Initiation of reepithelialization upon wounding is still poorly understood. To enhance this understanding, we focus here on changes in the adhesive state of desmosomes of cultured Madin-Darby canine kidney cells in response to wounding of confluent cell sheets. Previous results show that desmosomal adhesion in Madin-Darby canine kidney cells changes from a calcium-dependent state to calcium independence in confluent cell sheets. We show that this change, which requires culture confluence to develop, is rapidly reversed upon wounding of confluent cell sheets. Moreover, the change to calcium dependence in wound edge cells is propagated to cells hundreds of micrometers away from the wound edge. Rapid transition from calcium independence to calcium dependence also occurs when cells are treated with phorbol esters that activate PKC. PKC inhibitors, including the conventional isoform inhibitor Gö 6976, cause rapid transition from calcium dependence to calcium independence, even in subconfluent cells. The cellular location of the ␣ isoform of PKC correlates with the calcium dependence of desmosomes. Upon monolayer wounding, PKC␣ translocates rapidly to the cell periphery, becomes Triton X-100 insoluble, and also becomes concentrated in lamellipodia. The PKC␣ translocation upon wounding precedes both the increase in PKC activity in the membrane fraction and the reversion of desmosomes to calcium dependence. Specific depletion of PKC␣ with an antisense oligonucleotide increases the number of cells with calcium-independent desmosomes. These results show that PKC␣ participates in a novel signaling pathway that modulates desmosomal adhesion in response to wounding.

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1. The case is described of a child with retarded physical and mental development, recurrent megaloblastic anaemia, methylmalonic aciduria and abnormal homocysteine metabolism resulting from an inborn error in the metabolism of cobalamins. She died at the age of 7 years. At autopsy there was pulmonary fibrosis and the brain showed lesions typical of those seen in subacute combined degeneration of the cord. 2. A metabolic abnormality was present which resulted in an inability to maintain normal tissue concentrations of the two coenzyme forms of vitamin B12, methylcobalamin and adenosylcobalamin. Lack of methylcobalamin led to deficient activity of N5-methyltetrahydrofolate-homocysteine methyltransferase with reduced ability to methylate homocysteine, and lack of adenosylcobalamin to deficient activity of methylmalonyl-CoA mutase, which accounted for the methylmalonic aciduria. 3. Analyses of total vitamin B12 and of individual cobalamins by a chromatobioautographic technique showed that in organs sampled at autopsy, the content of total vitamin B12 and of methylcobalamin, adenosylcobalamin and hydroxocobalamin were all greatly reduced. The plasma had a high normal total vitamin B12 content, but showed a gross abnormality in the distribution of individual cobalamins, methylcobalamin being decreased and adenosylcobalamin and hydroxocobalamin increased. The erythrocytes showed a reduction in cobalamins resembling that in the solid organs, though less severe. 4. The underlying abnormality in this patient appeared to be either a defect in cellular uptake of vitamin B12, or a defect in a metabolic pathway leading to the formation of a common precursor of methylcobalamin and adenosylcobalamin. Abnormalities in transcobalamins I and II, plasma factors involved in plasma transport and cellular uptake of vitamin B12, were excluded. 5. The clinical and biochemical findings in the patient are compared with those described in three patients previously reported, who had methylmalonic aciduria and homocystinuria, and who were in some respects similar to this patient. The present case is unusual in that previous examples of errors in cobalamin metabolism have not had megaloblastosis or neurological changes typical of vitamin B12 deficiency. It is also the first case in which direct estimations of individual cobalamins have been made.

1. Serum from normal subjects has been chromatographed on Sephadex G-200 columns and the fractions containing transcobalamins 0, I and II have been identified. 2. The fractions corresponding to transcobalamin I contained, on average, 90% of the endogenous vitamin B12. Only 3% was attached to transcobalamin 0, and 7% was bound to transcobalamin II.

1. Methylcobalamin (Me-B, ,), adenosylcobalamin (Ado-B ,), hydroxocobalamin (OH-B,,) and cyanocobalamin (CN-Bl2) have been estimated by a chromatographicbioautographic technique in plasma, erythrocytes, leucocytes and bone marrow from normal subjects, hospital controls and patients with untreated pernicious anaemia.2. Estimates of concentrations of cobalamins have also been obtained in bile, cerebrospinal fluid, liver biopsies and in autopsy samples of liver, kidney, spleen, brain and pituitary.3. In normal and control subjects, Ado-B,, predominated in all samples except plasma, in which Me-B,, was the predominant form. Me-B,,, Ado-B,,, OH-B12 and CN-B,, were found in normal erythrocytes, leucocytes and bone marrow and the proportion of each cobalamin was fairly similar in all these tissues. In liver, kidney, spleen, brain and pituitary, the proportions of the cobalamins were more variable. No CN-B,, was detected in these organs. 4.In untreated pernicious anaemia, Me-B,, was disproportionately reduced in plasma, but not in erythrocytes, leucocytes or bone marrow. There was a small increase in the proportion of CN-B,, in plasma, blood cells and bone marrow in untreated pernicious anaemia.