A novel water-in-oil emulsion with a lecithin-modified bentonite prevents skin damage from urban dust and cedar pollen.

OBJECTIVE: Particulate matter (PM), such as air pollutants and pollens, are known to cause skin ageing through skin inflammation. It is important to develop formulations which protect the skin from PM. We previously developed a conventional water-in-oil emulsion with a synthetic surfactant, distearyldimonium chloride, modified bentonite (C-W/O), which protects skin from allergens. In the present study, we developed a novel water-in-oil emulsion with a natural surfactant, lecithin, modified bentonite (N-W/O). METHODS: The microarray analysis was performed using total RNA extracted from a reconstructed human epidermis (RHE) stimulated with urban aerosols or cedar pollen for 6 h in order to develop an epidermal inflammation model by PM for the evaluation of topical formulations. We then compared the efficacy of N-W/O and C-W/O to prevent epidermal degradation. Tissues and culture media were collected 24 h after the urban aerosol or cedar pollen stimulation for a histological assay, and the quantification of MMP1 and IL-8 secretion. RESULTS: The expression levels of proinflammatory cytokines and chemokines, such as IL1A and CXCL8, and matrix metalloproteinases, including MMP1, MMP3 and MMP9, were significantly up-regulated by the PM stimulation. As a result of ranking based on the pathway enrichment analysis, oxidative stress-related pathways, such as MAPK-mediated signalling, HIF-1 signalling, IL-1 signalling and ROS-induced cellular signalling, were ranked high in the urban dust- and cedar pollen-treated groups. A thickened stratum corneum, thinned vital layer and cleaved E-cadherin were observed by haematoxylin and eosin staining and immunohistochemical staining of E-cadherin in the PM treated groups. The secretion of MMP1 and IL-8 into the media was significantly increased by the PM stimulation. N-W/O prevented the degradation of epidermal integrity and secretion of inflammatory proteins more effectively than C-W/O. CONCLUSION: The present results showed that N-W/O made using natural surfactant is useful at protecting skin from PM, such as urban aerosols and cedar pollen.

OBJECTIF: Les particules en suspensions (PM), telles que les polluants atmosphériques et les pollens, sont connues comme des causes de vieillissement de la peau par inflammation cutanée. Il est essentiel de mettre au point des formules qui protègent la peau contre ces particules. Par le passé, nous avons mis au point une émulsion eau-dans-huile classique composée d'un tensioactif synthétique, de distearyldimonium chloride et de bentonites modifiées (E/H-C), qui protège la peau contre les allergènes. Dans la présente étude, nous avons conçu une nouvelle émulsion eau-dans-huile composée d'un tensioactif naturel, de lécithine et de bentonites modifiées (N-E/H). MÉTHODES: L'analyse des microréseaux a été réalisée à l'aide de l'ARN total extrait d'un épiderme humain reconstitué (EHR) stimulé par les aérosols urbains ou le pollen de cèdre pendant 6 h afin de mettre au point un modèle d'inflammation de l'épiderme par les particules en suspensions en vue de l'évaluation des formulations topiques. Nous avons ensuite comparé l'efficacité de la N-E/H et de l'E/H-C dans le but d'éviter la dégradation de la peau. Les milieux de culture tissulaire ont été collectés 24 h après stimulation par l'aérosol urbain ou par du pollen de cèdre pour un dosage histologique et une quantification de MMP-1 et des sécrétions de l'IL-8. RÉSULTATS: Les niveaux d'expression des cytokines pro-inflammatoires et des chimiokines, à l'instar de l'IL1A et du CXCL8, ainsi que des métalloprotéinases matricielles, notamment les MMP1, les MMP3 et les MMP9, étaient essentiellement régulés positivement par la stimulation des particules en suspensions. En raison du classement basé sur l'analyse d'enrichissement des voies, le stress oxydatif, telles que la signalisation médiée par MAPK, la signalisation HIF-1, la signalisation IL-1 et la signalisation cellulaire induite par les ROS ont été classés en tête pour les groupes traités par la poussière urbaine et par le pollen-de cèdre. Un stratum corneum épaissie, une couche vitale fine et une clivée d'E-cadhérine ont été observées par coloration à l'hématoxyline-éosine et par coloration immunohistochimique de l'E-cadhérine dans les groupes traités aux particules en suspensions. La sécrétion de MMP1 et de l'IL-8 dans les milieux a augmenté de façon significative par stimulation des particules en suspensions. La N-E/H a permis d'éviter une dégradation de l'intégrité de la peau et la sécrétion de protéines inflammatoires de manière plus efficace que l'E/H-C. CONCLUSION: Les résultats actuels ont révélé que la N-E/H produite grâce à l'utilisation d'un tensioactif naturel est utile pour la protection de la peau contre les particules en suspensions telles que les aérosols urbains et le pollen de cèdre.

Correlation of circulating dehydroepiandrosterone with activated protein C generation and carotid intima-media thickness in male patients with type 2 diabetes.

AIMS: Dehydroepiandrosterone exerts a protective effect against cardiovascular diseases. However, the relationship of dehydroepiandrosterone with the anticoagulant factor activated protein C, generated by the thrombin-thrombomodulin complex on vascular endothelial cells, remains unknown. This study aimed at studying the relationship between dehydroepiandrosterone and activated protein C generation in patients with Type 2 diabetes. METHODS: Sixty-two male patients with Type 2 diabetes were enrolled in this study. Data obtained from 40 healthy male subjects were used as controls. The plasma levels of dehydroepiandrosterone, the activated protein C-protein C inhibitor complex, high-sensitivity C-reactive protein and monocyte chemoattractant protein-1 were measured by enzyme immunoassays. Carotid intima-media thickness was measured by ultrasonography. RESULTS: The plasma levels of dehydroepiandrosterone (5.15 ± 2.81 vs. 3.76 ± 2.16 ng/ml; P < 0.005) and the activated protein C-protein C inhibitor complex (1.90 ± 1.07 vs. 1.02 ± 0.51 ng/ml; P < 0.001) were significantly lower in patients with diabetes than in normal subjects. Univariate analysis showed a significant correlation of the plasma level of dehydroepiandrosterone with that of the activated protein C-protein C inhibitor complex (r = 0.48, P < 0.001), high-sensitivity C-reactive protein (r = -0.30, P < 0.05) and with the mean intima-media thickness (r = -0.28, P < 0.05) in patients with diabetes. Stepwise multiple regression analysis showed that the plasma level of dehydroepiandrosterone is significantly correlated with the plasma levels of the activated protein C-protein C inhibitor complex (F = 18.06) and high-sensitivity C-reactive protein (F = 4.94). There was no correlation between the plasma levels of dehydroepiandrosterone and monocyte chemoattractant protein-1. CONCLUSIONS: These results suggest that lower circulating levels of dehydroepiandrosterone are associated with decreased activated protein C generation and higher intima-media thickness in patients with Type 2 diabetes.

The alpha subunit of GTP-binding protein G0 in neuroblastoma: correlation with advanced disease stage.

Tissue levels of the alpha subunit of G protein G0 (G0 alpha) were measured in solid tumors from pediatric patients by immunoassay. G0 alpha concentrations were determined in the supernatant obtained by centrifugation of tissue homogenates prepared in the presence (total G0 alpha) or absence of 2% sodium cholate (soluble G0 alpha). Mean G0 alpha concentrations (total G0 alpha and soluble G0 alpha) in neuroblastomas (7 ganglioneuromas, 13 ganglioneuroblastomas, and 50 neuroblastomas) were over 50-fold higher than those in other solid tumors from pediatric patients (n = 13). Mean total G0 alpha and soluble G0 alpha concentrations were 207.0 +/- 166.0 (SD) ng/mg of cholate-extractable protein and 58.6 +/- 47.0 ng/mg of soluble protein, respectively, in the neuroblastoma group (n = 70). Total G0 alpha concentration decreased with disease stage and was strongly correlated with outcome in patients with neuroblastoma. The mean total G0 alpha concentration in tumors from younger patients (< 1 year old) was 297.0 +/- 137.0 ng/mg of cholate-extractable protein, significantly higher than in tumors from older patients (140.0 +/- 155.0 ng/mg cholate-extractable protein, P < 0.0001). These results suggest that total G0 alpha levels in neuroblastoma may indicate the degree of malignancy.

Identification of a novel HLA-C allele, HLA-C*03:313, in a Japanese individual.

HLA-C*03:313 differs from HLA-C*03:04:01:01 by one non-synonymous amino acid exchange E161G.

Alteration of cGMP metabolism during chondrogenic differentiation of chondroprogenitor-like EC cells, ATDC5.

Guanosine 3',5'-cyclic monophosphate (cGMP) has been recently reported to be involved in bone formation. ATDC5 cells were used to investigate cGMP metabolism during chondrogenic differentiation. Natriuretic peptide receptor (NPR)-A and NPR-B coupled with guanylate cyclase (GC) mediate biological functions of NPs, whereas NPR-C uncoupled with GC is thought to be the clearance receptor for NPs. The amounts of NPR-A, NPR-B, and CNP transcripts were increased but the amount of NPR-C transcripts was decreased in association with the chondrogenic differentiation of ATDC5 cells. CNP, a specific ligand for NPR-B lets ATDC5 cells accumulate great amounts of cGMP, revealing NPR-B as a dominant biological receptor through differentiation. cGMP hydrolytic activities of PDE1 and PDE5 existed in ATDC5 cells, and the activity of PDE1, which is stimulated by Ca(2+) and calmodulin (CaM) was major of them. Total cGMP hydrolytic activities as well as the amounts of PDE1 and PDE5 transcripts were enhanced during chondrogenic differentiation. Therefore, cGMP production and hydrolysis, cGMP metabolism was considered to be activated in association with chondrogenic differentiation of ATDC5 cells. These observations may lead to a better understanding of cGMP in the chondrocytes where bone formation occurs.

The ABC-exporter genes involved in the lipase secretion are clustered with the genes for lipase, alkaline protease, and serine protease homologues in Pseudomonas fluorescens no. 33.

In Pseudomonas fluorescens no. 33, the lipase gene is clustered with the genes for alkaline protease, AprDEF exporter, and two homologue proteins of Serratia serine proteases (pspA and pspB). Secretion of the lipase and alkaline protease through AprDEF was shown in the Escherichia coli cells. Interestingly, the E. coli cells carrying the pspA gene secreted PspA to the media AprDEF-independently.

Divergence of a flagellin protein in Serratia marcescens.

A gene (hag) encoding the flagellin (Fla) protein was cloned from Serratia marcescens (Sm) 8000, the wild-type strain of Sr41. The hag gene codes for a 348-amino-acid (aa) protein of 36.7 kDa. The predicted aa sequence showed 79% homology compared with the Fla of Sm 274 which has been reported previously [Harshey et al., Gene 79 (1989) 1-8]. Dot-matrix analysis of the Sm 8000 Fla showed that the N- and C-terminal regions of this protein were highly similar to those of other bacterial Fla. However, the aa sequence of the middle portion was quite different from that of a variant strain of the same species, Sm 274.

Protein kinase C activation down-regulates natriuretic peptide receptor C expression via transcriptional and post-translational pathways.

Natriuretic peptide receptor C (NPR-C) mRNA expression and ANP-binding activity via NPR-C are significantly down-regulated in HeLa cells with phorbol myristate acetate (PMA) treatment. Stabilization of the NPR-C mRNA by PMA indicated that down-regulation of its mRNA was mediated through negative transcriptional regulation. Despite the significant loss of the mRNA, reduction of NPR-C-specific ANP-binding activity after PMA exposure (4 h) was accompanied by a slight decrease in total NPR-C protein (with a 5% loss) and was also produced in the presence of actinomycin D or cycloheximide. The inhibitory effect of a long PMA exposure (18 h) paralleled with a decrease in total NPR-C protein is suggested to be dependent on reduction of de novo NPR-C synthesis. PMA-induced transcriptional and post-translational down-regulation of NPR-C was effectively reversible in the presence of the protein kinase C inhibitor GF109203X. These findings demonstrate that protein kinase C activation down-regulated NPR-C expression through transcriptional and post-translational pathways and that immediate functional receptor loss was mediated via a post-translational mechanism, such as enhanced receptor internalization.

Site-directed mutation makes rabbit calcyclin dimer.

Unlike human, rat and mouse calcyclin, purified rabbit calcyclin did not form a dimer on Tricine SDS-PAGE under non-reduced conditions. Based on the internal peptide sequence of rabbit calcylin, we isolated and sequenced a cDNA clone encoding calcyclin. The sequence of this clone (pCalC) is 629 bp long and codes 90 amino acid residues of a protein with a molecular mass of 10,153 Da. By Northern blot analysis, a major band of 0.9 kbp and a minor band of 2.6 kbp were detected in the lung. The recombinant calcyclin mutated serine at the third position to cysteine was expressed in E. coli and made dimer formation under non-reduced conditions on SDS-PAGE. Whether or not this type of mutation which prevents dimer formation of calcyclin plays a physiological role in the rabbit lung is the subject of an ongoing study.

Age changes of myofibrils of human minor pectoral muscle.

Micro- and ultramicromeasuring studies have been made on the age changes of surgically resected minor pectoral muscles in 200 Japanese females of various ages (26-80 years). With advancement of age, muscle fibers of type I decreased in number and increased in size, and the age-related increase in size is not considered to be due to an increase in number of myofibrils but seems to be due to an increase in their volume resulting from an increase in the number of myofilaments.

Age changes in size and number of muscle fibers in human minor pectoral muscle.

Micromeasuring studies have been made on the senile changes of the surgically resected minor pectoral muscles in 200 Japanese females of various ages (26-80 years). The microscopic differentiation between type I (red) and type II (white) fibers were made by the histochemically demonstrated myosin-ATPase activity with frozen sections and also by the immunohistochemical demonstration of beta-enolase in the paraffin sections with indirect antibody method. The decrease with age in weight of the muscle was not markedly observed. Muscle fibers of type I were significantly decreased in number and increased in size after 60 years of age. However, size of muscle fibers of type II decreased gradually after 40 years of age. However, size of muscle fibers of type II decreased gradually after 40 years of age. Total volume of type I muscle fibers was not changed according to age, on the contrary, that of the type II fibers decreased significantly after 60 years of age.

Warm cells revealed by microfluorimetry of Ca2+ in cultured dorsal root ganglion neurons.

Warm cells were identified by Fura-PE3-based microfluorimetry of Ca2+ in cultured dorsal root ganglion (DRG) neurons. In response to a physiologically relevant stimulus temperature (43 degrees C), a subpopulation of small DRG neurons from new born rats increased the intracellular Ca2+ concentration ([Ca2+]i). Seven percent of the cells responded to the warm stimulus. The stimulus evoked elevation in [Ca2+]i from 52.5 +/- 9.5 nM (mean +/- S.D., n = 18) to 171.0 +/- 15.6 nM in cells between 15 and 25 microns in diameter. The depletion of extracellular Ca2+ diminished the Ca2+ elevation. The Na(+)-free condition also diminished the response. We concluded that the heat stimulation opens nonselective cation channels in putative warm cells from DRG neurons.

Isolation and analysis of lipase-overproducing mutants of Serratia marcescens.

We have isolated a lipase-overproducing mutant, GE14, from Serratia marcescens 8000 after three rounds of N-methyl-N'-nitro-N-nitrosoguanidine mutagenesis. The mutant GE14 produced 95 kU/ml of extracellular lipase in the lipase medium, which was about threefold higher than that of produced by the original strain 8000. Enzymatic characteristics including specific activity of purified lipases from culture supernatants of GE14 and 8000 were almost same. The lipase gene (lipA) of GE14 contained two base substitutions; one in the promoter region and another in the N-terminal region of the lipA gene without an amino acid substitution. Promoter analysis using lipA-lacZ fusion plasmids revealed that these substitutions were responsible for the increase in the lipA expression level, independently. In contrast, no base substitution was found in the genes encoding the lipase secretion device, the Lip system. In addition, the genes coding for metalloprotease and the cell surface layer protein which are both secreted through the Lip system and associated with extracellular lipase production, also contained no base substitution. The strain GE14 carrying a high-copy-number lipA plasmid produced a larger amount of the extracellular lipase than the recombinant strains of 8000 and other mutants also did, indicating that GE14 was not only a lipase-overproducing strain, but also an advantageous host strain for overproducing the lipase by a recombinant DNA technique. These results suggest that the lipase-overproducing mutant GE14 and its recombinant strains are promising candidates for the industrial production of the S. marcescens lipase.

Gastroesophageal reflux occurring after repair of congenital diaphragmatic hernia.

Over the past 2 decades, 110 patients with congenital diaphragmatic hernia (CDH) were treated in the authors' hospital. Eighty-six survived; of these, 10 patients (11.6%) had gastroesophageal reflux (GER) after repair of CDH. Seven occurred in the past 5 years, during which time advanced intensive care including extracorporeal membrane oxygenation (ECMO) was used. Vomiting started within 4 weeks after repair of CDH in eight cases, and hiatal hernia was demonstrated in six cases. Three patients responded to conservative therapy; the other seven required antireflux surgery. Several factors are believed to be possible causes of the development of GER in CDH cases. Among them, slow pulmonary expansion of the affected side was thought to be the most important. Namely, in a case of CDH associated with severe hypoplastic lung, the esophagus may be deviated to the affected side before the lung is expanded. After expansion, the abdominal esophagus shortens, and GER or a hiatal hernia can occur in severe cases. There were seven such patients in our series of 10. With the increase in the survival rate of CDH cases associated with severe hypoplastic lung, the number of such patients also may increase. Therefore, some additional procedure to prevent the lower esophagus from sliding will be necessary in the repair of diaphragmatic hernia.

Preservation of the umbilical cord at the primary fascial closure in infants with gastroschisis.

Recently, the survival of patients with gastroschisis has been dramatically improved and it has reached more than 90%. Over the last 10 years, 20 of 21 cases (95%) survived in our hospital. We have been using the primary fascial closure of the abdominal wall as a standard operative procedure. The umbilical cord was usually excised at the operation in order to secure the suture line and prevent wound infection. The survivors sometimes complained of the absence of the umbilicus. However, it was somewhat difficult to create a new umbilicus later by use of the surrounding skin. In the last five cases, we tried to carry out the primary fascial closure with preservation of the umbilical cord. All patients could obtain good cosmetic results with near-normal appearance. Omphalitis or cellulitis was never observed, but a small umbilical hernia occurred in one case.

Development in lung function of the affected side after repair of congenital diaphragmatic hernia.

The widespread use of newly developed techniques including extracorporeal membrane oxygenation (ECMO) has led to the survival of a number of patients with congenital diaphragmatic hernia (CDH) and associated hypoplastic lung. However, it is not fully recognized whether the hypoplastic and small lung of the affected side has the ability to develop its function after repair of CDH. The authors studied the lung function of 32 patients with CDH in whom these new methods were used. Two parameters, lung volume and pulmonary perfusion amount, were used to evaluate lung function. The former (checked by computed tomography scan) was used to evaluate the size of lung; the latter (checked by perfusion scintigram) was used to assess vascular density. The patients were divided into two groups, based on values of alveolar-arterial difference in oxygen content (AaDo2) at the time of admission. In group A (AaDo2 < 500 mm Hg; 12 cases), whose respiratory distress was mild and could be managed with ventilator care alone, the mean lung volume value for the affected side was 86% of the contralateral lung value from the initial study, and reached 93% at the time of follow-up study. The perfusion amount also exceeded 80% of the contralateral lung value from the initial study. Thus, it is likely that group A's affected-side lung is not small and has developed at a rate similar to that of the contralateral lung. However, in group B patients (AaDo2 > 500 mm Hg; 20 cases), who had severe respiratory distress at the admission and were managed with new techniques including ECMO, both lung volume and perfusion amount of the affected side initially were low in all cases (ie, mean values were 61% and 53% of contralateral-lung values, respectively). At the time of follow-up, the lung volume had increased in most cases (mean value, 88% of the contralateral lung value), but the perfusion amount of the affected side had not increased in most cases. It remained low, or decreased to below the initial value; the mean was 53% of the contralateral lung value. The initial mean perfusion: volume ratio (87%) had decreased significantly (to 62%) by the time of follow-up. This tendency was exaggerated in the 11 ECMO cases. These data might indicate that in most group B cases, the lung of the affected side has little ability to develop arterial branches, or certainly will be delayed in comparison to the contralateral lung, and that enlargement of lung volume may depend on overexpansion or emphysematous change rather than cellular growth. The present data also suggest that, in group B cases, total lung function will depend on the contralateral lung for a relatively long time.

Clinical, morphologic, and biochemical characteristics of Chediak-Higashi syndrome in fifty-six Japanese black cattle.

OBJECTIVE: To characterize Chediak-Higashi syndrome (C-HS) in Japanese Black cattle. ANIMALS: 56 of 200 cattle with a bleeding disorder and giant granules in leukocytes. PROCEDURE: Clinical observation, CBC, hemostatic screening test, platelet aggregometry, electron microscopy, platelet constituent analysis, and ophthalmoscopic examination were done. RESULTS: Affected Japanese Black cattle had increased bleeding tendency and abnormal granules in their leukocytes. Susceptibility to infection was not increased. Cutaneous albinism was evident in 6 new-born calves, but not in most affected cattle. In all affected cattle, the tapetal fundus was pale and the nontapetal fundus was almost devoid of pigment. By electron microscopy, a remarkable decrease in the number of dense granules in platelets was observed. Functionally, collagen-induced platelet aggregation was markedly reduced. CONCLUSIONS: This bleeding disorder was diagnosed as C-HS. With regard to susceptibility to infection, albinism, and mortality, clinical manifestations of C-HS in Japanese Black cattle were moderate, compared with C-HS in human beings and Hereford cattle. CLINICAL RELEVANCE: Because an autosomal recessive mode of inheritance was documented and recessive homozygotes could be easily detected, C-HS in Japanese Black cattle can be controlled.

[A case of successful operation for chronic aortic dissection (DeBakey 1 type) complicated with cerebral infarction].

We report a successful operation for a patient of chronic aortic dissection complicated with cerebral infarction. A 60-year-old woman has been treated suffering from cerebral infarction for 7 years. She was referred to our hospital for abnormal chest X-ray and complaint of dry cough. The enhanced CT scan and aortogram demonstrated chronic aortic dissection (DeBakey I) extending from ascending aorta to the abdominal aortic bifurcation with occlusion of right common carotid artery and dilated ascending aorta and aortic arch. The onset of aortic dissection seemed to be, from a clinical point of view, coincident with the onset of cerebral infarction seven years ago. The replacement of the ascending aorta and aortic arch with woven Dacron velour graft were performed under hypothermic and selective cerebral perfusion. The postoperative course was uneventful although complicated with transient recurrence nerve palsy and she has been doing well.

[Effect of CDDP/5'-DFUR combination chemotherapy for advanced or recurrent gastric cancer].

CDDP/5'-DFUR combination chemotherapy was performed on 17 patients with non-resected and recurrent gastric cancer (clinical stage were IVb in all patients). They were treated with 1,400 mg/m2 of 5'-DFUR on days 1-4 orally following by withdrawal 10 days, every 2 weeks repeatedly and 80 mg/m2 of CDDP (c. i. v., on day 5, every 4 weeks). This chemotherapy was performed for at least 2 courses on all patients. Eight of 17 patients achieved a partial response and the overall response rate was 47.1% (differentiated type 57.1%, undifferentiated type 45.5%). Response rates of each lesion were as follows: primary foci 42.9%, abdominal lymph nodes 57.1%, hepatic metastasis 60.0% and ascites 33.3%, respectively. Improvement of performance status was seen in 12 of 17 patients (70.6%). The overall median survival time was 227 days. The median outpatient period was 113 days. There was no high-grade toxicity over grade 2. Therapeutic toxicity of grade 2 was manifested as renal dysfunction (23.5%), nausea/vomiting (17.6%), leukopenia (5.9%) and anemia (5.9%). We evaluated the therapeutic effect by visual examination after completion of the second course. However, poor effect and high incidence of renal dysfunction were found in patients treated with this therapy over four times. Therefore, the maximum effect seemed to be revealed after completion of the fourth course. From the present study, CDDP/5'-DFUR combination chemotherapy seems to be effective for patients with high-grade advanced gastric cancer and improved their quality of life.