Calpains, the thiol proteinases of the calciumdependent proteolytic system, are regulated by a natural inhibitor, calpastatin, which is present in brain tissue in two forms. Although both calpastatins are highly active on human erythrocyte calpain, only one form shows a high inhibitory efficiency with both rat brain calpain isozymes. The second calpastatin form is almost completely inactive against homologous proteinases and can be converted into an active one by exposure to a phosphoprotein phosphatase, also isolated from rat brain. Phosphorylation of the active calpastatin by protein kinase C and protein kinase A promotes a decrease in its inhibitory efficiency. The interconversion between the two inhibitor forms seems involved in the adjustment of the level of intracellular calpastatin activity on specific cell requirements.

Key words: Proteolysis; Calcium; Calpain; activity and thus it can be considered as an active species of Activation process calpain. Materials and methods I. Introduction Purification of human erythrocyte calpainThe Ca2+-dependent proteinase, calpain, is normally locaHuman erythrocyte calpain was purified as previously described lised in the cytosol of the cells [1][2][3][4][5], together with its natural[15], modified as follows: packed red cells 50 ml were lysed in inhibitor calpastatin. In this cell localisation, calpain is pos-5 vols. of water containing 1 mM EGTA; the membranes were discarded by centrifugation at 25 000 ×g for 10 min and the supernatant tulated to be inactive, due to the high requirement for Ca 2+ was treated with 125 g (wet powder) of DE 32 previously extensively [6][7][8], in the absence of which the enzyme retains a native washed with 50 mM sodium acetate, pH 6.7, containing 0.1 mM conformation with an inaccessible active site. The transition EGTA (buffer A) and stirred for 10 min at 5°C. The resin was colfrom a high to low calcium requiring form results from aulected on a Buckner funnel, washed with 1 1 of buffer A and transferred on a glass column (2.5x 15 cm). The absorbed proteins were toproteolysis, which causes the removal of fragments at the Neluted in a single step with 0.2 M NaC1 dissolved in buffer A. The terminal region of both 80 kDa catalytic and 30 kDa subunits fractions containing calpain activity were collected, precipitated in [9][10][11][12]. The native catalytic subunit is then converted into a 75 50% saturated ammonium sulfate and centrifuged at 25000×g for kDa species, and the small subunit into a 18 kDa fragment.10 min. The pellet was suspended in buffer A and dialyzed for 4 h This new enzyme form expresses full catalytic activity at conin the same buffer solution. The dialyzed material was then loaded onto a column (2.5×3 cm) of Source 15Q (Pharmacia) previously centrations of calcium 50-100-times lower than that required equilibrated in buffer A. The proteins were eluted with a linear graby the native form, and has also been identified in red cells dient of 0~).3 M sodium chloride. The fractions containing calpain enriched with Ca 2+ following exposure of cells to a ionophore, activity were collected, dialyzed for 4 h against sodium borate (50 It seems therefore that autoproteolysis is an essential step that mM, pH 7.5) containing 0.1 mM EGTA (buffer B). To the dialyzed material was added sodium chloride at a final concentration of 0.3 M and the solution loaded onto a column (1 × 10 cm) containing PhenylSepharose CL 4B (Pharmacia) equilibrated with buffer B, containing *Corresponding author. Fax: (39) (10) 354415.0.3 M NaC1. The resin was washed and the bound proteins were 0014-57931961512.00

Four recombinant calpastatin forms, deduced from rat brain mRNAs and differing in the number of inhibitory repetitive domains from zero to four, were expressed and characterized for their inhibitory efficiency on W W-and m-calpain. Although the most effective one is a truncated calpastatin form composed of the N-terminal region (domain L) and a single inhibitory domain, all inhibitors are more active against W Wcalpain, but are preferentially degraded and inactivated by mcalpain. The protein form composed exclusively of a domain L is deprived of any inhibitory activity but prevents inhibition of calpain by the other calpastatin forms, indicating that this calpastatin region could be relevant in the recognition of the proteinase. A calpastatin form having molecular properties similar to those of the recombinant truncated calpastatin, has also been found in rat brain. It does not derive from proteolysis of a higher molecular mass precursor. The expression of multiple calpastatin forms may be relevant for the specific modulation of the different calpain isozymes normally present in a single cell type.z 1998 Federation of European Biochemical Societies.

Primary hypothyroidism is a chronic and insidious disease caused by failure of thyroid hormone production. We observed a 38-year-old woman admitted to our hospital due to progressive proximal weakness, muscle pain and fatigue during mild exercise. Laboratory tests showed features of rhabdomyolysis and hypothyroidism. After examination of the thyroid, we reached a diagnosis of Hashimoto’s thyroiditis and hypothyroid myopathy. Hypothyroidism should be considered as a differential diagnosis of creatine kinase elevation; actually, neuromuscular symptoms and signs occur in most newly diagnosed patients with thyroid diseases. Hypothyroidism presenting as muscle stiffness and pseudohypertrophy is called ‘Hoffman’s syndrome’.

The handling of biomaterials is crucial for precision medicine in advanced-stage lung patients with only cytology or small biopsies available. The main purpose of the study was to evaluate the quantity and quality of nucleic acids extracted from mixed stained slides (MSSs), including H&E, IHC and FISH, compared to the extraction from unstained slides (USs). A series of 35 lung adenocarcinoma surgical samples was selected to set up the method and the technical approach was validated in a series of 15 small biopsies and 38 cytological samples. DNA extracted from MSSs was adequate in all samples and the Real Time PCR was successful in 30/35 surgical samples (86%), 14/15 small biopsies (93%), and 33/38 cytological samples (87%). NGS using DNA extracted from MSSs was successful in 18/35 surgical samples (51%), 11/15 small biopsies (73%), and 26/38 cytological samples (68%). RNA extracted from MSSs was unsatisfactory in all cases showing an inadequate degree of fragmentation. Our technical approach based on the recovery of stained slides could represent a strategic way forward for DNA-based biomarker testing in lung cancer cases without biomaterials. The RNA extracted from MSSs did not represent a successful approach.