[Pulmonary artery stenting in chronic thromboembolic pulmonary hypertension: a report of 2 cases].

Chronic thromboembolic pulmonary hypertension (CTEPH) is a pulmonary vascular disease characterized by an insidious onset, progressive deterioration, and poor prognosis. It is distinguished by the thrombotic organization within the pulmonary arteries, leading to vascular stenosis or occlusion. This results in a progressive increase in pulmonary vascular resistance and pulmonary arterial pressure, ultimately leading to right heart failure. In recent years, balloon pulmonary angioplasty (BPA) has emerged as an effective treatment option for patients ineligible for pulmonary endarterectomy (PEA). However, the use of stents in patients with suboptimal balloon dilation remains controversial. This article describes two cases of chronic thromboembolic pulmonary hypertension (CTEPH) in which balloon angioplasty yielded unsatisfactory results, subsequently leading to stent placement. Following stent implantation, there was improved blood flow, significant reduction in pulmonary arterial pressure, and notable alleviation of patient symptoms. One-year follow-up showed no recurrence of stenosis within the stent, suggesting potential guidance for the use of pulmonary artery stenting as a treatment modality for CTEPH. This report provided new insights into the therapeutic approach for CTEPH.

Effects of CD133 on the biological features and in vivo oncogenicity of glioma cells.

This study aimed to investigate the effect of CD133 on the proliferation and migration of glioma cells and expressions of genes related to cancer stem cells/tumor stem cells (CSC/TSC) as well as their in-vivo oncogenicity. CD133-overexpressing U251-CD133 and U251-mock glioma cells were constructed. The effect of CD133 on in-vitro proliferation and the neurosphere-forming ability of glioma cells was determined by cell count and neurosphere formation assay. Real-Time PCR was performed to detect the expressions of CSC/TSC-related genes in the CD133-transfected cells. Nude mouse subcutaneous tumor formation assay was used to determine the effect of CD133 on the in-vivo oncogenicity of glioma cells. In serum-containing medium, human CD133 had no impact on the proliferation of U251 glioma cells, but the neural stem cells placed in serum-free medium promoted neurosphere formation. In the presence of CD133, the expressions of CSC/TSC-related genes were upregulated to varying degrees in glioma cells; CD133 greatly enhanced the in-vivo oncogenicity. In conclusion, CD133 promoted the upregulation of CSC/TSC-related genes in glioma cells, while enhancing the neurosphere-forming ability and in-vivo oncogenicity.

[A study of bacterial distribution and drug resistance in skin and soft tissue infection].

Objective: To investigate pathogenic bacteria and drug resistance in the patients with skin and soft tissue infection in order to provide the scientific evidences for clinical reasonable use of antibiotics. Methods: A retrospective analysis was performed on patients with skin and soft tissue infections in Department of Dermatology, Peking University Third Hospital from January 2012 to December 2017. Pus, secretions, skin lesions, urine, throat swabs, and alveolar lavage fluid were collected for bacterial culture, bacterial species were identified by VITEK2 Compact system and BD-Bruker MALDI Biotyper system. Drug resistance was detected by K-B agar diffusion method recommended by CLSI. Results: A total of 392 strains of bacteria were isolated from 327 patients distributed in 21 genus and 56 species, of which 225 were gram-positive cocci (57.40%), 114 were gram-negative rods (29.08%), 46 were gram-positive rods (11.73%), and 7 were gram-negative cocci (1.79%). The top 3 bacteria were Staphylococcus aureus 91(23.21%), Staphylococcus epidermidis 42 (10.71%), and Pseudomonas aeruginosa 24 (6.12%). Staphylococcus had a high rate of resistance to penicillin and erythromycin (>50%). Gram-negative rods were resistant to ampicillin (86.1%), and also had certain resistance to most second-generation and some third-generation cephalosporin (about 50%). There was no significant change in the drug resistance rate of MRSA compared to MSSA. Only the resistance rate to tetracycline was statistically different (P<0.05). Conclusion: The emergence of drug-resistant strains is an important factor leading to refractory infections. There are a wide range of pathogenic bacteria species among the skin and soft tissue infection patients, and antimicrobial drugs should be chosen wisely according to drug sensitivity.

[Diagnostic value of quantitative pharmacokinetic parameters and relative quantitative pharmacokinetic parameters in breast lesions with dynamic contrast-enhanced MRI].

Objective: To explore the differential between the value of dynamic contrast-enhanced MRI quantitative pharmacokinetic parameters and relative pharmacokinetic quantitative parameters in breast lesions. Methods: Retrospective analysis of 255 patients(262 breast lesions) who was obtained by clinical palpation , ultrasound or full-field digital mammography , and then all lessions were pathologically confirmed in Zhongda Hospital, Southeast University from May 2012 to May 2016. A 3.0 T MRI scanner was used to obtain the quantitative MR pharmacokinetic parameters: volume transfer constant (K(trans)), exchange rate constant (k(ep))and extravascular extracellular volume fraction (V(e)). And measured the quantitative pharmacokinetic parameters of normal glands tissues which on the same side of the same level of the lesions; and then calculated the value of relative pharmacokinetic parameters: rK(rans)、rk(ep) and rV(e).To explore the diagnostic value of two pharmacokinetic parameters in differential diagnosis of benign and malignant breast lesions using receiver operating curves and model of logistic regression. Results: (1)There were significant differences between benign lesions and malignant lesions in K(trans) and k(ep) (t=15.489, 15.022, respectively, P<0.05), there were no significant differences between benign lesions and malignant lesions in V(e)(t=-2.346, P>0.05). The areas under the ROC curve(AUC)of K(trans), k(ep) and V(e) between malignant and benign lesions were 0.933, 0.948 and 0.387, the sensitivity of K(trans), k(ep) and V(e) were 77.1%, 85.0%, 51.0% , and the specificity of K(trans), k(ep) and V(e) were 96.3%, 93.6%, 60.8% for the differential diagnosis of breast lesions if taken the maximum Youden's index as cut-off. (2)There were significant differences between benign lesions and malignant lesions in rK(trans), rk(ep) and rV(e) (t=14.177, 11.726, 2.477, respectively, P<0.05). The AUC of rK(trans), rk(ep) and rV(e) between malignant and benign lesions were 0.963, 0.903 and 0.575, the sensitivity of rK(trans), rk(ep) and rV(e) were 85.6%, 71.9%, 52.9% , and the specificity of rK(trans), rk(ep) and rV(e) were 94.5%, 92.7%, 60.6% for the differential diagnosis of breast lesions.(3)There was no significant difference in the area under the ROC curve between the predictive probability of quantitative pharmacokinetic parameters and the prediction probability of relative quantitative pharmacokinetic parameters(Z=0.867, P=0.195). Conclusion: There was no significant difference between the quantitative parameter values (K(trans,) k(ep)) and the relative quantitative parameter values (rK(trans,) rk(ep)) in diagnosis of breast lesions, which were important parameters in differential diagnosis of benign and malignant breast lesions.

[Effect of probucol on preventing contrast-induced nephropathy in patients undergoing percutaneous coronary intervention].

Objective: To investigate the preventive effect, possible mechanism and safety of probucol on contrast-induced nephropathy (CIN) after percutaneous coronary intervention (PCI) in patients with coronary heart disease (CHD). Methods: A total of 641 patients with coronary heart disease were consecutively enrolled from Department of Cardiology, in Tianjin Chest Hospital, Tianjin TEDA International Cardiovascular Hospital, Tianjin First Central Hospital, Tianjin Fourth Central Hospital. They were randomly divided into probucol group (n=321) and control group (n=320). The probucol group was given oral probucol 500 mg twice daily for day 0 to day 3 after PCI; the control group was given only conventional therapy. All patients were given intravenous drip 0.9% sodium chloride solution before 12 to 24 hours of operation. The levels of serum creatinine (Scr), blood urea nitrogen (BUN), evaluate glomerular filtration rate (eGFR), cystatin C (Cys-C), and high-sensitivity C-reactive protein (hs-CRP), neutrophil gelatinase associated lipocalin (NGAL), superoxide dismutase (SOD) and glutathione (GSH) were measured before and 72 h after the PCI operation in both groups. The incidence rates of CIN, the adverse events during hospitalization and postoperative 14-day follow-up were recorded in two groups. Results: There was no statistically significantly difference in the levels of Scr, BUN, eGFR, Cys-C, hs-CRP, NGAL, SOD and GSH between the two groups before PCI (P>0.05). The levels of serum Scr, BUN, Cys-C, hs-CRP, NGAL, SOD and GSH after operation in the two groups were higher than those before the operation (P<0.05). The levels of hs-CRP and NGAL in the probucol group were lower than those in the control group [(10±4) vs (11±4)mg/L, (25±8)vs (34±7)U/ml, P<0.05]. The levels of eGFR, SOD and GSH in probucol group were higher than those in control group [(80±27) vs (72±26) ml·min(-1)·1.73 m(-2,) (67±9) vs (58±8)U/ml, (4.6±0.9) vs (3.9±0.8)U/ml, P<0.05]. The incidence of CIN was 4.0% in the probucol group and 10.9% in the control group, and the difference was statistically significant (P<0.05, χ(2)=-3.31). Multivariate Logistic regression analysis showed that probucol was an independent protective factor for CIN (OR=0.334, 95%CI 0.172-0.648, P=0.001). There were no adverse events such as myasthenia gravis, abnormal liver function and cardiovascular events during the hospitalization and 14-day follow-up. Conclusions: Probucol can reduce the incidence of contrast-induced nephropathy after PCI. The protection mechanism is related with its anti-inflammatory and anti-oxidative stress effects, and it has good safety.

Homology-based analysis of the GRAS gene family in tobacco.

Members of the GRAS gene family are important transcriptional regulators. In this study, 21 GRAS genes were identified from tobacco, and were classified into eight subgroups according to the classification of Arabidopsis thaliana. Here, we provide a preliminary overview of this gene family in tobacco, describing the gene structure, gene expression, protein motif organization, phylogenetic analysis, and comparative analysis in tobacco, Arabidopsis, and rice. Using the sequences of 21 GRAS genes in Arabidopsis to search against the American tobacco genome database, 21 homologous GRAS genes in tobacco were identified. Sequence analysis indicates that these GRAS proteins have five conserved domains, which is consistent with their counterparts in other plants. Phylogenetic analyses divided the GRAS gene family into eight subgroups, each of which has distinct conserved domains and biological functions. Furthermore, the expression pattern of these 21 GRAS genes reveals that most are expressed in all six tissues studied; however, some have tissue specificity. Taken together, this comprehensive analysis will provide a rich resource to assist in the study of GRAS protein functions in tobacco.

YTHDF3 modulates the progression of breast cancer cells by regulating FGF2 through m6A methylation.

Introduction: Breast cancer (BC) is a prevailing malignancy among women, and its inconspicuous development contributes significantly to mortality. The RNA N6-methyladenosine (m6A) modification represents an emerging mechanism for gene expression regulation, with the active involvement of the YTH N6-methyladenosine RNA binding protein 3 (YTHDF3) in tumor progression across multiple cancer types. Nonetheless, its precise function in breast cancer necessitates further investigation. Methods: The expression of YTHDF3 in both cell lines and patient tissues was examined using Western blotting, reverse transcription quantitative PCR (RT-qPCR), and immunohistochemistry (IHC) techniques. Bioinformatics analysis of methylated RNA immunoprecipitation sequencing (MeRIP-seq) and transcriptome RNA sequencing (RNA-seq) data was employed to screen for the target genes of YTHDF3. The main focus of this study was to investigate the in vitro biological functions of YTHDF3. The specific binding of YTHDF3 to its target genes and its correlation with m6A methylation were studied through RNA pull-down, RNA immunoprecipitation, and co-immunoprecipitation experiments. The protein regulatory mechanisms of downstream genes of YTHDF3 were assessed using protein stability analysis. Furthermore, the biological functions of YTHDF3 and its target genes in breast cancer cells were validated through CRISPR-Cas9 technology and rescue experiments. Results: By constructing a risk model using the TCGA database, YTHDF3 was identified as a high-risk factor among m6A methylation factors. Subsequent investigations revealed its elevated expression in various subtypes of breast cancer, accompanied by poor prognosis. MeRIP-seq analysis further revealed fibroblast growth factor 2 (FGF2) as a downstream gene of YTHDF3. Knockdown of YTHDF3 in breast cancer cells led to significant inhibition of cell self-renewal, migration, and invasion abilities in vitro. Mechanistically, YTHDF3 specifically recognized the methylated transcript of FGF2 within its coding sequence (CDS) region, leading to the inhibition of FGF2 protein degradation. Moreover, depletion of FGF2 markedly suppressed the biological functions of breast cancer cells, while reducing FGF2 expression in YTHDF3-overexpressing breast cancer cell lines substantially alleviated the malignant progression. Conclusions: In summary, our study elucidates the role of YTHDF3 as an oncogene in maintaining FGF2 expression in BC cells through an m6A-dependent mechanism. Additionally, we provide a potential biomarker panel for prognostic prediction in BC.

[Reassessment of practice of Chinese surgeons since introduction of the watch and wait strategy after neoadjuvant therapy for rectal cancer].

Objective: To investigate perspectives and changes in treatment selection by Chinese surgeons since introduction of the watch-and-wait approach after neoadjuvant therapy for rectal cancer. Methods: A cross-sectional survey was conducted using a questionnaire distributed through the "Wenjuanxing" online survey platform. The survey focused on the recognition and practices of Chinese surgeons regarding the strategy of watch-and-wait after neoadjuvant therapy for rectal cancer and was disseminated within the China Watch-and-Wait Database (CWWD) WeChat group. This group targets surgeons of deputy chief physician level and above in surgical, radiotherapy, or internal medicine departments of nationally accredited tumor-specialist or comprehensive hospitals (at provincial or municipal levels) who are involved in colorectal cancer diagnosis and treatment. From 13 to 16 December 2023, 321 questionnaires were sent with questionnaire links in the CWWD WeChat group. The questionnaires comprised 32 questions encompassing: (1) basic physician characteristics (including surgical volume); (2) assessment methods and criteria for clinical complete response (cCR); (3) patients eligible for watch-and-wait; (4) neoadjuvant therapies and other measures for achieving cCR; (5) willingness to implement watch-and-wait and factors influencing that willingness; (6) risks and monitoring of watch-and-wait; (7) subsequent treatment and follow-up post watch-and-wait; (8) suggestions for development of the CWWD. Descriptive statistics were employed for data analysis, with intergroup comparisons conducted using the χ2 or Fisher's exact probability tests. Results: The response rate was 31.5%, comprising 101 responses from the 321 individuals in the WeChat group. Respondents comprised 101 physicians from 70 centers across 23 provinces, municipalities, and autonomous regions nationwide, 85.1% (86/101) of whom represented provincial tertiary hospitals. Among the respondents, 87.1% (88/101) had implemented the watch-and-wait strategy. The approval rate (65.6%, 21/32) and proportion of patients often informed (68.8%, 22/32) were both significantly higher for doctors in oncology hospitals than for those in general hospitals (27.7%, 18/65; 32.4%, 22/68) (χ2=12.83, P<0.001; χ2=11.70, P=0.001, respectively). The most used methods for diagnosing cCR were digital rectal examination (90.1%, 91/101), colonoscopy (91.1%, 92/101), and rectal T2-weighted magnetic resonance imaging (86.1%, 87/101). Criteria used to identify cCR comprised absence of a palpable mass on digital rectal examination (87.1%, 88/101), flat white scars or new capillaries on colonoscopy (77.2%, 78/101), absence of evident tumor signals on rectal T2-weighted sequences or T2WI low signals or signals equivalent to the intestinal wall (83.2%, 84/101), and absence of tumor hyperintensity on diffusion-weighted imaging with no corresponding hypointensity on apparent diffusion coefficient maps (66.3%, 67/101). As for selection of neoadjuvant regimen and assessment of cCR, 57.4% (58/101) of physicians preferred a long course of radiotherapy with or without induction and/or consolidation capecitabine + oxaliplatin, whereas 25.7% (26/101) preferred immunotherapy in combination with chemotherapy and concurrent radiotherapy. Most (96.0%, 97/101) physicians believed that the primary lesion should be assessed ≤12 weeks after completion of radiotherapy. Patients were frequently informed about the possibility of achieving cCR after neoadjuvant therapy and the strategy of watch-and-wait by 43.6% (44/101) of the responding physicians and 38.6% (39/101) preferred watch-and-wait for patients who achieved cCR or near cCR after neoadjuvant therapy for rectal cancer. Capability for multiple follow-up evaluations (70.3%, 71/101) was a crucial factor influencing physicians' choice of watch-and-wait after cCR. The proportion who patients who did not achieve cCR and underwent surgical treatment was lower in provincial tertiary hospitals (74.2%, 23/31) than in provincial general hospitals (94.5%, 52/55) and municipal hospitals (12/15); these differences are statistically significant (χ2=7.43, P=0.020). The difference between local recurrence and local regrowth was understood by 88.1% (89/101) of respondents and 87.2% (88/101) agreed with monitoring every 3 months for 5 years. An increase in local excision or puncture rates to reduce organ resections in patients with pCR was proposed by 64.4% (65/101) of respondents. Conclusion: Compared with the results of a previous survey, Chinese surgeons' awareness of the watch-and-wait concept has improved significantly. Oncologists in oncology hospitals are more aware of the concept of watch-and-wait.

Hepatitis C virus genotype diversity in Ningxia Hui autonomous region, Northwestern China.

Hepatitis C virus (HCV) genotypes vary greatly in different regions. The aim of this study is to investigate the distribution of HCV genotypes in HCV infected patients, in Ningxia Hui Autonomous Region. Nucleic acid extraction and amplification were performed with test kits on 153 HCV infected patients serum samples. The HCV viral load was measured using reverse transcriptase PCR (RT-PCR) and HCV genotypes were determined. Among the 153 HCV-infected patients, 56 had genotype (GT)1b (36.60%), 45 had GT2a (29.40%), 23 had GT3a (15.00%), 14 had GT3b (9.20%),13 had GT6a (8.50%), 1 had GT1g (0.70%), 1 had GT6xa (0.70%). In GT1b, 21.40% were female and 78.60% were male; in GT2a, 42.20% were female and 57.80% were male;Males were most prevalent in genotypes 1b(39.30%), while female were most prevalent in genotype 2a(46.30%). Rare GT1g and GT6xa were also detected in males. The 41-50 year age group had the highest HCV prevalence of 32.00%. HCV GT1b is the predominant HCV genotype in Ningxia Hui Autonomous Region.

[Total neoadjuvant therapy followed by watch and wait approach or organ preservation for MRI stratified low-risk rectal cancer: early result from a prospective, single arm trial].

Objective: To explore the safety and efficacy of watch and wait strategy and organ preservation surgery after total neoadjuvant treatment for MRI stratified low-risk rectal cancer. Methods: A prospective single arm phase Ⅱ trial developed at Department of Gastrointestinal Cancer, Peking University Cancer Hospital & Institute was preliminarily analyzed. Subjects were enrolled from August 2016 to January 2019. Low-risk rectal cancer with following MRI features were recruited: mid-low tumor, mrT2-3b, MRF (-), EMVI (-), CRM (-), differentiation grade 1-3. Patients received intensity-modulated radiotherapy (IMRT) 50.6 Gy/22f with concurrent capecitabine and 4 cycles of consolidation CAPEOX. Patients with cCR/near-cCR confirmed by physical examination, rectal MRI, endoscopy, and serum CEA were recommended for watch & wait approach or local excision (LE). The main study outcomes were 2-year organ preservation rate (OPR) and sphincter preservation rate (SPR). Results: Thirty-eight patients were eligible for analysis, including 24 males and 14 females with median age of 56 years; 9 cases of mrT2 (23.7%), 14 cases of mrT3a (36.8%) and 15 cases of mrT3b (39.5%); 5 cases of well differentiated adenocarcinoma (13.2%), 32 cases of moderately differentiated adenocarcinoma (84.2%) and 1 case of mucinous adenocarcinoma (2.6%). Carcinoemobryonic antigen (CEA) was elevated before treatment in 1 case. One case (2.6%) of grade 3 radiation dermatitis occurred during IMRT; 18 cases (47.4%) occurred grade 3 to 4 adverse events during consolidation chemotherapy. After total neoadjuvant treatment, the cCR and near-cCR rates were 42.1% (16/38) and 23.7% (9/38), respectively, while non-cCR rate was 34.2% (13/38). Twenty patients (20/38, 52.6%) of cCR or near-cCR underwent watch & wait approach, with a local regrowth rate of 20% (4/20). Four patients received LE, including one salvage LE. Thirteen patients (4 were ypCR) received radical resection, including 10 cases of initial low anterior resections (LAR), 1 cases of initial abdominal perineal resection (APR) and 2 cases of salvage LAR, four patients refused operation. The median follow-up time was 23.5 (8.5-38.3) months. At the last interview of follow-up, the OPR and SPR were 52.6% (20/38) and 84.2% (32/38), respectively. Only one patient developed lung metastasis and no local recurrence occurred after radical resection or LE. Conclusion: Total neoadjuvant treatment for low-risk rectal cancer achieves high cCR/near-cCR rate, with increased probability of receiving watch and wait approach and organ preservation in this subgroup.

The 50- and 58-kdalton keratin classes as molecular markers for stratified squamous epithelia: cell culture studies.

The keratins are a highly heterogeneous group of proteins that form intermediate filaments in a wide variety of epithelial cells. These proteins can be divided into at least seven major classes according to their molecular weight and their immunological reactivity with monoclonal antibodies. Tissue-distribution studies have revealed a correlation between the expression of specific keratin classes and different morphological features of in vivo epithelial differentiation (simple vs. stratified; keratinized vs. nonkeratinized). Specifically, a 50,000- and a 58,000-dalton keratin class were found in all stratified epithelia but not in simple epithelia, and a 56,500- and a 65-67,000-dalton keratin class were found only in keratinized epidermis. To determine whether these keratin classes can serve as markers for identifying epithelial cells in culture, we analyzed cytoskeletal proteins from various cultured human cells by the immunoblot technique using AE1 and AE3 monoclonal antikeratin antibodies. The 56,500- and 65-67,000-dalton keratins were not expressed in any cultured epithelial cells examined so far, reflecting the fact that none of them underwent morphological keratinization. The 50,000- and 58,000-dalton keratin classes were detected in all cultured cells that originated from stratified squamous epithelia, but not in cells that originated from simple epithelia. Furthermore, human epidermal cells growing as a monolayer in low calcium medium continued to express the 50,000- and 58,000-dalton keratin classes. These findings suggest that the 50,000- and 58,000-dalton keratin classes may be regarded as "permanent" markers for stratified squamous epithelial cells (keratinocytes), and that the expression of these keratin markers does not depend on the process of cellular stratification. The selective expression of the 50,000- and 58,000-dalton keratin classes, which are synthesized in large quantities on a per cell basis, may explain the high keratin content of cultured keratinocytes.

Differentiation-related expression of a major 64K corneal keratin in vivo and in culture suggests limbal location of corneal epithelial stem cells.

In this paper we present keratin expression data that lend strong support to a model of corneal epithelial maturation in which the stem cells are located in the limbus, the transitional zone between cornea and conjunctiva. Using a new monoclonal antibody, AE5, which is highly specific for a 64,000-mol-wt corneal keratin, designated RK3, we demonstrate that this keratin is localized in all cell layers of rabbit corneal epithelium, but only in the suprabasal layers of the limbal epithelium. Analysis of cultured corneal keratinocytes showed that they express sequentially three major keratin pairs. Early cultures consisting of a monolayer of "basal" cells express mainly the 50/58K keratins, exponentially growing cells synthesize additional 48/56K keratins, and postconfluent, heavily stratified cultures begin to express the 55/64K corneal keratins. Cell separation experiments showed that basal cells isolated from postconfluent cultures contain predominantly the 50/58K pair, whereas suprabasal cells contain additional 55/64K and 48/56K pairs. Basal cells of the older, postconfluent cultures, however, can become AE5 positive, indicating that suprabasal location is not a prerequisite for the expression of the 64K keratin. Taken together, these results suggest that the acidic 55K and basic 64K keratins represent markers for an advanced stage of corneal epithelial differentiation. The fact that epithelial basal cells of central cornea but not those of the limbus possess the 64K keratin therefore indicates that corneal basal cells are in a more differentiated state than limbal basal cells. These findings, coupled with the known centripetal migration of corneal epithelial cells, strongly suggest that corneal epithelial stem cells are located in the limbus, and that corneal basal cells correspond to "transient amplifying cells" in the scheme of "stem cells----transient amplifying cells----terminally differentiated cells."

The role of keratin subfamilies and keratin pairs in the formation of human epidermal intermediate filaments.

The four major keratins of normal human epidermis (molecular mass 50, 56.5, 58, and 65-67 kD) can be subdivided on the basis of charge into two subfamilies (acidic 50-kD and 56.5-kD keratins vs. relatively basic 58-kD and 65-67-kD keratins) or subdivided on the basis of co-expression into two "pairs" (50-kD/58-kD keratin pair synthesized by basal cells vs. 56.5-kD/65-67-kD keratin pair expressed in suprabasal cells). Acidic and basic subfamilies were separated by ion exchange chromatography in 8.5 M urea and tested for their ability to reassemble into 10-nm filaments in vitro. The two keratins in either subfamily did not reassemble into 10-nm filaments unless combined with members of the other subfamily. While electron microscopy of acidic and basic keratins equilibrated in 4.5 M urea showed that keratins within each subfamily formed distinct oligomeric structures, possibly representing precursors in filament assembly, chemical cross-linking followed by gel analysis revealed dimers and larger oligomers only when subfamilies were combined. In addition, among the four major keratins, the acidic 50-kD and basic 58-kD keratins showed preferential association even in 8.5 M urea, enabling us to isolate a 50-kD/58-kD keratin complex by gel filtration. This isolated 50-kD/58-kD keratin pair readily formed 10-nm filaments in vitro. These results demonstrate that in tissues containing multiple keratins, two keratins are sufficient for filament assembly, but one keratin from each subfamily is required. More importantly, these data provide the first evidence for the structural significance of specific co-expressed acidic/basic keratin pairs in the formation of epithelial 10-nm filaments.

Differentiation-dependent changes in the solubility of a 195-kD protein in human epidermal keratinocytes.

We have prepared a monoclonal antibody, AE11, that recognizes specifically a 195-kD protein (pI 5.4) of human keratinocytes. This antigen constitutes approximately 0.01-0.1% of total protein in keratinocytes of skin, esophagus, and cornea, and is readily detectable in these cells by immunofluorescent staining and immunoblotting. However, it is barely detectable in MCF mammary carcinoma cells and HeLa cells, and is undetectable in nonepithelial cell types. Results from serial extraction experiments have shown that this protein exists in two distinct pools: a Tris-soluble, and a Tris-insoluble but urea- or SDS-soluble one. The distribution of the 195-kD protein between these two pools appears to be differentiation-related, since relatively undifferentiated cells selected by a low-calcium medium contain primarily the soluble form, while highly differentiated cells contain mainly the insoluble form. Data from immunofluorescent staining and trypsin-sensitivity experiments suggest that the soluble form is cytoplasmic, whereas the insoluble form is submembranously located at the cell periphery of upper, differentiated cells. The insoluble, cell peripheral form of the 195-kD antigen increases progressively during epidermal differentiation; its insolubility appears to be related to the formation of disulfide-bond(s). These results indicate that the 195-kD protein, which has recently been suggested to be involved in cornified envelope formation (Simon, M., and H. Green, 1985, Cell, 36:827-834), undergoes significant changes in its solubility characteristics and intracellular location during keratinocyte maturation.

Classification of epidermal keratins according to their immunoreactivity, isoelectric point, and mode of expression.

Human epidermal keratinocytes express under various growth conditions a total of at least nine keratins that can be divided into two subfamilies. Subfamily A comprises 40-, 46-, 48-, 50-/50'-, and 56.5-kilodalton (kd) keratins which are relatively acidic (pI less than 5.5) and, with the exception of 46-kd keratin, are recognized by AE1 monoclonal antibody. Subfamily B comprises 52-, 56-, 58-, and 65-67-kd keratins which are relatively basic (pI greater than 6) and are recognized by AE3 monoclonal antibody. Within each keratin subfamily, there is a constant member (50-/50'- and 58-kd keratins of the subfamilies A and B, respectively) that is always expressed. The other seven keratins of both subfamilies are variable members whose expression depends upon the cellular differentiated state, which is in turn modulated by the growth environment. The 56.5-kd keratin (subfamily A) and the 65-67-kd keratins (subfamily B) are coordinately expressed during keratinization. In contrast, the 40-, 46-, and 48-kd keratins (subfamily A) and the 52- and 56-kd keratins (subfamily B) are characteristic of cultured epidermal cells forming nonkeratinized colonies. These results demonstrate that human epidermal keratins can be classified according to their reactivity with monoclonal antikeratin antibodies, isoelectric point, and mode of expression. The classification of keratins into various subgroups may have important implications for the mechanisms of epidermal differentiation, the evolution of keratin heterogeneity, and the use of keratin markers for tumor diagnosis.

Monoclonal antibody analysis of keratin expression in epidermal diseases: a 48- and 56-kdalton keratin as molecular markers for hyperproliferative keratinocytes.

The polypeptide composition of epidermal keratin varies in disease. To better understand the biological meaning of these variations, we have analyzed keratins from a number of human epidermal diseases by the immunoblot technique using AE1 and AE3 monoclonal antikeratin antibodies. The results reveal a continuous spectrum of keratin expression ranging from one closely resembling the normal in vivo pattern to one almost identical to cultured epidermal keratinocytes. Specifically, a 50-kilodalton (kd) (AE1-positive) and a 58-kd (AE3-positive) keratin are present in all diseases, supporting the concept that they represent "permanent" markers for keratinocytes. A 56.5-kd (AE1) and a 65-67-kd (AE3) keratin, previously shown to be markers for keratinization, are expressed only by lesions retaining a keratinized morphology. A 48-kd (AE1) and a 56-kd (AE3) keratin are present in all hyperproliferative (para- or nonkeratinized) disorders, but not in normal abdominal epidermis or in ichthyosis vulgaris which is a nonhyperproliferative disease. These two keratins have previously been found in various nonepidermal keratinocytes undergoing hyperproliferation, suggesting that these keratins are not epidermis-specific and may represent markers for hyperproliferative keratinocytes in general. In various epidermal diseases, there is a reciprocal expression of the (keratin) markers for hyperproliferation and keratinization, supporting the mutual exclusiveness of the two cellular events. Moreover, our results indicate that, as far as keratin expression is concerned, cultured human epidermal cells resemble and thus may be regarded as a model for epidermal hyperplasia. Finally, the apparent lack of any major, disease-specific keratin changes in the epidermal disorders studied so far implies that keratin abnormalities probably represent the consequence, rather than the cause, of these diseases.

The fibrillar substructure of keratin filaments unraveled.

We show that intermediate-sized filaments reconstituted from human epidermal keratins appear unraveled in the presence of phosphate ions. In such unraveling filaments, up to four "4.5-nm protofibrils" can be distinguished, which are helically twisted around each other in a right-handed sense. Lowering the pH of phosphate-containing preparations causes the unraveling filaments to further dissociate into "2-nm protofilaments." In addition, we find that reconstitution of keratin extracts in the presence of small amounts of trypsin yields paracrystalline arrays of 4.5-nm protofibrils with a prominent 5.4-nm axial repeat. Limited proteolysis of intact filaments immobilized on an electron microscope grid also unveils the presence of 4.5-nm protofibrils within the filament with the same 5.4-nm axial repeat. These results, together with other published data, are consistent with a 10-nm filament model based on three distinct levels of helical organization: (a) the 2-nm protofilament, consisting of multi-chain extended alpha-helical segments coiled around each other; (b) the 4.5-nm protofibril, being a multi-stranded helix of protofilaments; and (c) the 10-nm filament, being a four-stranded helix of protofibrils.

Association of a basic 25K protein with membrane coating granules of human epidermis.

Keratinocytes of the upper granular layers contain unique round-to-oval granules, 100-500 nm in diameter, in their peripheral cytoplasm. These granules (known as membrane coating granules [MCG], or lamellar granules) fuse with the apical cell surface of uppermost granular cells and discharge their contents into the intercellular space, where they are believed to play a role in establishing the permeability barrier of the epidermis and possibly in regulating the orderly desquamation of terminally differentiated keratinocytes. Using two monoclonal antibodies originally prepared against hair follicle antigens, we have identified a 25K epidermal protein in association with both MCG-like granules in the peripheral cytoplasm of granular cells as well as MCG-derived intercellular material. This protein is relatively basic (pI greater than 8), largely aqueous soluble, methionine deficient, and is relatively abundant in epidermis (comprising up to approximately 0.1% of soluble proteins). Its distribution is restricted to the granular layer of keratinized (cornified) stratified squamous epithelia. The identification of this protein component opens new avenues for studying the molecular mechanisms underlying the establishment of permeability barrier and/or regulation of desquamation.

Expression of keratin and vimentin intermediate filaments in rabbit bladder epithelial cells at different stages of benzo[a]pyrene-induced neoplastic progression.

Rabbit bladder epithelium, grown on collagen gels and exposed to the chemical carcinogen benzo[a]pyrene, produced nontumorigenic altered foci as well as tumorigenic epithelial cell lines during 120-180 d in culture. Immunofluorescence studies revealed extensive keratin filaments in both primary epithelial cells and benzo[a]pyrene-induced altered epithelial foci but showed no detectable vimentin filaments. The absence of vimentin expression in these cells was confirmed by two-dimensional gel electrophoresis. In contrast, immunofluorescence staining of the cloned benzo[a]pyrene-transformed rabbit bladder epithelial cell line, RBC-1, revealed a reduction in filamentous keratin concomitant with the expression of vimentin filaments. The epithelial nature of this cell line was established by the observation that cells injected into nude mice formed well-differentiated adenocarcinomas. Frozen sections of such tumors showed strong staining with antikeratins antibodies, but no detectable staining with antivimentin antibodies. These results demonstrated a differential expression of intermediate filament type in cells at different stages of neoplastic progression and in cells maintained in different growth environments. It is apparent that the expression of intermediate filaments throughout neoplastic progression is best studied by use of an in vivo model system in parallel with culture studies.