We have investigated the functional interchangeability of EF hands I and III or II and IV, which occupy structurally analogous positions in the native I-II and III-IV EF hand pairs of calmodulin. Our approach was to functionally characterize four engineered proteins, made by replacing in turn each EF hand in one pair by a duplicate of its structural analog in the other. In this way functional determinants we define as unique were localized to the component EF hands in each pair. Replacement of EF hand I by III reduces calmodulin-dependent activation of cerebellar nitric oxide synthase activity by 50%. Replacement of EF hand IV by II reduces by 60% activation of skeletal muscle myosin light chain kinase activity. There appear to be no major unique determinants for activation of these enzyme activities in the other EF hands. Replacement of EF hand III by I or IV by II reduces by 50 -80% activation of smooth muscle myosin light chain kinase activity, and replacement of EF hand I by III or II by IV reduces by 90% activation of this enzyme activity. Thus, calmodulindependent activation of each of the enzyme activities examined, even the closely related kinases, is dependent upon a distinct pattern of unique determinants in the four EF hands of calmodulin. All the engineered proteins examined bind four Ca 2؉ ions with high affinity. The crystal structure of the multifunctional regulatory protein, calmodulin (CaM), 1 shows an elongated molecule in which two globular lobes are joined by an extended central helix (1). Each globular region is composed of a pair of EF hand Ca 2ϩ -binding domains, which include all amino acid residues in CaM apart from residues 1-8 at the N terminus and 76 -81 in the central helix (2). We shall refer to these regions as the Nterminal leader and the central helix linker, respectively. CaM appears to recognize in its enzyme targets basic amphiphilic helix-forming segments that are generally termed CaM-binding domains; there is no consensus amino acid sequence for these regions (3-5). The simplest model for CaM-dependent enzyme activation is one in which the CaM-binding domain also functions as an intrasteric inhibitor whose influence is relieved when it is bound by CaM. In a more complex model, occupancy of the CaM-binding domain is insufficient for enzyme activation and further, secondary, CaM-target interactions are required. Data for CaM-dependent activation of myosin light chain kinase activity suggest that this latter model may apply, while much of the available data for CaM-dependent activation of nitric oxide synthase and cyclic nucleotide phosphodiesterase activities are consistent with the former (6 -11). Comparison of the CaHigh resolution structures have been determined for the complexes between CaM and peptides representing the CaMbinding domains in either skeletal or smooth muscle myosin light chain kinase or in CaM-dependent protein kinase II (12, 13). Given the considerable internal structural homology seen in CaM, it is perhaps not surprising that these complexes each exhibit ...

Small hepatocellular carcinomas of 2.0 cm or less in diameter should be resected with an adequate surgical margin. However, surgical margin was not a significant factor in the resection of hepatocellular carcinomas larger than 2.0 cm.

Over the past 3 decades, ending in 1979, resection has been performed in 7,220 patients with gastric carcinoma. Synchronous multiple gastric carcinoma, according to the diagnostic criteria of Moertel, was found in 468, of which 178 (38%) were "early." There were 135 double, 33 triple, and 10 quadruple early earcinomas. Fewer than one-third of the smaller tumors were diagnosed preoperatively, and 61.5% of the smaller lesions were less than 10 mm in diameter. In 53% of the patients, the smaller lesions were in the lower one-third of the stomach with the main lesion in the upper one-third, whereas in 13% the smaller neoplasms were iocated in the upper one-third of the stomach with the main lesion in the Iower one-third. The clinical significance of the smaller lesions was their location relative to the resection line. When planning treatment of gastric carcinoma, it is important to evaluate the whole stomach before and during the operation and after examining the resected specimen. The 5-year survival rate for patients with multiple early gastric carcinoma was 85.8%.The concept of the management of early gastric carcinoma bas evolved since the first description by Verse in 1908 [1]. Recently, we have achieved an increase in the detection of early gastric carcinoma by improved diagnosis principally due to fiberoptic instruments. In 1980 Ohta et al. [2] reported that more than one-third of the resected gastric carcinomas (108 of 277 cases) at the Cancer Institute Hospital in Tokyo were "early." The frequency of early cancer has also increased in the United States [3] and in Europe [4]. This increase has contributed significantly to the recent improvement in survival

It has been proposed that alcohols and anesthetics selectively inhibit proteins containing easily disrupted motifs, e.g., alpha-helices. In this study, the calcineurin/calmodulin/Ca(2+) enzyme system was used to examine the effects of alcohols on calmodulin, a protein with a predominantly alpha-helical structure. Calcineurin phosphatase activity and Ca(2+) binding were monitored as indicators of calmodulin function. Alcohols inhibited enzyme activity in a concentration-dependent manner, with two-, four- and five-carbon n-alcohols exhibiting similar leftward shifts in the inhibition curves for calmodulin-dependent and -independent activities; the former was slightly more sensitive than the latter. Ca(2+) binding was measured by flow dialysis as a direct measure of calmodulin function, whereas, with the addition of a binding domain peptide, measured calmodulin-target interactions. Ethanol increased the affinity of calmodulin for Ca(2+) in the presence and absence of the peptide, indicating that ethanol stabilizes the Ca(2+) bound form of calmodulin. An increase in Ca(2+) affinity was detected in a calmodulin binding assay, but the affinity of calmodulin for calcineurin decreased at saturating Ca(2+). These data demonstrate that although specific regions within proteins may be more sensitive to alcohols and anesthetics, the presence of alpha-helices is unlikely to be a reliable indicator of alcohol or anesthetic potency.

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