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.

CDPKs are a family of calcium (Ca2+)-dependent protein kinases which are defined by a carboxyl-terminal calmodulin-like domain. Mutational analysis indicates that the junction domain, which joins the kinase and calmodulin-like domains, contains an autoinhibitor. CDPK isoform AK1 from Arabidopsis was expressed in Escherichia coli as a fusion protein sandwiched between glutathione S-transferase and six consecutive histidines at the N- and C-terminal ends, respectively. This fusion, called AK1-6H, was purified and displayed kinase activity which was stimulated up to 127-fold by Ca2+, with a typical specific activity of 2000 nmol min-1 mg-1, using syntide-2 as peptide substrate. A truncation which deletes the calmodulin-like domain, as in mutant delta C-6H, disrupts Ca2+ activation and leaves the enzyme with a basal level of activity. Delta C-6H could be activated 87-fold by preincubation with a purified polyclonal IgG which was raised against a junction domain fusion. A further deletion of the junction domain, as in mutant delta JC, results in a constitutively active enzyme. This indicates that the junction domain in delta C-6H can function as an autoinhibitor. Its function as an autoinhibitor in a full-length enzyme was confirmed by site-specific mutagenesis, as shown by mutant KJM23-6H, which had a six-residue substitution in the junction domain between A422 and A432. Both delta JC and KJM23-6H encoded Ca(2+)-independent enzymes which had specific activities greater than 70% that of a fully active AK1-6H and displayed equivalent Km values for ATP and syntide-2. Inhibition studies on delta JC, using peptides based on the autoinhibitory domains of Ca2+/calmodulin-dependent protein kinases, are consistent with a model where the junction domain contains a similar pseudosubstrate-type autoinhibitor.

Transrectal ultrasound (TRUS) is becoming more widely used as a method of investigating prostatic disease. This study investigated the acceptance of this technique in 89 patients undergoing evaluation for suspected malignant disease. The true morbidity associated with TRUS and TRUS-guided biopsy was evaluated. Serious complications were rare, but minor complications were frequent. Careful counselling is recommended prior to the procedure in order to minimise the patients' anxiety and ensure that if complications do occur they are dealt with swiftly and appropriately.

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Objective-To observe the effects of introducing an acute pain service to the general surgical wards of a large teaching hospital.