Masamitsu Yamamoto

ADP-ribosylation of the rho/rac proteins induces growth inhibition, neurite outgrowth and acetylcholine esterase in cultured PC-12 cells.

Botulinum ADP-ribosyltransferase C3 (C3 exoenzyme) was purified to homogeneity and added to cultured rat pheochromocytoma PC-12 cells. Incubation with this exoenzyme caused inhibition of cell growth and induced neurites as well as acetylcholine esterase in these cells. These changes were dependent on the amount of the enzyme added to the culture, which correlated with the in situ ADP-ribosylation of the rho/rac proteins in the cells. Preincubation with a specific anti-C3 exoenzyme monoclonal antibody inhibited both the ADP-ribosyltransferase activity and the neurite-inducing activity of the enzyme preparation. These results suggest that C3 exoenzyme affected the cellular function of the rho/rac proteins by ADP-ribosylation to induce these changes in the cells.

Aleukemic leukemia cutis

A 39-year-old man had multiple nodules on the skin. The appearance of atypical monocytes in a skin biopsy specimen preceded the onset of overt acute monocytic leukemia by 14 months.

Expression of thromboxane A2 receptor in cultured human erythroleukemia cells and its induction by 12-O-tetradecanoylphorbol-13-acetate

Using [125I]I-S-145-OH, a radiolabeled derivative of a thromboxane (TX) A2 receptor antagonist, we have studied the expression of the TXA2 receptor in several lines of cultured leukemia cells. Specific binding of the ligand was observed in cells of two human erythroleukemia cell lines, K562 and HEL. However, only negligible binding was seen in HL-60 human promyelocytic leukemia cells and L-1210 murine leukemia cells. Scatchard analyses revealed a curvilinear plot which indicated the existence of two classes of binding sites in these cells. The Kd and Bmax values of the high and low affinity bindings in HEL cells were 2.4 nM and 24 fmol/10(6) cells, and 58 nM and 360 fmol/10(6) cells, respectively. These values in K562 cells were 2.8 nM and 16 fmol/10(6) cells, and 18 nM and 46 fmol/10(6) cells, respectively. The addition of 12-O-tetradecanoyl-phorbol-13-acetate (TPA) to the cultures of K562 and HEL cells caused a concentration- and time-dependent increase in the binding activity. TPA at 10(-8) M increased the Bmax values of both high and low affinity bindings approximately 3 times without significant change in their Kd values and these increases were inhibited by the addition of actinomycin D. Both classes of the binding in cells of each cell line were specifically displaced by several TXA2/prostaglandin (PG) H2 analogues, although the relative specificities to the analogues were different in the two classes. These results suggest that both HEL and K562 cells express the TXA2 receptor on their cell surface and TPA strongly induces this expression in these cells.

Late onset erythropoietic porphyria

A 51‐year‐old Japanese man and his 56‐year‐old sister of consanguineous parents had skin lesions with areas of dark‐brown pigmentation and blisters with minimal trauma on sunexposed skin which resembled those seen in porphyria cutanea tarda. Their fresh urine was wine‐red in colour and fluoresced with ultraviolet light. The peripheral blood contained fluorocytes and porphyrin analysis of the red blood cells, urine and faeces of the patients revealed an increase of the isotype I of uro‐ and coproporphyrin and normal concentrations of δ‐aminolaevulinate and porphobilinogen, suggesting the diagnosis of erythropoietic porphyria. No other members of this family had symptoms or biochemical findings suggestive of porphyria. We consider these two cases to be that of late onset erythropoietic porphyria.

Increased serum levels of squamous cell carcinoma-related antigen in pemphigus

Serum levels of squamous cell carcinoma‐related antigen (SCC‐RAG) were measured in five cases of pemphigus, five cases of bullous pemphigoid and 18 cases of benign and malignant dermatoses other than SCC. The SCC‐RAG titres were significantly raised in four of five patients with pemphigus, while they remained within the normal range in the other dermatoses except in one case. In three pemphigus cases in whom serial measurements were made, SCC‐ RAG levels seemed to be related to disease activity. The SCC‐RAG levels in blister fluids were much higher than those in serum, suggesting that the skin is a major source of serum SCC‐ RAG.

Sneddon Syndrome with Multiple Cerebral Infarctions 12 years after the Onset of Livedo Vasculitis : A Possible Involvement of Platelet Activation

Sneddon syndrome is characterized by livedo reticularis and multiple cerebral infarctions. Skin and central nervous system symptoms usually have a synchronous onset and at times initial symptoms affect one of them, the other lagging several years behind. We here report a patient with Sneddon syndrome who developed multiple cerebral infarctions more than 10 years after the onset of livedo reticularis. While the neurological symptoms were apparent, the patient did not display active skin manifestations. Laboratory findings excluded collagen diseases, antiphospholipid antibody syndrome, and inherited quantitative deficiency of protein C, protein S and antithrombin III. Abnormal findings included extremely elevated levels of β‐thromboglobulin and platelet factor‐4 in the blood, although these acute phase markers of thrombosis were examined several years after the onset of cerebral infarctions. Platelet activation may have caused Sneddon syndrome in the present case.

Effect of a cytotoxic prostaglandin, Δ12-prostaglandin J2 on E-cadherin expression in transformed epidermal cells in culture

The cyclopentenone prostaglandins (PGs), such as delta 12-PGJ2 and PGA1, are potent inhibitors of growth in a variety of cultured cells, including human epidermal cells. To clarify the mechanism of the cytotoxicity of these PGs, we examined the effects of delta 12-PGJ2 on the function and expression of E-cadherin, which plays a major role in the maintenance of intercellular adhesion, in transformed human epidermal cells in culture (HSC-1). A 12-h incubation with 5 micrograms/ml of delta 12-PGJ2 did not affect the cell-binding activity of E-cadherin expressed in HSC-1 cells. Immunoblot analysis using a monoclonal antibody specific to human E-cadherin revealed that a 12-h incubation with 5 micrograms/ml of delta 12-PGJ2 induced E-cadherin expression in HSC-1 cells. Immunofluorescence using a monoclonal antibody against human E-cadherin demonstrated that E-cadherin was localized to the cell-cell contact regions in HSC-1 cells. Following a 12-h incubation with 5 micrograms/ml of delta 12-PGJ2, E-cadherin was also detected in a uniform pattern along cell junctions, although cell morphology was changed by the presence of cytotoxic PGs. These results suggest that the cytotoxicity of cyclopentenone PGs is related, at least in part, to E-cadherin expression in transformed human epidermal cells.

Effect of botulinum C3 exoenzyme on cell growth and cytoskeleton organization in transformed human epidermal cells in culture: a possible role for rho protein in epidermal cells.

We examined the role of rho gene products (rho proteins) on cell growth and cytoskeleton organization in transformed human epidermal cells in culture (HSC-1), using recombinant botulinum C3 exoenzyme which specifically ADP-ribosylates rho proteins. Incubation of HSC-1 cell lysates with C3 exoenzyme revealed a single [32P]ADP-ribosylated protein with a molecular weight of 23,000. This protein was identified as rhoA protein by isoelectric focusing (pI 6.0). Addition of C3 exoenzyme to the culture medium of HSC-1 cells changed the shape of HSC-1 cells to a round form with beaded processes in a time- and dose-dependent manner. Moreover, C3 treatment reduced the cell growth rate; 72-h treatment with C3 exoenzyme at 1, 3, 10, 30 and 60 micrograms/ml culture medium resulted in 9.0 +/- 1.8%, 20 +/- 2.9%, 26 +/- 2.3%, 50 +/- 1.4% and 40 +/- 2.0% inhibition of the growth rate relative to controls, respectively. Under this condition, actin stress fibers were disassembled, as revealed using fluorescent-labeled phallacidin, whereas keratin intermediate filaments were not affected, visualized by immunofluorescence using anti-keratin antibody. These results suggest that rho proteins are closely related to cell growth and that these proteins regulate, at least in part, the assembly of actin stress fibers in transformed human epidermal cells.