[Interleukin-9 promotes the activation of hepatic stellate cells in mice infected with Schistosoma japonicum].

OBJECTIVE: To investigate the effects of interleukin-9 (IL-9) in the activation of hepatic stellate cells (HSCs) in mice infected with Schistosoma japonicum. METHODS: Primary HSCs were isolated from mice 7 weeks post-infection with S. japonicum using the in situ liver perfusion and density gradient centrifugation, and cultured in vitro. HSCs were randomly assigned to the PBS control group and IL-9 stimulation group (stimulation with 20 ng/mL IL-9). HSCs were harvested 48 h and 72 h poststimulation, and the expression of α-smooth muscle actin (α-SMA), type I collagen (Col I) and type III collagen (Col III) was determined in HSCs using Western blotting. RESULTS: Following stimulation with 20 ng/mL IL-9 for 48 h, the expression of α-SMA [(0.87 ± 0.02) vs. (0.69 ± 0.01); t = 17.39, P < 0.01], Col I [(0.74 ± 0.02) vs. (0.65 ± 0.01); t = 9.56, P < 0.01] and Col III [(0.94 ±0.04) vs. (0.75 ± 0.03); t = 6.15, P < 0.01] was significantly greater in HSCs in the IL-9 stimulation group than in the PBS control group. Following stimulation with 20 ng/mL IL-9 for 72 h, the expression of α-SMA was significantly greater in HSCs in the IL-9 stimulation group than in the PBS control group[(0.76 ± 0.02) vs. (0.58 ± 0.02); t = 12.52, P < 0.01]; however, there were no significant differences between the two groups in terms of Col I [(0.68 ± 0.02) vs. (0.66 ± 0.02); t = 1.15, P > 0.05] or Col III expression [(0.75 ± 0.01) vs. (0.72 ± 0.02); t = 2.22, P > 0.05]. CONCLUSIONS: IL-9 promotes the activation of HSCs in mice infected with S. japonicum.

[Application of acellular bovine pericardium patch in implant based immediate breast reconstruction].

Objective: To examine the clinical effect of acellular bovine pericardium patch in implant based immediate breast reconstruction. Methods: The clinicopathological information of 141 breast cancer patients, who admitted to Department of Breast Reconstruction and Oncoplastic Surgery, Tianjin Medical University Cancer Hospital, underwent immediate mammoplasty with implants combined with acellular bovine pericardium patches were analyzed from June 2016 to October 2019. All patients were female, with the age of (38.8±8.5) years (range: 13 to 60 years). The body mass index was (21.9±2.5) kg/m2 (range: 16.0 to 32.3 kg/m2). There were 39 cases of duct carcinoma in situ, 46 cases of stage Ⅰ, 40 cases of stage Ⅱ and 16 cases of stage Ⅲ. All patients received nipple-areola-sparing mastectomy or skin-sparing mastectomy with sentinel lymph node biopsy or axillary lymph node dissection, and prosthesis implantation with sub-pectoralis combined with breast patch. The correlation of clinicopathological characters and complications was assessed by t test, χ2 test, Fisher's exact probability method and Logistic regression. Pre-and post-operative aesthetic, quality of life scores were recorded. Results: The operation time (M(IQR)) was 3.6(1.5) hours (range: 3.0 to 6.5 hours). The early postoperative complication rate was 22.0% (31/141), prosthesis removal was the main postoperative complication, accounting for 64.5% (20/31) of the total complications, of which 15 cases occurred in the first 30 patients. The follow-up time was 28(8) months (range: 20 to 53 months), The most frequent long-term complications were capsular contracture and implant displacement, with the incidence of 11.2% (14/125) and 10.4% (13/125), respectively. Multivariate analysis showed that prosthesis volume ≥300 ml (OR=8.173, 95%CI: 1.302 to 51.315, P=0.021) and peri-areolar incision (OR=7.809, 95%CI: 2.162 to 28.211, P<0.01) were independent relative factors for the occurrence of short-term postoperative local complications. After 2 years of operation, the score of breast appearance satisfaction was 71.7±15.5, postoperative effect satisfaction was 90.4±9.5, psychological satisfaction was 90.7±17.1, sexual satisfaction was 70.1±25.1. The immediate postoperative satisfaction rate at discharge was 95.4% (134/141), and 17.6% (22/125) of patients had the intention to received revision surgery. Conclusions: Prosthesis volume ≥300 ml and peri-areolar incision were independent realtive factors for short-term local complications after bovine pericardium patch combined with prosthesis implantation in the immediate breast reconstruction. After completing the learning curve, the postoperative complications of the procedure could be decreased.

[Advances in application of functional magnetic resonance imaging in patients with painful temporomandibular disorders].

Temporomandibular disorders (TMD), characterized by pain and dysfunction of the temporomandibular joint, are the most common chronic orofacial pain. However, the etiologies and pathologies of TMD related chronic pain are poorly understood. Functional magnetic resonance imaging (fMRI) measures brain activity by detecting changes associated with blood flow without invasiveness, and has been widely used in chronic pain research. We reviewed recent fMRI studies exploring the brain changes of patients with painful TMD to investigate the role of central nervous system in abnormal pain perception and impaired pain modulation, and to summarize the effects of splint therapy, in the hope of facilitating the clinical diagnosis and treatment of TMD.

[Three-dimensional visual assessment and virtual reality study of centrally located hepatocellular carcinoma on the axis of blood vessels].

Objective: To explore a novel method for preoperative precision assessment of centrally located hepatocellular carcinoma(HCC) with blood vessel as axis based on three-dimensional(3D) visualization and virtual reality(VR) technology and its application values. Methods: High-quality thin-layer enhanced CT data were collected from 20 patients with centrally located HCC who treated at First Department of Hepatobiliary Surgery, Zhujiang Hospital, Southern Medical University from March 2017 to August 2018 diagnosed by preoperative examination. There were 18 males and 2 females, aged 28 to 69 years, all of Child-Pugh grade A. First of all, 3D reconstruction was performed by a 3D visualization software; then, the reconstructed 3D image was imported into VR development engine for VR research; afterwards, the analysis and evaluation system with blood vessel as axis was established based on 3D visualization classification of centrally located HCC; therefore, the relationship of the tumor to its major peripheral blood vessels was accurately judged and the surgical planning was formulated. Two images were brought into the operating room for navigation in surgery. The assessments results of preoperative data (CT and (or) MRI) and three-dimensional visualization of blood vessels in VR environment were compared; the values of the preoperative and postoperative hemoglobin, serum albumin and bilirubin were recorded and compared. Chi-square test, t-test and non-parametric test were used for the analysis of counting data, continuous measurement data and non-normal distribution measurement data, respectively. Results: 3D visualization modeling was completed in all of the 20 patients with centrally located HCC. According to the results of 3D visualization classification of centrally located HCC, there were 3 cases of type Ⅰ,1 case of type Ⅱ,4 cases of type Ⅲ,7 cases of type Ⅳ and 5 cases of type Ⅴ; according to the assessment and classification based on blood vessel as the axis, there were 6 cases of type Ⅰa,2 cases of type Ⅰb,2 cases of type Ⅱa,9 cases of type Ⅱb and 1 case of type Ⅱc. All patients underwent successful resection of tumor under the guidance of 3D visualization and VR technology. There were 15 cases whose assessment results based on preoperative CT/MRI were consistent with intraoperative findings, with a coincidence rate of 75.0%(15/20); while in VR environment, the assessment results of 3D visualization with blood vessel as axis were all consistent with the intraoperative findings, with coincidence rate of 100%(20/20). There was a statistically significant difference between the groups (χ(2)=5.714, P=0.017). There was no red blood cell transfusion in all patients during the operation. The preoperative hemoglobin was (128.8±14.9)g/L, and it was (119.8±12.5)g/L on postoperative day 1. There was no significant difference between these two sets of data (t=2.07, P=0.054). No death during the perioperative period and no complications such as hepatic failure, hemorrhage and biliary fistula after operation occurred. Conclusion: Preoperative evaluation based on 3D visualization and VR technology with blood vessel as the axis has significant clinical value for preoperative planning and surgical navigation of centrally located HCC.

Association of meteorological factors with seasonal activity of influenza A subtypes and B lineages in subtropical western China.

The seasonality of individual influenza subtypes/lineages and the association of influenza epidemics with meteorological factors in the tropics/subtropics have not been well understood. The impact of the 2009 H1N1 pandemic on the prevalence of seasonal influenza virus remains to be explored. Using wavelet analysis, the periodicities of A/H3N2, seasonal A/H1N1, A/H1N1pdm09, Victoria and Yamagata were identified, respectively, in Panzhihua during 2006-2015. As a subtropical city in southwestern China, Panzhihua is the first industrial city in the upper reaches of the Yangtze River. The relationship between influenza epidemics and local climatic variables was examined based on regression models. The temporal distribution of influenza subtypes/lineages during the pre-pandemic (2006-2009), pandemic (2009) and post-pandemic (2010-2015) years was described and compared. A total of 6892 respiratory specimens were collected and 737 influenza viruses were isolated. A/H3N2 showed an annual cycle with a peak in summer-autumn, while A/H1N1pdm09, Victoria and Yamagata exhibited an annual cycle with a peak in winter-spring. Regression analyses demonstrated that relative humidity was positively associated with A/H3N2 activity while negatively associated with Victoria activity. Higher prevalence of A/H1N1pdm09 and Yamagata was driven by lower absolute humidity. The role of weather conditions in regulating influenza epidemics could be complicated since the diverse viral transmission modes and mechanism. Differences in seasonality and different associations with meteorological factors by influenza subtypes/lineages should be considered in epidemiological studies in the tropics/subtropics. The development of subtype- and lineage-specific prevention and control measures is of significant importance.

[Research advances in the role of CX3CL1 and its receptor in liver diseases].

In recent years, an increasing number of studies have found that the interaction between CX3CL1 and its receptor plays an important role in a series of inflammatory, infectious, and neurological processes and in various cancers. Therefore, its therapeutic targeting represents a promising treatment strategy for diseases. This review summarizes the structure, biological function, and mechanism of signal transduction of CX3CL1 and its receptor, and introduces the research progress in CX3CL1 and its receptor in hepatitis, hepatic fibrosis, cirrhosis, and hepatoma.

[Hydrogen peroxide induces oxidative stress and the mitochondrial pathway of apoptosis in RAT intestinal epithelial cells (IEC-6)].

UNLABELLED: In order to investigate the mechanism of apoptosis in rat intestinal epithelial cells (IEC-6) induced by hydrogen peroxide (H(2)O(2)), IEC-6 cells were subjected to 20 µmol/L H(2)O(2) and cell proliferation activity was determined using 3-(4,5-dimethyl-2-yl)-2,5-diphenyltetrazolium bromide. Cell morphology was observed by microscopy and cell apoptosis was detected by acridine orange and ethidium bromide staining and the portion of apoptotic cells was measured by flow cytometry. Genes and proteins related to cell apoptosis were detected by RT-PCR and Western blotting, and the mitochondrial membrane potential was evaluated by fluorescence probes. RESULTS: Significant morphology damage was caused by exposure to H(2)O(2), and results showed that ROS generation significantly increased (P < 0.01). The activity of superoxide dismutase decreased significantly (P < 0.05), malondialdehyde content increased (P < 0.05), and expression of both catalase and glutathione peroxidase decreased significantly (P < 0.05) in the H(2)O(2) treatment group. Mitochondrion membrane potential was reduced, cytochrome released into the cytoplasm and caspase-9 and caspase-3 were significantly increased (P < 0.01) after treatment with H(2)O(2). Moreover, the ratio of Bax/Bcl-2 and apoptosis were significantly increased (P < 0.01) in the H(2)O(2) group. In conclusion, the present study indicated that the mitochondrial pathway plays a vital role in H(2)O(2) induced IEC-6 cell apoptosis.

Altered neural activation pattern during teeth clenching in temporomandibular disorders.

OBJECTIVE: The aim was to explore the neural activations during teeth clenching in TMDs patients pre- and post-treatment. SUBJECTS AND METHODS: Thirty TMDs patients and 20 controls underwent clinical evaluations and functional magnetic resonance imaging with a teeth clenching task. Eleven patients received repeat evaluation and imaging after wearing a stabilization splint for 3 months. RESULTS: During teeth clench, the TMDs patients showed decreased positive activity in the left precentral gyrus, right and left inferior temporal gyrus, and left cerebellum and increased negative activations in the right medial prefrontal cortex (P < 0.05 after AlphaSim correction). The 11 TMDs patients after treatment showed a return to normal neural activity in these areas. No brain areas in TMDs patients showed differences in activation after treatment compared with the controls, except for an increase in activation in the right cerebellum in the 11 TMDs patients (P < 0.05 after AlphaSim correction). CONCLUSION: Decreased activations in cerebral areas associated with motor and cognitive functions in TMDs patients during teeth clenching were observed. Normalized activations of these areas happened in patients after routine treatment. These findings may facilitate the understanding of TMDs pathogenesis and the therapeutic mechanisms of the stabilization splint.

Spontaneous neural activity alterations in temporomandibular disorders: a cross-sectional and longitudinal resting-state functional magnetic resonance imaging study.

The involvement of the central nervous system in the pathophysiology of temporomandibular disorders (TMD) has been noticed. TMD patients have been shown dysfunction of motor performance and reduced cognitive ability in neuropsychological tests. The aim of this study is to explore the spontaneous neural activity in TMD patients with centric relation (CR)-maximum intercuspation (MI) discrepancy before and after stabilization splint treatment. Twenty-three patients and twenty controls underwent clinical evaluations, including CR-MI discrepancy, Helkimo indices and chronic pain, and resting state functional magnetic resonance imaging scans at baseline. Eleven patients repeated the evaluations and scanning after the initial wearing (T1) and 3months of wearing (T2) of the stabilization splint. The fractional amplitude of low-frequency fluctuation (fALFF) was calculated to compare the neural functions. At baseline, the patients showed decreased fALFF in the left precentral gyrus, supplementary motor area, middle frontal gyrus and right orbitofrontal cortex compared with the controls (P<0.05, AlphaSim corrected). Negative correlations were found between the fALFF in the left precentral gyrus and vertical CR-MI discrepancy of bilateral temporomandibular joints of patients (P<0.05, two-tailed). At T2, the symptoms and signs of the patients were improved, and a stable condylar position on the CR was recovered, with increased fALFF in the left precentral gyrus and left posterior insula compared with pretreatment. The fALFF decrease in the patients before treatment was no longer evident at T2 compared with the controls. The results suggested that TMD patients with CR-MI discrepancy showed significantly decreased brain activity in their frontal cortexes. The stabilization splint elicited functional recovery in these cortical areas. These findings provided insight into the cortical neuroplastic processes underlying TMD with CR-MI discrepancy and the therapeutic mechanisms of stabilization splint.

Comparison of anchorage capacity between implant and headgear during anterior segment retraction.

OBJECTIVE: To compare the anchorage effects of the implants and the headgear for patients with anterior teeth retraction in terms of incisor retraction, anchorage loss, inclination of maxillary incisors, positional change of maxillary basal bone, and treatment duration. MATERIALS AND METHODS: An electronic search for relative randomized controlled trials (RCTs) prospective and retrospective controlled trials was done through the Cochrane Central Register of Controlled Trials (CENTRAL), PubMed, Embase, Medline, and CNKI, regardless of language of study. Study selection, methodological quality assessment, and data extraction were performed by two reviewers independently. Meta-analysis was performed when possible; otherwise descriptive assessment was done. RESULTS: The search yielded 35 articles, of which eight met the inclusion criteria and were categorized into five groups according to types of intervention. For the midpalatal implant, the anchorage loss was much less than for the headgear group, with insignificant differences in terms of anterior teeth retraction, maxillary incisor inclination, positional change of basal bone, and treatment duration. For the mini-implant, greater anterior teeth retraction and less anchorage loss were demonstrated, with inconsistent results for the other measures. For the onplant, less anchorage loss was noted, with insignificant differences for the other measures. CONCLUSIONS: The skeletal anchorage of the midpalatal implant, mini-implant, and onplant offer better alternatives to headgear, with less anchorage loss and more anterior teeth retraction. There were inconsistent results from the included studies in terms of maxillary incisor inclination, positional change of maxillary basal bone, and treatment duration. More qualified RCTs are required to provide clear recommendations.

Medical radiation usage and exposures from medical X ray diagnosis in Shandong province of China.

Shandong province is situated in the eastern part of China with a population of 88 million. A collaborative survey was conducted in 1999 to investigate the medical radiation usage and the patients' exposures from medical X ray diagnosis in this province. There were three stages in this survey: general survey, sampling survey and dose survey. Patients' entrance surface doses (ESD) were measured using thermoluminescence dosemeters (TLDs) attached to the patient's skin. All the hospitals in this province and 2153 patients were included in this survey. The frequencies of tele- and brachytherapy were 1.450 and 1.685 per 1000 population in 1996 and 1998 respectively. The frequency of diagnostic nuclear medicine was 0.325 and 0.412 per 1000 population in 1996 and 1998 respectively; for therapeutic nuclear medicine, it was 0.015 and 0.021. The total annual frequencies of every type of X ray examination and interventional radiology (IVR) in 1996 and 1998 were 179.8 and 200.0 per 1000 population respectively. The main type of X ray examination was chest fluoroscopy, which made up about 39% of the total. The average entrance surface doses were comparatively high for the examinations of CT, gastrointestinal tract, lumbarspine, urography, angiography, hip and cerebral angiography.

Cholesterol sulfate stimulates involucrin transcription in keratinocytes by increasing Fra-1, Fra-2, and Jun D.

Lipids that are synthesized de novo in the epidermis, including fatty acids, oxysterols, 1,25-dihydroxyvitamin D(3), and farnesol, can regulate the differentiation of normal human keratinocytes (NHK). Cholesterol sulfate (CS), an epidermal lipid that is produced in the upper nucleated layers of the epidermis coincident with terminal differentiation, has been shown to play a role in the regulation of the late stages of keratinocyte differentiation, including formation of the cornified envelope. In the present study, we determined i) whether CS regulates involucrin (INV), an early keratinocyte differentiation marker, and ii) the mechanism by which CS regulates differentiation. mRNA and protein levels of INV, a precursor protein of the cornified envelope, increased 2- to 3-fold in NHK incubated in the presence of CS. In contrast, cholesterol had no effect on INV protein or mRNA levels. Transcriptional regulation was assessed in NHK transfected with INV promoter-luciferase constructs. CS increased luciferase reporter activity approximately 2- to 3-fold in NHK transfected with a 3.7-kb INV promoter construct. Deletional analysis revealed a CS-responsive region of the INV promoter located between bp --2452 and --1880. A 5-base pair (bp) mutation of the AP-1 site (bp --2117 to --2111) within this responsive region abolished CS responsiveness, suggesting a role for the AP-1 complex in the regulation of INV transcription by CS. Electrophoretic mobility shift analysis demonstrated increased binding of nuclear extracts isolated from CS-treated NHK to AP-1 DNA as compared with vehicle-treated controls. Incubation of the nuclear extract with the appropriate antibodies showed that the AP-1 DNA-binding complex contained Fra-1, Fra-2, and Jun D. Western blots demonstrated that CS treatment increased the levels of Fra-1, Fra-2, and Jun D, and Northern analyses revealed that CS increased mRNA levels for these same AP-1 factors. These data indicate that CS, an endogenous lipid synthesized by keratinocytes, regulates the early stages of keratinocyte differentiation, and may do so through its ability to modulate levels of AP-1 proteins. -- Hanley, K., L. Wood, D. C. Ng, S. S. He, P. Lau, A. Moser, P. M. Elias, D. D. Bikle, M. L. Williams, and K. R. Feingold. Cholesterol sulfate stimulates involucrin transcription in keratinocytes by increasing Fra-1, Fra-2, and Jun D. J. Lipid Res. 2001. 42: 390--398.

Oxysterols induce differentiation in human keratinocytes and increase Ap-1-dependent involucrin transcription.

Ligands and activators of the nuclear hormone receptor superfamily are important in the regulation of epidermal development and differentiation. Previously, we showed that naturally occurring fatty acids, as well as synthetic ligands for the peroxisome proliferator-activated receptor, induce keratinocyte differentiation in vitro. Here we asked whether oxysterols, another class of lipids formed de novo in the epidermis and that activate liver X-activated receptor, regulate keratinocyte differentiation. mRNA and protein levels of involucrin and transglutaminase 1, markers of differentiation, increased 2- to 3-fold in normal human keratinocytes incubated in the presence of 25- or 22R-hydroxycholesterol in low calcium. In high calcium, which alone induces differentiation, mRNA levels were further increased by oxysterols. Rates of cornified envelope formation, an indicator of terminal differentiation, also increased 2-fold with oxysterol treatment. In contrast, the rate of DNA synthesis was inhibited approximately 50% by oxysterols. Transcriptional regulation was assessed in keratinocytes transfected with either transglutaminase 1 or involucrin promoter-luciferase constructs. 22R-hydroxycholesterol increased transglutaminase 1 and involucrin promoter activity 2- to 3-fold. Either deletion of the -2452 bp to -1880 bp region of the involucrin promoter, or mutation of the AP-1 site within this region, abolished oxysterol responsiveness. Moreover, increased AP-1 DNA binding was observed in oxysterol-treated keratinocytes by gel shift analyses. Finally, we demonstrated the presence of liver X-activated receptor alpha and beta mRNAs, and showed that oxysterols stimulate a liver X-activated receptor response element transfected into keratinocytes. These data suggest that oxysterols induce keratinocyte differentiation, in part through increased AP-1-dependent transcription of the involucrin gene, an effect that may be mediated by liver X-activated receptor.

Farnesol stimulates differentiation in epidermal keratinocytes via PPARalpha.

The isoprenoids farnesol and juvenile hormone III (JH), metabolites of the cholesterol biosynthetic pathway, have been shown to stimulate fetal epidermal development in rodents. In this study we determined whether this effect might be attributed to a direct induction of keratinocytes differentiation and examined the mechanisms responsible for these effects. Rates of cornified envelope formation, a marker of keratinocyte terminal differentiation, as well as protein and mRNA levels of two proteins required for cornified envelope formation, involucrin (INV) and transglutaminase, increased 2- to 3-fold in normal human keratinocytes (NHK) treated with either farnesol or JH, even at low calcium concentrations (0.03 mM), which otherwise inhibit differentiation. In contrast, neither cholesterol nor mevalonate affected INV or transglutaminase mRNA levels. Effects of farnesol and JH on INV and transglutaminase mRNA levels were additive with high calcium concentrations (1.2 mM) that independently stimulate keratinocyte differentiation. In contrast, keratinocyte DNA synthesis was inhibited by these compounds. Both farnesol and JH stimulated INV and transglutaminase promoter activity, suggesting regulation at the transcriptional level. A series of truncation and deletion experiments revealed a farnesol-responsive region (-2452 to -1880 base pairs (bp)) in the INV gene. This region contained an AP-1 site. A single base pair mutation of the AP-1 site at -2116 to -2110 bp abolished farnesol responsiveness, identical to effects by peroxisome proliferator-activated receptor (PPARalpha) activators. Farnesoid X-activated receptor mRNA was not detected in NHK, but farnesol treatment increased activities of both a PPAR response element and PPARalpha mRNA levels in NHK. Furthermore, the increase in PPRE activity by farnesol was dependent upon PPARalpha in CV-1 cells. Finally, topical applications of farnesol increased mRNA and protein levels of the differentiation-specific genes, profilaggrin and loricrin, determined by immunohistochemistry and in situ hybridization, in wild-type but not in PPARalpha-/- murine epidermis. These findings suggest a novel role for selected isoprenoid cholesterol intermediates in the regulation of differentiation-specific gene transcription and a convergence of PPARalpha with the cholesterol synthetic pathway.

Fetal epidermal differentiation and barrier development In vivo is accelerated by nuclear hormone receptor activators.

Nuclear receptors which interact with the retinoid X receptor are involved in the regulation of epidermal differentiation and development. We have recently shown that activators of the peroxisome proliferator-activated receptor and of the farnesoid X-activated receptor accelerate epidermal barrier maturation in fetal rat skin in vitro. In this study we asked whether cutaneous development in utero was affected by peroxisome proliferator-activated receptor or farnesoid X-activated receptor activators, or by an activator of another retinoid X receptor partner, liver X receptor. Activators of the peroxisome proliferator-activated receptor (clofibrate or linoleic acid), farnesoid X-activated receptor (farnesol or juvenile hormone III), or liver X receptor (22R-hydroxycholesterol), were injected into the amniotic fluid of fetal rats on gestational day 17. Fetal epidermal barrier function and morphology was assessed on day 19. Whereas vehicle-treated fetal rats displayed no measurable barrier (transepidermal water loss > 10 mg per cm2 per h), a measurable barrier was induced by the intra-amniotic administration of all activators tested (transepidermal water loss range 4.0-8.5 mg per cm2 per h). By light microscopy, control pups lacked a well-defined stratum corneum, whereas a distinct stratum corneum and a thickened stratum granulosum were present in treated pups. By electron microscopy, the extracellular spaces of the stratum corneum in control pups revealed a paucity of mature lamellar unit structures, whereas these structures filled the stratum corneum interstices in treated pups. Additionally, protein and mRNA levels of loricrin and filaggrin, two structural proteins of stratum corneum, were increased in treated epidermis, as were the activities of two lipid catabolic enzymes critical to stratum corneum function, beta-glucocerebrosidase and steroid sulfatase. Finally, peroxisome proliferator-activated receptor-alpha and -delta and liver X receptor-alpha and -beta mRNAs were detected in fetal epidermis by reverse transcriptase-polymerase chain reaction and northern analyses. The presence of these receptors and the ability of their activators to stimulate epidermal barrier and stratum corneum development suggest a physiologic role for peroxisome proliferator-activated receptor and liver X receptor and their endogenous ligands in the regulation of cutaneous development.

Keratinocyte differentiation is stimulated by activators of the nuclear hormone receptor PPARalpha.

Peroxisome proliferator activated receptors (PPAR) belong to the superfamily of nuclear hormone receptors that heterodimerize with the retinoid X receptor and regulate transcription of several genes involved in lipid metabolism and adipocyte differentiation. Because of the role of 1,25-dihydroxyvitamin D3 and retinoic acid working through similar receptors (the vitamin D receptor and retinoic acid receptor, respectively) on keratinocyte differentiation, we have examined the effects of activators of PPARalpha on keratinocyte differentiation. The rate of cornified envelope formation was increased 3-fold in keratinocytes maintained in low calcium (0.03 mM) and incubated in the presence of clofibric acid, a potent PPARalpha activator. Involucrin, a cornified envelope precursor, and the cross-linking enzyme transglutaminase, were increased at both the message level (2-7-fold) and the protein level (4-12-fold) by clofibric acid. Furthermore, physiologic doses of the fatty acids oleic acid, linoleic acid, and eicosatetraynoic acid, which are also activators of PPARalpha, also induced involucrin and transglutaminase protein and mRNA. In contrast, the PPARgammaligand prostaglandin J2 had no effect on protein or mRNA levels of involucrin or transglutaminase. Levels of involucrin and transglutaminase mRNA and protein were induced by clofibric acid in keratinocytes incubated in 1.2 mM calcium, a concentration which by itself induces keratinocyte differentiation. Finally, PPARalpha activators inhibit DNA synthesis. This study demonstrates that PPARalpha activators, including putative endogenous ligands such as fatty acids, induce differentiation and inhibit proliferation in keratinocytes, and suggests a regulatory role for the PPARalpha in epidermal homeostasis.

High performance liquid chromatography separation and simultaneous fluorometric detection of trace amount of polyamines in cell culture media.

A highly sensitive and precise method for the determination of the polyamines (including putrescine, spermidine and spermine) in cell culture media is described. The samples were concentrated by C18 column and the polyamines were converted to fluorometric derivatives with DNS-Cl. The polyamine derivatives were then completely separated by HPLC and determined by simultaneous fluorometric detection. The CV of intragroup and intergroup were 2.49%-4.26% and 4.29%-5.16%, respectively. The rate of recovery was 103%-99%. There was trace amount of polyamines detected by this method in the media of F12, 8900, RPMI-1640, DMEM and M199 even without incubation with cells. So this method can be used for detecting the changes of polyamines in a medium before and after incubation with cells. It is helpful for the researches on the regulation of cell proliferation by polyamines.

The micro-determination of serum gamma-glutamyl transferase.

The method of serum gamma-glutamyl transferase colorimeter assay was modified by changing the wave length from 420 nm to 400 nm, the incubated temperature from 37 degrees C to 50 degrees C, the sample amount from 30 microliters to 6 microliters. The modified method was proved to be more sensitive, with CV of intra-group being 2.06% and accuracy 97.4%. The method is suitable for clinical application with small-amount blood samples collected from earlobe or finger tip.

Use of fluorescent substrate 4-MUP in the detection of biotin-labeled DNA probes.

To improve the sensitivity of detecting biotin-labeled DNA Probes, a new fluorescent substrate of alkaline phosphatase, 4-methylum belliferylphosphate (4-mup) was studied instead of conventional BCIP-NBT. The result of dot-blot hybridization demonstrates that this new substrate can be used for the colorimetric detection of biotin-labeled probes after hybridization to immobilized nucleic acids. The sensitivity is about one order of magnitude higher than that of BCIP-NBT system, and the time required for color development is very short, only about five min. It is suggested that the Bio-SA-Bio-AP-4-MUP colorimetric detection system can be widely used in gene diagnosis.

Changes of serum cholinesterase isozyme patterns in liver diseases.

A simple method was established for the separation and identification of serum cholinesterase (SChE) isozymes by using gradient polyacrylamide gel electrophoresis and densitometric scanning. With this method, SChE isozyme patterns of 39 healthy adults and 232 patients with liver diseases were studied. 9 to 11 bands of SChE were demonstrated in healthy adults. The activities increasing in Band 1 and decreasing or disappearing in Bands 2, 3, 4 and 5 were found in most patients with liver diseases in comparing with the control. It showed that the activity decreasing is correlated closely with the severity of liver diseases.