Pseudo-acylceramide with linoleic acid produces selective recovery of diminished cutaneous barrier function in essential fatty acid-deficient rats and has an inhibitory effect on epidermal hyperplasia.

Pseudo-acylceramides with different acyl properties were investigated for their capacity to restore diminished barrier function in essential fatty acid-deficient rats. Daily topical applications of synthetic pseudo-acylceramides containing ester-linked linoleic acid caused a dose-dependent, significant reduction of transepidermal water loss (TEWL). Both other pseudo-acylceramides with ester-linked oleic acid or saturated alkyl chains and ordinary ceramides exhibited a poor effect on recovery of TEWL. Furthermore, pseudoceramide containing ether-linked linoleic acid, which is biologically inactive in terms of degradation by hydrolytic enzymes, also induced a significant and similar increase in the barrier function. This restoration of barrier function by pseudo-acylceramides with linoleic acid was accompanied by suppressed DNA synthesis in the EFAD rat epidermis. In UVB-irradiated guinea pig skin, topical applications of the pseudo-acylceramides with linoleic acid immediately after the exposure significantly reduced epidermal hyperplasia, secondary to markedly diminished barrier disruption, whereas linoleic acid itself did not. A comparison of both the anti-hyperplasia and the barrier recovery effects in the series of pseudo-ceramide derivatives examined revealed that the suppressive effect on the induced epidermal hyperplasia was paralleled by the recovery of the barrier defect in EFAD rats. These findings directly suggest that acylceramide with an ester-linked linoleic acid has an essential role in the epidermal permeability barrier.

Developmental outcome of very low birth weight twins conceived by assisted reproduction techniques.

OBJECTIVE: To evaluate the differences in developmental outcomes between very low birth weight twins conceived by assisted reproduction techniques and those conceived spontaneously. STUDY DESIGN: Twenty-two sets of very low birth weight twins were evaluated by the Kyoto Scale for Psychological Development at 36 months of corrected age. Total developmental quotient and developmental quotient (DQ) for three subscales, posture-motor, cognition-adaptation and language-social, were evaluated. RESULTS: Twins conceived with medical assistance demonstrated a higher incidence of total DQ below 85 with lower DQ for cognition-adaptation and language-social skills than spontaneously conceived twins, whereas the quotient for posture-motor skills in medically assisted twins was comparable to that of spontaneously conceived twins. CONCLUSION: At 3 years of age very low birth weight twins conceived by assisted reproduction techniques demonstrated lower cognitive and language skills than twins conceived naturally.

Induction of melanization within hair bulb melanocytes in chinchilla mutant by melanogenic stimulants.

In an attempt to clarify the mechanisms underlying the lack of melanin formation in hair bulb melanocytes of chinchilla mice (genotype a/a, cch/cch, strain PW), we studied the effect of exogenous melanogenic stimulants such as theophylline (Tp), dibutyryl cyclic AMP (db-cAMP), and alpha-melanocyte-stimulating hormone (alpha-MSH) on the induction of melanization. Skin explants excised from the dorsa of chinchilla or lethal yellow C57BL/6J, Ay/a) mice at 7 to 9 days of age were cultured in the presence of Tp (2 mM), db-cAMP (2 mM), or alpha-MSH (1.0 microgram/ml). After 2 to 5 days, melanin formation was induced in hair bulb melanocytes of chinchilla mutant in response to both Tp and db-cAMP, but alpha-MSH did not produce new melanin formation. In contrast, yellow mutant increased the melanin formation in response to all stimulants. Electron microscopic studies demonstrated that while non-treated hair bulb melanocytes of chinchilla mutant contain a large number of stage II-III melanosomes without melanin deposition, a hair bulb treated with Tp exhibits the new formation of melanin within melanosomes that appears both as typical eumelanosomes with striated longitudinal matrices and as pheomelanosomes with vacuolar melanization. Quantitative analysis of melanin has revealed that in chinchilla mutant, Tp and db-cAMP induce a severalfold increase in the formation of both eumelanin [pyrrole-2,3,5-tricarboxylic acid (PTCA)] and pheomelanin (aminohydroxyphenylalanine), whereas alpha-MSH does not stimulate production of either melanin. In yellow mutant, db-cAMP induced a remarkable increase in eumelanin (PTCA), in contrast to the fewfold increase induced by alpha-MSH and Tp. All stimulants induced a slight increase in pheomelanin to a similar extent. These different reactions to melanogenic stimulation suggest a possible defect in the tyrosinase activation system within hair bulb melanocytes in chinchilla mutants.

Allergic contact dermatitis releases soluble factors that stimulate melanogenesis through activation of protein kinase C-related signal-transduction pathway.

Phenylazo-naphthol (PAN) allergy induces visibly well-defined and late-appearing hyperpigmentation of brownish yellow guinea pig skin in clear contrast to dinitrochlorobenzene (DNCB) allergy, which has very low incidence of hyperpigmentation. Skin extract from PAN allergy at 20-29 d post-challenge exhibited marked melanogenic stimulatory effects (3H2O release and 14C-thiouracil incorporation) when added to cultured guinea pig melanocytes. The time course in the appearance of melanogenic factor was definitely consistent with the induction pattern of visible pigmentation. By contrast, the addition of DNCB-challenged skin extract demonstrated no significant stimulating effect on melanogenesis in either assay system on any of the post-challenge days tested. Assay of intracellular inositol 1,4,5-trisphosphate formed through incubation with the melanocytes demonstrated that the PAN-allergy skin extract at day 28, which contains definite melanogenic factors, stimulated the formation of inositol 1,4,5-trisphosphate that occurs around 50 seconds in contrast to no or little increase with extracts obtained at days 0 and 1 post-challenge. Gel chromatographic analysis revealed that the PAN-allergy skin extract at day 28 contained a newly generated melanogenic fraction with a molecular weight of approximately 9000 Da which was also capable of stimulating DNA synthesis and activating the signal-transduction process (inositol trisphosphate formation) when added to guinea pig melanocytes. Both stimulations of melanogenesis and DNA synthesis by the 9000 Da fraction were completely abolished by the prior and simultaneous addition of protein kinase C (PKC) inhibitor (H-7) or its down-regulatory agent, phorbol 12,13-dibutyrate (PdBu). Taken together, these results suggest that PAN allergy provides a new mechanism of hypermelanization in which endogenous factors synthesized within skin induce the activation of signal-transduction pathways such as phosphoinositide turnover through ligands-receptor binding, resulting in the stimulation of melanocytes possibly through the activation of PKC.

Endothelin-1 as a new melanogen: coordinated expression of its gene and the tyrosinase gene in UVB-exposed human epidermis.

We previously demonstrated that human keratinocytes produce and secrete endothelins (ET), which can be strong mitogens for human melanocytes. Ultraviolet B (UVB) exposure highly stimulated the paracrine linkage of endothelins between keratinocytes and melanocytes, indicating that they are keratinocyte-derived intrinsic mitogens in UVB-induced pigmentation. In this study, the role of ET-1 as a melanogen in UVB melanogenesis was investigated in vitro and in vivo. In the conditioned medium of keratinocytes exposed to UVB, melanin synthesis by human melanocytes, as measured by 14C-thiouracil incorporation, was significantly accentuated. This stimulatory effect was reduced by anti-ET-1 to the level of that in the non-UVB-exposed control, suggesting an essential role of ET-1 as an intrinsic melanogen in UVB-induced melanogenesis. In a parallel study, the addition of 10 nM ET-1 induced an increase in tyrosinase activity in cultured human melanocytes and was accompanied by elevated levels of tyrosinase and tyrosinase-related protein-1 mRNA expression as shown by Northern blotting. Reverse transcription-polymerase chain reaction of RNA isolated from the epidermis of human skin exposed to UVB revealed that, whereas in non-exposed sites ET-1, IL-1 alpha, and tyrosinase mRNA signals were scarcely detected, UVB-irradiation, with a dose of twice the minimal erythema dose, caused a significant increase in the expressions of the three genes 5 d after irradiation. These findings suggest that ET-1 is an important mediator for UVB-induced pigmentation in the epidermis in vivo.

Sphingosylphosphorylcholine is a potent inducer of intercellular adhesion molecule-1 expression in human keratinocytes.

We recently reported that the epidermis of patients with atopic dermatitis contains an abnormally expressed sphingomyelin deacylase that yields a large amount of sphingosylphosphorylcholine (SPC) rather than ceramide. In this study, we characterize inflammatory roles of newly discovered chemicals in the epidermis by elucidating biologic effects of SPC on intercellular adhesion molecules-1 (ICAM-I) expression by human keratinocytes in culture in comparison with other sphingolipids. Using fluorescence-activated cell sorter analysis, we found that SPC treatment at concentrations of 10-20 microM significantly enhanced the expression of ICAM-I by cultured human keratinocytes in a dose-dependent manner after incubation for 15-24 h, and, using northern blot analysis, that this was accompanied by increased expression of ICAM-1 mRNA within 4 h of incubation. Transforming necrosis factor-alpha (TNF-alpha) levels in the medium of keratinocytes treated at a 10 microM concentration of SPC were significantly increased by 200%. Furthermore, the SPC-induced ICAM-1 expression was partially abolished by the concomitant addition of anti-TNF-alpha, suggesting a partial autocrine involvement of TNF-alpha in ICAM-1 expression. Assay of mitogen-activated protein kinase revealed that 10 microM SPC induced a rapid activation of mitogen-activated protein kinase in human keratinocytes, including an increase in its phosphorylation within 5 min, which then declined to the baseline control level after 30 min. In contrast, sphingomyelin or sphingosine had no significant potential to activate mitogen-activated protein kinase at the same concentration. These findings suggest that SPC plays an important role in the inflammatory process of epidermis in skin diseases, such as atopic dermatitis, with high expression of sphingomyelin deacylase.

Abnormal expression of sphingomyelin acylase in atopic dermatitis: an etiologic factor for ceramide deficiency?

Previously, we demonstrated that there is a marked reduction in the amount of ceramide in the stratum corneum of both lesional and nonlesional forearms in atopic dermatitis (AD), suggesting that an insufficiency of ceramides in the stratum corneum is an etiologic factor in atopic dry and barrier-disrupted skin. In this study, we investigated, as a possible mechanism involved in the ceramide deficiency, whether sphingomyelin (SM) metabolism is altered in AD as compared to normal controls. In stripped stratum corneum and biopsied whole epidermis of patients with AD, SM hydrolysis as measured at pH 4.7 using [choline-methyl-14C]sphingomyelin as a substrate were markedly increased by 27- and 7-fold, respectively. Radio-thin-layer chromatography of the reaction products revealed that, whereas the SM hydrolysis in age-matched normal controls were associated with sphingomyelinase (SMase) that degrades SM to yield ceramides and phosphorylcholine (PC), most of the SM hydrolysis detected in AD were attributable not to the SMase but to a hitherto undiscovered epidermal enzyme, SM acylase, which releases free fatty acid and sphingosyl-PC (Sph-PC) instead of ceramides. The potential of this acylase-like enzyme to generate Sph-PC through SM hydrolysis was corroborated by thin-layer chromatographic analysis of the reaction products obtained using porcine kidney acylase, followed by high-performance liquid chromatography-mass spectrometry. Furthermore, Sph-PC was also detected by high-performance liquid chromatography-mass spectrometry after incubation of SM with atopic stratum corneum samples. On the other hand, the stratum corneum of patients with contact dermatitis or chronic eczema exhibited neither increased SM hydrolysis nor the generation of Sph-PC upon radio-thin-layer chromatographic analysis. These findings suggest that SM metabolism is altered in AD, resulting in a decrease in levels of ceramides, which could be an etiologic factor in the continuous generation of atopic dry and barrier disrupted skin observed in AD.

Inhibition by cyclic AMP of guanine nucleotide-induced activation of phosphoinositide-specific phospholipase C in human platelets.

Phosphoinositide-specific phospholipase C (PLC) activity of human platelet membranes was activated by the nonhydrolyzable guanine nucleotide GTP gamma S. This activation did not occur in either membranes prepared from dibutyryl cyclic AMP-pretreated platelets (A-membranes) or those prepared from untreated cells and subsequently incubated with cyclic AMP (cAMP) (B-membranes). This cAMP-mediated inhibition was abolished in the presence of inhibitors of cAMP-dependent protein kinase (A-kinase), suggesting that the inhibition was due to phosphorylation of (a) protein component(s). No significant differences were observed in the basal PLC activity and the extent of pertussis toxin-catalyzed ADP-ribosylation among control membranes and the two types of phosphorylated membranes (A- and B-membranes). GTP-binding activities of Gs, Gi and GTP-binding proteins of lower molecular masses were not altered by the phosphorylation of the membranes. These findings suggest that a GTP-binding protein is involved in the GTP gamma S-mediated activation of PLC and that cAMP (plus A-kinase) inhibits this activation by phosphorylating a membrane protein (probably a 240-kDa protein), rather than the GTP-binding protein or PLC itself. It is likely that this phosphorylation uncouples the GTP-binding protein from PLC.

High thoracic neurinoma mimicking femoral neuralgia.

We report a case of spinal neurinoma at a high thoracic level, whose main presentation was intractable pain in a body part innervated by the right femoral nerve. Sensations of pain and temperature were impaired in the right thigh, but usual symptoms of myelopathy were undetectable. In conjunction with the other reports, this case suggests that spinal tumors at high thoracic levels can produce remote symptoms mimicking peripheral neuropathy such as femoral or sciatic neuralgia.

Augmentation of anti-tumor activity by immunization with Mycobacterium tuberculosis (Tbc) and tuberculin-coupled tumor cells.

The anti-tumor effect of immunization with heat-killed Mycobacterium tuberculosis (Tbc) and Tuberculin (PPD)-coupled syngeneic tumor cells was examined in vivo. Three tumor cell lines were employed. Immunization of Tbc-primed BALB/c mice with PPD-coupled syngeneic Meth-A tumor cells displayed a potent anti-tumor effect on viable Meth-A cells inoculated subcutaneously. Neither PPD-coupled LLC (Lewis Lung Carcinoma) cells nor sonicated PPD-coupled Meth-A cells were capable of immunizing these mice. PPD-coupled syngeneic whole tumor cells were indispensable for induction of this tumor-specific resistance. Immunization of Tbc-primed C3H/He mice with PPD-coupled syngeneic MH134 tumor cells did not elicit anti-tumor activity against MH134, but additional pretreatment of mice with cyclophosphamide brought on an anti-tumor effect. Antimetastatic reactivity was investigated in C57BL/6 mice bearing LLC, with a reduction in metastases noted. This antimetastatic effect was observed even when the mice were immunized with PPD-coupled LLC cells three days after removal of the initial tumor. Immunization with Tbc and PPD-coupled Meth-A cells together with intraperitoneal administration of murine or rat interleukin 2 (IL 2) further augmented anti-Meth-A resistance. Murine IL 2 further inhibited tumor growth during the early stage, while rat IL 2 showed an anti-tumor effect throughout the course of tumor growth.

[A severe case of reexpansion pulmonary edema in an asthmatic patient].

A 60-year-old man with poorly controlled bronchial asthma was proposed for an emergency appendectomy. His preoperative chest X-P revealed that his left lung was completely collapsed with pneumothorax, but its onset was unclear. Following the left thoracocentesis, appendectomy was performed under general anesthesia (oxygen-halothane). About one hour after the thoracocentesis, pinkish foamy tracheal secretion was massively drained and its protein concentration was 3.8 g.dl-1.PaCO2 was 95 mmHg and PaO2 was 69 mmHg (FIO2 1.0). His chest X-P showed signs of pulmonary edema in his left lung and infiltrating shadow was observed in his right lung. IMV with PEEP, aminophylline and prednisolone improved his respiratory status and on the 11 th day he was weaned from the respirator. In a case of pneumothorax with unclear duration like ours, it is necessary to consider the possibility of the reexpansion pulmonary edema.

[Survival of patients with gastric cancer treated with intra-lymph nodal injection of activated carbon particles absorbed mitomycin C].

We studied the efficacy of the intra-lymph nodal injection of the activated carbon particle absorbed mitomycin C (MMC-CH40) for gastric cancer. Ninety-five patients with gastric cancer underwent gastrectomy with D1 or D2 lymph node dissection. Of these, 38 patients were treated with intra-lymph nodal injection of MMC-CH 40 (MMC-CH 40 group). The other 57 patients were classified into the control group. The survival of MMC-CH group was significantly higher than that of the control group using generalized Wilcoxon method. However, since the percentage of stage IV was higher in the control group than in MMC-CH 40 group, the survivals of subgroups of stage I-III were compared. Although the percentage of the early gastric cancer was higher in the control group of stage I-III (n = 35) than in the MMC-CH group of stage I-III (n = 38), the survival curves of MMC-CH group were higher than in the control group. The difference in survival between the two groups was significant at 8 months after surgery. These results indicate that this new therapy improves survival in patients with stage I-III gastric cancer.

Hemophagocytic lymphohistiocytosis in a newborn infant born to a mother with Sjögren syndrome antibodies.

We encountered a neonatal patient with hemophagocytic lymphohistiocytosis (HLH) whose mother was positive for anti-Ro/SSA and anti-La/SSB antibodies. Complete atrioventricular block was found in a male patient at 29 weeks of gestation. The patient was born at 40 weeks of gestation. He showed severe circulatory disturbance at 22 h after the birth, and he also had elevated serum levels of aspartate aminotransferase (1027 IU l(-1)), alanine aminotransferase (121 IU l(-1)), lactic dehydrogenase (3490 IU l(-1)), ferritin (9769.7 ng ml(-1)) and soluble interleukin-2 (IL-2) receptor (3230 U ml(-1)). We could not find any known HLH genetic abnormality in the patient, but he fulfilled seven of the eight criteria for HLH. Serum levels of IL-6 and IL-8 had been already elevated in his cord blood, and serum levels of granulocyte-macrophage colony-stimulating factor and IL-8 were significantly increased on the second day of life. His symptoms regressed with the administration of hydrocortisone. We presumed that transplacental transfer of maternal antibodies could be related to the occurrence of HLH.

Neutron activation analysis of a particle returned from asteroid Itokawa.

A single grain (~3 micrograms) returned by the Hayabusa spacecraft was analyzed by neutron activation analysis. This grain is mainly composed of olivine with minor amounts of plagioclase, troilite, and metal. Our results establish that the Itokawa sample has similar chemical characteristics (iron/scandium and nickel/cobalt ratios) to chondrites, confirming that this grain is extraterrestrial in origin and has primitive chemical compositions. Estimated iridium/nickel and iridium/cobalt ratios for metal in the Itokawa samples are about five times lower than CI carbonaceous chondrite values. A similar depletion of iridium was observed in chondrule metals of ordinary chondrites. These metals must have condensed from the nebular where refractory siderophile elements already condensed and were segregated.

Purification and biochemical characterization of membrane-bound epidermal ceramidases from guinea pig skin.

Ceramidase (CDase) catalyzes the hydrolysis of ceramides to yield sphingosine and fatty acid. In this paper, two forms of membrane-bound alkaline ceramidase, have been, for the first time, purified from guinea pig epidermis by chromatography on DEAE-cellulose, phenyl-Superose, HCA-hyroxyapatite, isoelectric focusing, Mono Q, and TSK-3000SW column. One species (CDase-I) migrated upon SDS-polyacrylamide gel electrophoresis as a single band with an apparent molecular mass of 60 kDa; the other (CDase-II) was only partially purified with apparent M(r) of about 148,000 estimated by gel filtration. The specific activities of the two species increased by 1.130- (for CDase-I) and 400-fold (for CDase-II) over the original tissue extract. The activity of both enzymes for ceramide species decreased in the order of linoleoyl > oleoyl > palmitoylsphingosine. The optimal pH for enzyme activity was approximately 7.0-9.0 for CDase-I and 7.5-8.5 for CDase-II. Interestingly, both enzymes were inhibited by the reaction product sphingosine with a concentration for half-maximal inhibition (ID50) of 100-130 microM, compared to the apparent kinetic parameters with CDase-I (Km = 90 microM, Vmax = 0.62 unit) and CDase-II (Km = 140 microM, Vmax = 0.50 units). Some lipids, such as phosphatidylcholine and sphingomyelin, are also inhibitory with IC50 values of 50-250 microM, suggesting well controlled CDase activity by sphingolipid metabolites. These studies begin to elucidate a regulatory mechanism for the balance of the ratio of ceramide/sphingosine which can serve as an intracellular effector molecule in epidermis.

Purification and characterization of membrane-bound phospholipase C specific for phosphoinositides from human platelets.

Two peaks (mPLC-I and mPLC-II) of phosphatidylinositol 4,5-bisphosphate (PIP2)-hydrolyzing activity were resolved when 1% sodium cholate extract from particulate fractions of human platelet was chromatographed on a heparin-Sepharose column. The major peak of enzyme activity (mPLC-II) was purified to homogeneity by a combination of Fast Q-Sepharose, heparin-Sepharose, Ultrogel AcA-44, Mono Q, Superose 6-12 combination column, and Superose 12 column chromatographies. The specific activity increased 2,700-fold as compared with that of the starting particulate fraction. The purified mPLC-II had an estimated molecular weight of 61,000 on sodium dodecyl sulfate-polyacrylamide gels. The minor peak of enzyme activity (mPLC-I) was partially purified to 430-fold. Both enzymes hydrolyzed PIP2 at low Ca2+ concentration (0.1-10 microM) and exhibited higher Vmax for PIP2 than for phosphatidylinositol. PIP2-hydrolyzing activities of both enzymes were enhanced by various detergents and lipids, such as deoxycholate, cholate, phosphatidylethanolamine, and dimyristoylphosphatidylcholine. The mPLC-I and mPLC-II activities were increased by Ca2+, but not by Mg2+, while Hg2+, Fe2+, Cu2+, and La3+ were inhibitory. GTP-binding proteins (Gi, Go, and Ki-ras protein) had no significant effects on the mPLC-II activity.

Signalling mechanisms of endothelin-induced mitogenesis and melanogenesis in human melanocytes.

To understand the signalling mechanisms involved in the dual stimulatory effects of endothelin-1 (ET-1) on DNA synthesis and melanization in cultured human melanocytes, we analysed the biological profile of ET-1 receptor and determined the effects of ET-1 on the protein kinase C, cyclic AMP system and mitogen-activated protein kinase (MAP kinase) in comparison with their relevant stimulants. The photoaffinity labelling of ET-1 receptors with Denny-Jaff reagents revealed an ET-1 receptor with a molecular mass of 51 kDa in human melanocytes. The ET(A) receptor subtype-sensitive antagonist BQ123(50 nM) or pertussis toxin (100 ng/ml) significantly suppressed the ET-1-induced intracellular calcium mobilization, indicating the presence of pertussis toxin-sensitive G-protein-coupled ET(A) receptors. An assay of protein kinase C activity revealed that 10nM ET-1 translocated cytosolic protein kinase C to membrane-bound protein kinase C within 5 min of the start of incubation. In contrast, receptor-mediated melanocyte activation by ET-1 was accompanied by an elevated level of cyclic AMP (4-fold over control) after 10-60 min of incubation, whereas 60 min of incubation of human melanocytes with c-Kit or c-Met ligands such as stem cell factor (10 nM) or basic fibroblast growth factor (10 nM) did not elevate the cyclic AMP level. We have also demonstrated that a specific tyrosine kinase inhibitor, tyrphostin B-42 (10 microM), inhibited the ET-1-induced growth stimulation, suggesting the involvement of the tyrosine kinase pathway in growth stimulation. Consistently, an assay of MAP kinase revealed that ET-1 caused a 10-fold activation of MAP kinase after 5 min of incubation with human melanocytes in a similar way to tyrosine kinase ligands such as stem cell factor and hepatocyte growth factor. Further, the DNA synthesis stimulated by the c-Kit ligand stem cell factor at a concentration of 1 nM was synergistically enhanced by 5 nM ET-1. These results suggest that ET-induced dual cellular events in human melanocytes are closely associated with cross-talk between the protein kinase C and A and tyrosine kinase pathways.