blood ; pathology ; Male ; Middle Aged ; Neoplasm Staging ; RNA, Messenger ; blood ; genetics ; Reverse Transcriptase Polymerase Chain Reaction