Robust genotyping tool for autosomal recessive type of limb-girdle muscular dystrophies.

BACKGROUND: Limb-girdle muscular dystrophies are characterized by predominant involvement of the shoulder and pelvic girdle and trunk muscle groups. Currently, there are 31 genes implicated in the different forms of limb-girdle muscular dystrophies, which exhibit similar phenotypes and clinical overlap; therefore, advanced molecular techniques are required to achieve differential diagnosis. METHODS: We investigated 26 patients from Latvia and 34 patients from Lithuania with clinical symptoms of limb-girdle muscular dystrophies, along with 565 healthy unrelated controls from general and ethnic populations using our developed test kit based on the Illumina VeraCode GoldenGate genotyping platform, Ion AmpliSeq Inherited Disease Panel and direct sequencing of mutations in calpain 3 (CAPN3), anoctamin 5 (ANO5) and fukutin related protein (FKRP) genes. RESULTS: Analysis revealed a homozygous CAPN3 c.550delA mutation in eight patients and three heterozygous variants in controls: dysferlin (DYSF) c.5028delG, CAPN3 c.2288A > G, and FKRP c.135C > T. Additionally, three mutations within FKRP gene were found: homozygous c.826C > A, and two compound - c.826C > A/c.404_405insT and c.826C > A/c.204_206delCTC mutations, and one mutation within CLCN1 gene - c.2680C > T p.Arg894Ter. ANO5 c.191dupA was not present. CONCLUSIONS: Genetic diagnosis was possible in 12 of 60 patients (20%). The allele frequency of CAPN3 gene mutation c.550delA in Latvia is 0.0016 and in Lithuania - 0.0029. The allele frequencies of CAPN3 gene mutation c.2288A > G and DYSF gene mutation c.4872delG are 0.003.

Repetitive or continuous thyrotropin (TSH)-releasing hormone administration induces a biphasic rise in plasma TSH in euthyroid but not in hypothyroid rats.

In euthyroid rats, repetitive bolus injections of 0.1, 1, or 5 micrograms/100 g BW TRH every 15 min for 2 h produced a biphasic rise in the plasma TSH concentration. After an initial peak at 15 min, plasma TSH fell at 60 min to a nadir 50-80% of the 15-min peak, then rose again by 90 min to a plateau with approximately the same amplitude as the initial peak. Plasma TSH remained at this level until TRH injections were stopped at 2 h, then fell to the pre-TRH baseline by 3 h. A similar biphasic rise in plasma TSH was produced by constant infusion of 0.01, 0.1, or 1 microgram/min TRH for 3 h. If the dose of bolus TRH injected was increased at 45 min, the dip in plasma TSH at 60 min was significantly decreased. A single iv injection of 1 microgram/100 g BW T4 immediately or 4 h before the bolus TRH injections did not abolish the biphasic TSH response. However, the T4 injection 4 h before TRH significantly attenuated the amplitude of the TSH response. In hypothyroid rats, either repetitive bolus injections or constant infusion of TRH induced only a single peak of plasma TSH at 15-30 min, after which plasma TSH fell to and remained at the pre-TRH baseline. If the hypothyroid rats were injected with 2 micrograms/100 g BW T4 for 4 days before TRH bolus injections, a biphasic TSH response to continual TRH, identical to that in euthyroid rats, was produced. The pituitary TSH content of the hypothyroid rats was significantly subnormal. T4 treatment for 4 days restored both plasma and pituitary TSH levels to the euthyroid range. Our data indicate that 1) constant or repetitive exposure to TRH induces a biphasic rise in plasma TSH in euthyroid, but not in hypothyroid, rats; 2) this biphasic phenomenon is not produced by negative feedback of T4 on the thyrotroph; and 3) the rapid development of refractoriness to TRH in hypothyroid rats is not dependent on continuous exposure to a constant concentration of TRH, but may be related to the reduced TSH content of the hypothyroid thyrotroph.

Hyposmolar stimulation of in vitro pituitary secretion of luteinizing hormone: a potential clue to the secretory process.

Diluting the perifusion medium with water caused a striking prompt increase in LH secretion from perifused, acutely dispersed adenohypophseal cells. The minimum effective proportion of water was 4%; the quantity of hormone secreted was proportional to the dilution of the medium up to greater than 50% water. Secretion was not induced if the dilution was made with 5% aqueous mannitol to maintain isotonicity. The LH secretory responses to hyposmolarity or to LHRH were qualitatively indistinguishable. We suggest that expansion of the outer cell membrane may be an important initial component of the mechanism of secretion from adenohypophyseal cells.

A second consensus sequence of ATP-requiring proteins resides in the 21-kDa C-terminal segment of myosin subfragment 1.

Previous comparisons of sequence homologies of ATP-requiring enzymes have defined three consensus sequences which appear to be involved in the binding of the nucleotide. One of these was identified in the N-terminal 27-kDa segment of the myosin heavy chain but the other two sequences have not hitherto been located in myosin. The present paper proposes that one of these other two consensus sequences is in the 21-kDa C-terminal portion of S1 and that it may contribute to the ATP binding domain.

A unique loop contributing to the structure of the ATP-binding cleft of skeletal muscle myosin communicates with the actin-binding site.

Actin binding to skeletal muscle myosin subfragment-1 (S1) increases the dissociation rate of reaction products from the myosin ATPase site; conversely, ATP binding facilitates dissociation of complexed acto-S1. However, details of the molecular mechanism by which the ATP- and actin-binding sites communicate with each other is still obscure. We present evidence that the effect of actin is mediated by a conformational change in the loop containing amino acids from 677 to 689 [loop M (677-689)], a segment of the 20-kDa tryptic fragment that contributes to the structure of the ATP-binding cleft. Initially, a fluorescent ADP analogue, methylanthranyloyl-8-azido-ADP (Mant-8-N3-ADP), was covalently crosslinked to loop M (Mant-S1), perhaps at Lys 681. Actin-activated Mg2+-ATP hydrolysis by Mant-S1 was accelerated approximately 6 times over that by unmodified S1, suggesting that the ATPase site is not blocked by the ADP analogue crosslinked in the loop M (677-689). Nevertheless, analysis of Mant-group fluorescence polarization and acrylamide-induced quenching showed the crosslinked probe to be entrapped within the ATP-binding cleft at a location where Mant-group rotational mobility was hindered, and where it was relatively inaccessible to the solvent. Exposing Mant-S1 to Mg2+-ATP and/or actin elicited similar decreases in fluorescence polarization, indicating increased rotational mobility of the Mant-group and movement of crosslinked Mant-8-N3-ADP to a less hindered position. Stern-Volmer quench curves showed that Mant-8-N3-ADP was translocated to a site where it was more accessible to dissolved quencher, perhaps outside the ATP-binding cleft. Since actin does not bind to the ATPase site, actin-induced translocation of Mant-8-N3-ADP crosslinked to loop M (677-689) probably results from a conformational change in loop M (677-689). These results suggest that loop M acts as a signal transducer mediating communication between the ATP- and actin-binding sites.

Conformational changes in the unique loops bordering the ATP binding cleft of skeletal muscle myosin mediate energy transduction.

Myosin has three highly-conserved, unique loops [B (320-327), M (677-689), and N (127-136)] at the entrance of the ATP binding cleft, and we previously showed that the effects of actin are mediated by a conformational change in loop M [Maruta and Homma (1998) J. Biochem. 124, 528-533]. In the present study, loops M and N were photolabeled respectively with fluorescent probes Mant-8-N(3)-ADP and Mant-2-N(3)-ADP in order to study conformational changes in the loops related to energy transduction. The effect of actin on the conformation of loop N was examined by analyzing fluorescence polarization and acrylamide quenching; the results were then compared with those previously reported for loop M. In contrast to loop M, the fluorescence polarization and the value of K(sv) of the Mant-groups crosslinked to loop N were slightly affected by actin binding. To study conformational changes in loops M and N during the ATPase cycle, FRET was analyzed using TNP-ADP.BeFn and TNP-ADP. AlF(4)(-) as FRET acceptors of Mant fluorescence. The resultant estimated distances between loop M and the active site differed for the Mant-S1.TNP-ADP.BeFn and Mant-S1.TNP-ADP.AlF(4)(-) complexes, whereas the distances between loop N and the active site differed slightly. These findings indicate that the conformation of loop M changes during the ATPase cycle, suggesting that Loop M acts as a signal transducer mediating communication between the ATP- and actin-binding sites. Loop N, by contrast, is not significantly flexible.

Analysis of conformational changes at the unique loop adjacent to the ATP binding site of smooth muscle myosin using a fluorescent probe.

Recent crystallographic studies have shown that smooth muscle myosin has three highly conserved unique loops, loop B (320-327), loop M (687-699), and loop N (125-134), similar to other myosins, skeletal muscle and dictyostelium myosins. We previously demonstrated that the effect of actin is mediated by a conformational change in one of the loops, loop M comprising amino acids 677 to 689 of skeletal muscle myosin [Maruta and Homma (1998) J. Biochem. 124, 528-533]. In the present study, in order to clarify the role of these smooth muscle myosin loops in energy transduction, we specifically labeled the loops with a fluorescent photoreactive ADP analogue, 3'-O-(N-methylanthraniloyl)-8-azido-ADP (Mant-8-N(3)-ADP), and then measured the fluorescent polarization. When Mant-8-N(3)-ADP was trapped by aluminium fluoride or vanadate into the ATPase site, Mant-8-N(3)-ADP was covalently incorporated into loop N (125-134). In contrast, Mant-8-N(3)-ADP trapped by beryllium fluoride was covalently incorporated into both loop M (687-699) and loop N (125-134) at an almost equimolar ratio. Actin binding to smooth muscle myosin S1 (SMO-S1) labeled at only loop N (125-134) increased the polarization due to the viscosity of actin. In contrast, S1 labeled at both loops N and M showed a much smaller increase in polarization. Our results indicate that the probe at loop M (687-699) of smooth muscle myosin moved to a less hindered region, suggesting that actin binding induces conformational changes at loop M (687-699) similar to those of the corresponding loop (677-689) in skeletal muscle myosin, as previously demonstrated in our laboratory.

Conformational changes at the highly reactive cystein and lysine regions of skeletal muscle myosin induced by formation of transition state analogues.

Myosin forms stable ternary complexes with Mg2+-ADP and phosphate analogues of aluminum fluoride (AlF4-), beryllium fluoride (BeFn), and scandium fluoride (ScFn). These complexes are distinct from each other and may mimic different transient states in the ATPase cycle [Maruta et al. (1993) J. Biol. Chem. 268, 7093-7100]. Regions of skeletal muscle myosin containing the highly reactive residues Cys 707 (SH1), Cys 697 (SH2), and lysine 83 (RLR) dramatically alter their local conformation when myosin hydrolyzes ATP, and these changes may reflect formation of a series of transient intermediates during ATP hydrolysis. We used the fluorescent probes 4-fluoro-7-sulfamoylbezofurazan, 2-(4'-maleimidylanilino)naphthalene-6-sulfonic acid, and trinitrobenzene-sulfonate, which bind to SH1, SH2, and RLR, respectively, to examine differences in local conformations within myosin.ADP.phosphate analogue (BeFn, Vi, AlF4-, and ScFn) complexes. It was observed that the ternary complexes had SH1 conformations similar to those seen on S-1 in the presence of ATP. In contrast, local conformations in the SH2 and RLR regions of S-1.ADP.BeFn were different from those in corresponding regions of S-1.ADP.AlF4- or ScFn. These results suggest that SH1 and SH2 move distinctly during ATP hydrolysis and that the local conformations of the SH2 and RLR regions more sensitively reflect different transient states.

ADP/vanadate mediated photocleavage of myosin light chain kinase at the autoinhibitory region.

The vanadate (Vi)-mediated photocleavage reaction was used to study the interaction between the regulatory segment and the catalytic site of smooth muscle myosin light chain kinase (MLCK). When MLCK was irradiated with long-wave UV (366 nm) in the presence of ADP and Vi, kinase activity was substantially decreased, and the MLCK polypeptide of 130 kDa was cleaved into several smaller fragments with apparent molecular masses of 100, 70, 60, 32, and 28 kDa. Inhibition of kinase activity and photocleavage were both competitively antagonized by the addition of ATP. Inconsistency between the observed maximum levels of UV-induced inhibition of MLCK-mediated phosphorylation (80%) and photocleavage (15-20%) suggested that the photocleavage reaction proceeds as a two-step process. Monoclonal antibodies recognizing the C-terminus of MLCK labeled the 60- and 28-kDa fragments, indicating that MLCK was cleaved at two sites, at 28 and 60 kDa from the C-terminus, within what are believed to be the autoinhibitory region and the catalytic site, respectively. Moreover, Ca2+-calmodulin (Ca2+-CaM) protected against cleavage at the site at 28 kDa from the C-terminus. Analysis of the amino acid composition of the fragment revealed that the cleavage site at 28 kDa from C-terminus occurred at Lys 799 +/- 3 amino acid residues, which is in a region where the CaM-binding and pseudosubstrate regions overlap. These results suggest that the three-dimensional structure of MLCK brings the regulatory segment into direct contact with the ATP-binding site. Moreover, the binding of Ca2+-CaM displaces the regulatory segment away from the catalytic site.

Formation of the myosin.ADP.gallium fluoride complex and its solution structure by small-angle synchrotron X-ray scattering.

In the presence of MgADP, a novel phosphate analogue of gallium fluoride (GaFn) forms a ternary complex with the myosin subfragment-1 (S-1), in the same way that has been previously reported with aluminum fluoride (AlF4-), beryllium fluoride (BeFn), scandium fluoride (ScFn), and vanadate (Vi), and this complex formation may mimic different states along the ATPase kinetic pathway. This novel complex has been characterized and compared with other complexes to ascertain whether it forms a transition-state analogue of myosin ATPase. The complex formed quickly, although several times slower than the BeFn complex. The half-life of the myosin.ADP.GaFn complex was about 50 h at 4 degreesC. The formation of the myosin.ADP.GaFn complex was accompanied by an increase in tryptophane fluorescence, similar to that observed upon the addition of ATP, but slightly lower than that of the M**.ADP.Pi complex. Upon addition of GaFn to acto-myosin.ADP, acto-myosin did not dissociate, and the S-1.ADP.GaFn complex was scarcely decomposed by actin, like the AlF4- and ScFn complexes but unlike the BeFn and Vi complexes. The conformations at the localized region of SH1, SH2, and RLR, which are very accessible to the binding of ATP, were studied by fluorescent labeling and chemical modification, and the results suggested that these conformations are very similar to that of the M**.ADP.Pi state. Small-angle X-ray solution scattering showed that the radius of gyration value decreases by about 3 A when S-1 forms an S-1.ADP.GaFn complex, suggesting that the shape of the complex becomes compact or rounded in shape, similar to that in the presence of ATP or complexes with other phosphate analogues, and thus mimics the myosin**.ADP.Pi state closely. The overall results may indicate that the complex mimics a somewhat different transient state from that of other complexes but has a similar global conformation along the ATPase kinetic pathway.

Amino acid sequence of the essential light chain of adductor muscle myosin from Ezo giant scallop, Patinopecten yessoensis.

The amino acid sequence of the essential light chain (abbreviated as SHLC) of adductor muscle myosin from Ezo giant scallop (Patinopecten yessoensis) was determined by conventional methods. The light chain was composed of 156 amino acid residues with proline and lysine as its amino and carboxyl termini, respectively. Comparing this sequence with that of the SHLC from bay scallop (Aquipecten irradians), only 5 amino acid substitutions were recognized. The sequence homology between scallop and squid SHLCs was 53.7%. On the other hand, a partially fragmented SHLC "modified SHLC" reported by Konno and Watanabe (J. Biochem. 98, 141-148 (1985) was prepared by chymotryptic digestion of the scallop myosin in the presence of EDTA, and was assigned as the carboxyl-terminal 106-residue peptide of the SHLC. This may suggest that the regulatory light chain covers the amino-terminal region of the SHLC in the myosin molecule.

Isolation and amino acid sequence of a peptide containing an epoxide-reactive residue from the thermolysin-digest of Scytalidium lignicolum acid protease B.

Scytalidium lignicolum acid protease B, a pepstatin-insensitive acid protease, was modified by 1,2-epoxy-3-(p-nitrophenoxy)propane (EPNP) with the concomitant loss of its enzyme activity, and an EPNP-labeled peptide was isolated from the thermolysin-digest of the modified enzyme by HPLC. The amino acid sequence of the peptide was determined to be Ile-Leu-Glu-Thr-Gly, which corresponds to the sequence of residue Nos. 51-55 of the enzyme. The results of treatment of the labeled peptide with hydroxylamine suggested that the EPNP moiety is ester-linked to Glu53 of the enzyme. The amino acid sequence around Glu53 of the acid protease B showed high homology with those around the active site Asp residues of calf chymosin and porcine pepsin. These results show that it is highly possible that Glu53 of the acid protease B is one of the amino acid residues involved in its catalytic activity.

Solution structure of myosin-ADP-MgFn ternary complex by fluorescent probes and small-angle synchrotron X-ray scattering.

In the presence of excess amounts of fluorine, a physiological divalent cation, magnesium (Mg(2+)), forms a novel phosphate analogue, magnesium fluoride (MgFn). Park et al. [Biochim. Biophys. Acta 1430, 127-140 (1999)] previously demonstrated that MgADP. MgFn forms a complex with myosin subfragment-1 (S-1), and the S-1.ADP. MgFn ternary complex mimics a transient state in the activity cycle of ATPase. In the present study, localized conformations in the regions of highly reactive cysteine and lysine residues, Cys 707 (SH1), Cys 697 (SH2), and Lys 83 (RLR), which change their conformations markedly during ATP hydrolysis, were studied using fluorescent probes and chemical modification. The global shape of the complex was also studied using small angle X-ray solution scattering and compared it with other previously reported myosin.ADP. fluorometal ternary complexes. The results suggest that the overall conformation and localized functional regions of the complex are quite similar to those in the presence of ATP, indicating that the complex mimics the M(**).ADP.P(i) steady state.

Complete amino acid sequence of Scytalidium lignicolum acid protease B.

The acid protease B (SLB) of Scytalidium lignicolum was reduced and carboxymethylated and then subjected to tryptic digestion. Five fragments were isolated and some of them were further digested with alpha-chymotrypsin, thermolysin, and dilute acetic acid. The sequence analysis of these fragments and the peptides by conventional methods established the complete amino acid sequence of SLB. The enzyme was composed of 204 amino acid residues with threonine and valine as its amino- and carboxyl-termini, respectively. Locations of three disulfide bridges were also established to be Cys47-126, Cys140-163, and Cys192-201 by enzymatic fragmentation of the denatured and unmodified SLB. Only a slight homology was found in the sequences of SLB and other acid proteases hitherto reported.

A case of early cancer in cystic intrahepatic duct associated with congenital choledochal cyst.

Herein, we report an extremely rare case of early cancer localized in the cystic intrahepatic duct, which is associated with the choledochal cyst including both the intra- and extrahepatic duct (type IV-A cyst). A small polypoid lesion in the cyst was detected by computed tomography. Extensive resection of the choledochal cyst with lobectomy was performed on the tumor in a 23-year-old woman. Tubular adenocarcinoma localized in the wall of the cystic left hepatic duct was histologically confirmed. Awareness of the diagnostic and treatment modalities, with a focus on potential malignancy in the cystic intrahepatic duct, in cases with anomalous arrangement of the pancreatobiliary duct is necessary.

[A case of necrosis of a hepatocellular carcinoma, caused by spasm of the celiac artery].

A 54-year-old man was admitted to our hospital complaining of back pain and right hypochondrial pain. Ultrasonography and celiac angiography revealed a large tumor sized 9.4 X 8.1 cm. The tumor appeared hypervascular on angiogram. During the second angiography, an attempt at superselective hepatic angiography for the purpose of infusing a combination of Adriamycin and Lipiodol, spasm of the celiac artery occurred. High fever continued for 11 days after the spasm and serum transaminase was elevated. At the third angiography, the nature of the tumor was seen to have changed remarkably to one of hypovascularity. Percutaneous transhepatic tumor biopsy was done. Pathological diagnosis was necrosis of hepatocellular carcinoma. Due to heart disorders, ligation of the right hepatic artery was performed instead of hepatic resection. Postoperatively, the size of the tumor decreased further. It is thought that this patient had a tendency to suffer from vasospasm and that the tumor had a relatively low resistance to ischemia.