Surgical management of trapezius palsy.

BACKGROUND: Injury to the spinal accessory nerve in the posterior cervical triangle leads to paralysis of the trapezius muscle. The aim of this study was to determine the indications for nerve repair or reconstructive surgery according to the etiology, the duration of the preoperative delay, and specific patient characteristics. METHODS: Of twenty-seven patients with a trapezius palsy, twenty were treated with neurolysis or surgical repair (direct or with a graft) of the spinal accessory nerve and seven were treated with the Eden-Lange muscle transfer procedure. Lymph node biopsy was the main cause of the nerve injury. The nerve repairs were performed at an average of seven months after the injury, and the reconstructive procedures were done at an average of twenty-eight months. Nerve repair was performed for iatrogenic injuries of the spinal accessory nerve, within twenty months after the onset of symptoms, and in one patient with spontaneous palsy. Reconstructive surgery was performed for cases of trapezius palsy secondary to radical neck dissection, for spontaneous palsies, and after failure of nerve repair or neurolysis. The mean follow-up period was thirty-five months. The functional outcome was assessed clinically on the basis of active shoulder abduction, pain, strength of the trapezius on manual muscle-testing, and level of subjective patient satisfaction. RESULTS: The results were good or excellent in sixteen of the twenty patients treated with nerve repair and in four of the seven patients treated with the Eden-Lange procedure. Poor results were seen in older patients and in patients with a previous radical neck dissection. CONCLUSIONS: Good results can be expected from a repair of the spinal accessory nerve if it is performed within twenty months after the injury, as the nerve is basically a purely motor nerve and the distance from the injury to the motor end plates is short. Muscle transfer should be performed in patients with spontaneous trapezius palsy, when previous nerve surgery has failed, or when the time from the injury to treatment is over twenty months. Treatment is less likely to succeed when the patient is older than fifty years of age or the palsy was due to a radical neck dissection, penetrating injury, or spontaneous palsy.

Surgical management of trapezius palsy.

BACKGROUND: Injury to the spinal accessory nerve in the posterior cervical triangle leads to paralysis of the trapezius muscle. The aim of this study was to determine the indications for nerve repair or reconstructive surgery according to the etiology, the duration of the preoperative delay, and specific patient characteristics. METHODS: Of twenty-seven patients with a trapezius palsy, twenty were treated with neurolysis or surgical repair (direct or with a graft) of the spinal accessory nerve and seven were treated with the Eden-Lange muscle transfer procedure. Lymph node biopsy was the main cause of the nerve injury. The nerve repairs were performed at an average of seven months after the injury, and the reconstructive procedures were done at an average of twenty-eight months. Nerve repair was performed for iatrogenic injuries of the spinal accessory nerve, within twenty months after the onset of symptoms, and in one patient with spontaneous palsy. Reconstructive surgery was performed for cases of trapezius palsy secondary to radical neck dissection, for spontaneous palsies, and after failure of nerve repair or neurolysis. The mean follow-up period was thirty-five months. The functional outcome was assessed clinically on the basis of active shoulder abduction, pain, strength of the trapezius on manual muscle-testing, and level of subjective patient satisfaction. RESULTS: The results were good or excellent in sixteen of the twenty patients treated with nerve repair and in four of the seven patients treated with the Eden-Lange procedure. Poor results were seen in older patients and in patients with a previous radical neck dissection. CONCLUSIONS: Good results can be expected from a repair of the spinal accessory nerve if it is performed within twenty months after the injury, as the nerve is basically a purely motor nerve and the distance from the injury to the motor end plates is short. Muscle transfer should be performed in patients with spontaneous trapezius palsy, when previous nerve surgery has failed, or when the time from the injury to treatment is over twenty months. Treatment is less likely to succeed when the patient is older than fifty years of age or the palsy was due to a radical neck dissection, penetrating injury, or spontaneous palsy.

Decreased expression of high-molecular-weight calmodulin-binding protein and its correlation with apoptosis in ischemia-reperfused rat heart.

A cardiac high-molecular-weight calmodulin-binding protein (HMWCaMBP) was previously identified as a homologue of the calpain inhibitor, calpastatin. In the present study, we investigated the expression of HMWCaMBP and calpains in rat heart after ischemia and reperfusion. Western blot analysis of normal rat heart extract with a polyclonal antibody raised against bovine HMWCaMBP indicated a prominent immunoreactive band of 140kDa. Both the expression and the activity of HMWCaMBP were decreased by ischemia reperfusion. Immunohistochemical studies showed strong-to-moderate HMWCaMBP immunoreactivity in normal heart and poor immunoreactivity in ischemia-reperfused heart muscle. However, the expression of micro-calpain and m-calpain in ischemia-reperfused heart was increased as compared to normal heart. The calpain inhibitory activity of ischemia-reperfused heart tissues was significantly lower as compared to normal heart tissues. The pre-ischemic and post-ischemic perfusion of hearts with a cell-permeable calpain inhibitor suppressed the increase in calpain expression but increased the HMWCaMBP expression. In-vitro HMWCaMBP was proteolyzed by micro-calpain and m-calpain. We also measured apoptosis in normal and ischemia-reperfused tissues. An increase in the number of apoptotic bodies was observed with increased duration of ischemia and reperfusion. Bcl-2 expression did not change in any of the groups, whereas Bax expression increased with ischemia-reperfusion and correlated well with the degree of apoptosis. Our findings suggest that HMWCaMBP may sequester calpains from its substrates in the normal myocardium, but it is susceptible to proteolysis by calpains during ischemia-reperfusion. Thus, decreased expression of HMWCaMBP may play an important role in myocardial injury.

Effect of glycemic control and vitamin E supplementation on total glutathione content in non-insulin-dependent diabetes mellitus.

Thirty patients with non-insulin-dependent diabetes mellitus were selected for the study. 15 age-matched healthy volunteers served as controls. Serum malonaldehyde, total glutathione, and vitamin E levels were estimated before and after glycemic control and after 4 weeks of vitamin E supplementation. Both total glutathione and vitamin E levels increased after glycemic control and showed an increase after vitamin E supplementation. Malonaldehyde levels lowered after glycemic control, but remained higher than controls. Since vitamin E supplementation significantly decreased oxidative stress in the present study, it may play a role in reducing free-radical-induced oxidant injury in diabetes mellitus.

Altered expression of high-molecular-weight calmodulin-binding protein in human ischaemic myocardium.

A high-molecular-weight calmodulin-binding protein (HMWCaMBP) was previously identified and purified from the cytosolic fraction of bovine heart. Based on the sequence homology, amino acid analysis, antibody reactivity, and calpain inhibition, HMWCaMBP has been identified as a homologue of the calpain inhibitor calpastatin. In the present study the expression of HMWCaMBP was investigated in normal and ischaemic human myocardium. Western blot analysis of normal human cardiac muscle extract with the polyclonal antibody raised against bovine HMWCaMBP indicated a prominent immunoreactive band with a molecular mass of 140 kD. HMWCaMBP was localized in the cytoplasm and myofilaments of cardiac myocytes. Furthermore, Western blot analysis of normal and ischaemic cardiac tissues indicated a decrease in the expression of HMWCaMBP in ischaemic tissues. These studies were further substantiated by immunohistochemical studies, indicating strong to moderate HMWCaMBP immunoreactivity in normal cardiac muscle and poor to negative immunoreactivity in ischaemic muscle. The results obtained from the rat ischaemic model suggested that the expression of cardiac HMWCaMBP was significantly decreased during ischaemia/reperfusion. In addition, micro-calpain and m-calpain expression was higher in ischaemic cardiac tissue samples than in normal controls. The calpain inhibitory activity of ischaemic cardiac tissues was significantly lower than normal cardiac tissue samples. In some cases of cardiac ischaemia, HMWCaMBP highlighted the contraction band necrosis seen at the margins of a myocardial infarct. In vitro, HMWCaMBP was proteolysed by micro-calpain and m-calpain. These results indicate that HMWCaMBP could be susceptible to proteolysis by calpains during ischaemia or reperfusion and may play a contributory role in myocardial injury.

Increased expression of N-myristoyltransferase in gallbladder carcinomas.

BACKGROUND: Activated Src, which has intrinsic protein tyrosine kinase activity, has been found in human solid tumors such as colorectal and breast carcinomas. The Src gene encodes a cytoplasmic tyrosine kinase p60src, which attaches to the inner surface of the membrane after N-terminal myristoylation and is implicated in transduction of signals to the nucleus. N-myristoyltransferase (NMT) catalyzes the biochemical modification process called N-myristoylation. To investigate whether, through Src, NMT contributes to the pathogenesis of gallbladder carcinoma, the authors investigated expression of NMT and p53 in in situ and invasive carcinomas. METHODS: One hundred cases of documented gallbladder carcinoma were reviewed, and 30 cases were selected randomly to evaluate expression of NMT and p53 by immunohistochemistry in both in situ and in invasive tumor components. RESULTS: Eighteen cases (60%) of gallbladder carcinoma showed moderate to strong cytoplasmic positivity for NMT with increased intensity in the invasive component, and 12 cases (40%) were negative. The in situ component revealed mild to moderate cytoplasmic staining in 20 cases (67%), whereas the normal gallbladder mucosa showed weak to negative cytoplasmic staining. Moderate to strong p53 staining was observed in 17 in situ cases (63%) and 24 invasive cases (80%). The in situ staining patterns of p53 were unrelated to the clinical outcome of the tumor. However, moderate to strong staining of the invasive component as observed in 15 cases (50%) was associated with a mean survival of 8.8 months. Amplification of intron-8 in normal gallbladder mucosa and invasive carcinoma were similar in intensity, suggesting the absence of NMT gene amplification in these tumors. CONCLUSIONS: The increased expression of NMT in these tumors could be due to transcriptional activation. Tumors with increased expression of NMT and p53 were associated with poor clinical outcomes as evidenced by their mean survival times. NMT is likely to play a pathogenic role in gallbladder carcinoma.

N-myristoyltransferase.

Myristoylation refers to the co-translational addition of a myristoyl group to an amino-terminal glycine residue of a protein by an ubiquitously distributed enzyme myristoyl-CoA:protein N-myristoyltransferase (NMT, EC 2.3.1.97). This review describes the basic enzymology, molecular cloning and regulation of NMT activity in various pathophysiological processes such as colon cancer and diabetes.

Evaluation of oxidative stress before and after control of glycemia and after vitamin E supplementation in diabetic patients.

The present study evaluates the presence of oxidative stress in the uncontrolled diabetic state. Glycemic control reduced the oxidative stress, but total normalization of the parameters of oxidative stress was not achieved, indicating continued oxidant injury despite optimal control of the diabetes. Vitamin E supplementation for 4 weeks in these patients further reduced the oxidative stress, suggesting that vitamin E supplementation might be helpful in reducing free-radical-induced oxidant injury.

Calmodulin-dependent cyclic nucleotide phosphodiesterase (PDE1).

Ca2+/calmodulin-dependent cyclic nucleotide phosphodiesterase (PDE1) is one of the key enzymes involved in the complex interactions between the cyclic nucleotide and Ca2+ second messenger systems. Currently, three genes encode PDE1, and alternate splicing of these genes gives rise to functionally different isozymes which exhibit distinct catalytic and regulatory properties. Some isozymes have similar kinetic and immunological properties but are differentially regulated by Ca2+ and calmodulin. These isozymes also differ in their mechanism of regulation by phosphorylation. Analysis of various regulatory reactions involving Ca2+ and cyclic adenosine monophosphate (cAMP) has revealed the importance of the time dependence of these reactions during cell activation; however, no measurement is available for the time of occurrence of specific regulatory reactions. cAMP-signalling systems provide a pivotal centre for achieving crosstalk regulation by various signalling pathways. It has been proposed that polypeptide sequences enriched in proline (P), glutamate (E), serine (S) and threonine (T), known as PEST motifs, serve as putative intramolecular signals for rapid proteolytic degradation by calpains. Calpains are Ca(2+)-dependent cysteine proteases that regulate various enzymes, transcription factors and structural proteins through limited proteolysis. Isozyme PDE1A2 has a PEST motif and acts as a substrate for m-calpain. In this paper, we have described PDE1A2 regulation by calpains and its physiological implications. cAMP is an important component of the signal transduction pathway and plays an integral role in various physiological processes such as gene transcription, various neuronal functions, cardiac muscle contraction, vascular relaxation, cell proliferation and a host of other functions. It is important to identify the cellular processes where PDE isoform(s) and cAMP response are altered. This will lead to better understanding of the pathology of disease states and development of novel therapeutics. The different PDE1 isozymes, although similar in kinetic properties, can be distinguished by various pharmacological agents. Our recent understanding of the role of PDE1 inhibitors such as ginseng, dihydropy-ridine antagonists and antiparkinsonian agents are described in this review. The exact function of PDE1 isozymes in various pathophysiological processes is not clear because most of the studies have been carried out in vitro; therefore, it is essential that further research be directed to in vivo studies.

An evaluation of oxidative stress in diabetes mellitus during uncontrolled and controlled state and after vitamin E supplementation.

OBJECTIVE: The study was conducted on 50 patients (10 insulin dependent diabetes mellitus (IDDM) and 40 non-insulin dependent diabetes mellitus (NIDDM) of recently diagnosed diabetes mellitus. The main objectives of the study were: 1. To evaluate oxidative stress at uncontrolled stage. 2. To evaluate the effect of optimal control on oxidative stress irrespective of type of drug therapy used. 3. To further evaluate the effect of vitamin E supplementation on oxidative stress after achieving optimal control. This was done in order to explore anti-oxidant effect of vitamin E. METHODS: Fifty patients of uncontrolled diabetes of less than 1 year duration and without any overt complications were studied. The parameters of oxidative stress included malonyl-di-aldehyde (MDA), reduced glutathione and vitamin E levels in the blood. They were done at three stages i.e. (a) In uncontrolled stage, (b) At controlled stage and (c) After 4 weeks of vitamin E supplementation in dosage of 400 mg daily. The parameters of control included fasting blood sugar < or = 140 mg%, post prandial < or = 200 mg and HbA1c < or = 7% (analysed by prepared kit). RESULTS: The significantly raised levels of MDA and decreased levels of reduced glutathione and vitamin E during uncontrolled stage of diabetes indicated free radical stress inducing lipid peroxidation. The significant fall of MDA and rise in reduced glutathione and vitamin E levels in blood after optimal control revealed its beneficial effect on oxidative stress. The levels were not normalised but still stayed higher than controls. After 4 weeks of vitamin E supplementation, further fall in MDA and rise in reduced glutathione suggested beneficial effect of vitamin E over and above the optimal control. Vitamin E estimation in blood at this stage did not constitute parameter of oxidative stress as it was provided from outside but was done to know the compliance of patients. Normalisation or near normalisation was not achieved with vitamin E therapy indicating persistence of oxidative stress. CONCLUSION: There was an evidence of oxidative stress in diabetes which decreased with optimal control and further declined after vitamin E supplementation indicating anti-oxidant effect of vitamin E alone. Normalisation of oxidative stress was not achieved. A further study is desired to study the effect of vitamin E for longer period at least 3-6 months before a definite conclusion is drawn.

Increased oxidative stress in rat liver and pancreas during progression of streptozotocin-induced diabetes.

1. Oxygen free radicals have been suggested to be a contributory factor in complications of diabetes mellitus. There are many reports indicating the changes in parameters of oxidative stress in diabetes mellitus. In this study we aimed to identify whether oxidative stress occurs in the liver and pancreas in the initial stages of development of diabetes. 2. We therefore investigated the lipid peroxide level (thiobarbituric acid-reactive substances, TBARS) and activities of antioxidant enzymes [superoxide dismutase (SOD), catalase and glutathione peroxidase] in liver and pancreas of control and streptozotocin-induced diabetic rats at various stages of development of diabetes. 3. Male Sprague-Dawley rats were divided into two groups: group I, control (n = 42) and group II, diabetic (n = 42). Each group was further subdivided into seven groups consisting of six rats each. Rats in these subgroups were studied at weekly intervals (0 to 6 weeks). Plasma glucose levels, TBARS levels and activities of antioxidant enzymes were measured in liver and pancreas at various time intervals. 4. There was a significant (P < 0.05) and progressive increase in TBARS levels of liver and pancreas in the diabetic group. Total SOD and Cu-Zn-SOD activity increased (P < 0.05) with progression of diabetes while Mn-SOD activity showed no significant change in either tissue. Catalase and glutathione peroxidase activities increased significantly (P < 0.05) in liver and pancreas. 5. Immunohistochemical study of pancreatic islet revealed a decrease in the expression of insulin with progression of diabetes. However, glucagon and somatostatin showed an increase in immunoreactivity and a difference in their distribution pattern. 6. The findings of the present study suggest that oxidative stress starts at early onset of diabetes mellitus and increases progressively. In conclusion, the structural damage to these tissues or complications of diabetes mellitus may be due to oxidative stress.

In vitro generation of an active calmodulin-independent phosphodiesterase from brain calmodulin-dependent phosphodiesterase (PDE1A2) by m-calpain.

In the present study we have shown that bovine brain 60-kDa calmodulin-dependent cyclic nucleotide phosphodiesterase isozyme (CaMPDE - PDE1A2) is proteolyzed by a Ca2+-dependent cysteine protease, m-calpain. The proteolysis of PDE1A2 by m-calpain results in its conversion to a totally calmodulin (CaM)-independent form accompanied by degradation of PDE1A2 into a 45-kDa catalytic fragment and a 15-kDa fragment. The activity of PDE1A2 is unaffected by the presence or absence of CaM during cleavage, suggesting that the interaction between CaM and PDE1A2 does not alter substrate recognition by calpain. Furthermore, we provide evidence, based on the studies of CaM overlay and phosphorylation, that the cleavage site is not present either in the CaM-binding domain or phosphorylation site. N-terminal sequence analysis of the 45-kDa fragment indicated that cleavage occurs between residues 126Gln and 127Ala, and eliminates the CaM-dependent activity of carboxy termini PDE1A2. The present findings suggest that limited proteolysis in the brain through calpains could be an alternate mechanism for activating CaMPDE(s) and for regulating intracellular levels of cAMP.

Myristoyl-coA:protein N-myristoyltransferase from bovine cardiac muscle: molecular cloning, kinetic analysis, and in vitro proteolytic cleavage by m-calpain.

Myristoyl-CoA:protein N-myristoyltransferase (NMT) catalyzes the attachment of myristate onto the amino terminal glycine residue of select polypeptides. Cardiac tissue expresses high levels of cAMP-dependent protein kinase whose catalytic subunit is myristoylated; however, cardiac muscle extracts were found to contain low NMT activities. Northern blot analysis of bovine heart poly(A)+ RNA probed with bovine spleen NMT cDNA revealed a 1.7-kb mRNA. Western blot analysis of cardiac muscle extracts with human NMT antibody indicated a prominent immunoreactive band with a molecular mass of 50 kDa. The expression of mRNA and protein levels in cardiac muscle is not correlated with NMT activities, suggesting the presence of regulators of the enzyme activity. We have isolated the cDNA encoding bovine cardiac muscle NMT (cNMT) by reverse transcription polymerase chain reaction. The single long open reading frame of 1248 bp of bovine cNMT specifies a protein of 416 amino acids with a predicted mass of 46,686 Da. The cDNA clone expressed in Escherichia coli resulted in the production of functionally active 50-kDa NMT. Ultrastructural and immunolocalization of NMT utilizing the immunogold labeling technique demonstrated cytoplasmic distribution with occasional mitochondrial and myofilaments localization of the NMT antibody. Cardiac muscle NMT has a higher affinity for myristoyl-CoA than toward palmitoyl-CoA. Substrate specificity indicated that cNMT has a higher affinity toward pp60src and M2 gene segment of reovirus type 3-derived peptide substrates than toward cAMP-dependent protein kinase-derived peptide. Primary translational product of cNMT sequence contained several regions rich in proline, glutamic acid, serine, and threonine, which are known as "PEST" regions. PEST-FIND analysis of the amino acid sequences indicated eight PEST regions were present in the cNMT. These PEST regions are suggested to be recognized by specific proteases, particularly Ca(2+)-dependent neutral proteases, calpains, which are responsible for the degradation of PEST-containing proteins. We have demonstrated the abolishment of NMT activity and NMT protein degradation in vitro by m-calpain. The proteolysis of cNMT by m-calpain and the abolishment of NMT activity was prevented by the calpain inhibitor, calpastatin. These observations indicate that calpains may regulate NMT activity.

Recombinant bovine spleen myristoyl CoA: protein N-myristoyltransferase.

Myristoyl-CoA:protein N-myristoyltransferase (NMT) is an essential eukaryotic enzyme that catalyzes the co-translational transfer of myristate to the NH2-terminal glycine residue of a number of important proteins of diverse function. Recently, we have isolated full length cDNA encoding bovine spleen NMT [27] the full length cDNA was cloned and expressed in E. coli, resulting in the expression of functionally active 50 kDa NMT. Using the combination of SP-Sepharose fast flow and Mono S fast protein liquid chromatography, the enzyme was purified 20-fold with a high yield. The spleen NMT (sNMT) fusion protein exhibited an apparent molecular weight of 53 kDa on SDS-PAGE. Upon cleavage by the Enterokinase the sNMT exhibited an apparent molecular weight of 50 kDa without loss of catalytic activity. The two synthetic peptide substrates based on the N-terminal sequence of pp60src (GSSKSKMR) and cAMP dependent protein kinase (GNAAAKKRR) have different kinetic parameters of Km values of 40 and 200 microM. Recombinant sNMT was also potently inhibited by Ni2+ (histidine binder) in a concentration dependent manner with a half maximal inhibition of 280 microM. The E. coli expressed sNMT was homogenous and showed enzyme activity.

Biological significance of phosphorylation and myristoylation in the regulation of cardiac muscle proteins.

Post-translational modification has long been recognized as a way in which the properties of proteins may be subtly altered after synthesis of the polypeptide chain is complete. Amongst the moieties most commonly encountered covalently attached to proteins are oligosaccharides, phosphate, acetyl, formyl and nucleosides. Protein phosphorylation and dephosphorylation is one of the most prevalent and best understood modifications employed in cellular regulation. The bovine heart calmodulin-dependent cyclic nucleotide phosphodiesterase (CaMPEDE) can be phosphorylated by cAMP-dependent protein kinase, resulting in a decrease in the enzyme's affinity for Ca2+ and calmodulin (CaM). The phosphorylation of CaMPDE is blocked by Ca2+ and CaM and reversed by the CaM-dependent phosphatase (calcineurin). The dephosphorylation is accompanied by an increase in the affinity of the phosphodiesterase for CaM. Analysis of the complex regulatory properties of CaMPDE has led to the suggestion that fluxes of cAMP and Ca2+ during cell activations are closely coupled and that the CaMPDE play a key role in the signal coupling phenomenon. The high molecular weight calmodulin binding protein (HMWCaMBP) was phosphorylated by cAMP-dependent protein kinase. Phosphorylation of HMWCBP was higher in the absence of Ca2+/CaM then in the presence of Ca2+/CaM and reversed by the CaM-dependent phosphatase. Recently, it has become apparent that the binding of myristate to proteins is also widespread in eukaryotic cells and viruses and certainly is of great importance to the correct functioning of an organism. Myristoyl CoA:protein N-myristoyltransferase (NMT) catalyses the attachment of myristate to the amino-terminal glycine residue of various signal transduction proteins. Cardiac tissue express high levels of cAMP-dependent protein kinase whose catalytic subunit is myristoylated. The subcellular localization of bovine cardiac muscle NMT indicated a majority of the activity was localized in cytoplasm. Under native conditions the enzyme exhibited an apparent molecular mass of 50 kDa. Recovery of NMT activity, from both cytosol and particulate fractions, was found to be higher than the total activity in crude homogenates, suggesting that particulate fraction may contain an inhibitory activity towards NMT. Research in our laboratory has been focusing on the covalent modification of proteins and regulation of various signal transduction proteins. This special review is designed to summarize some aspects of the current work on co- and post-translational modification of proteins in cardiac muscle.

Cardiac high molecular weight calmodulin binding protein contains calpastatin activity.

A high molecular weight calmodulin binding protein (HMWCaMBP) was previously identified and purified from bovine heart cytosolic fraction [Sharma, R.K. (1990) J. Biol. Chem. 265, 1152-1157]. In this study, we report the biological function of this protein. HMWCaMBP was subjected to peptide mapping and three peptides were sequenced. Two of the three peptide sequences were shown to be highly homologous to the calpain inhibitor, calpastatin. However, the third peptide did not show homology to any known proteins. The Western blot analysis of HMWCaMBP and purified calpastatin from bovine cardiac muscle showed immunoreactivity with polyclonal antibody raised against HMWCaMBP. Furthermore, HMWCaMBP inhibited calpain II and calpain I activities in a dose dependent fashion. Our data based on sequence homology, amino acid analysis, antibody reactivity and calpain inhibition suggests that HMWCaMBP is homologous to calpastatin and may be a CaM-binding form of calpastatin.

Tyrosine phosphorylation in contraction of opossum esophageal longitudinal muscle in response to SNP.

Sodium nitroprusside (SNP) has been shown to elicit a guanosine 3',5'-cyclic monophosphate (cGMP)-mediated, indomethacin-sensitive contraction of the opossum esophageal longitudinal muscle. We examined the role of tyrosine phosphorylation in the signal transduction pathway of contractions induced by SNP and cGMP in longitudinal muscle strips in vitro. Force of isometric contractions was expressed as the percentage of responses to KCl (73 mM). SNP (100 microM)-induced contractions were 75 +/- 5% before and 3 +/- 2% after 50 microM genistein (P < 0.005) and 86 +/- 16% before and 0 +/- 0% after 50 microM tyrphostin B46. Contractions in response to 8-bromo-cGMP (8-BrcGMP; 1 mM) were 74 +/- 15% before and 3 +/- 2% after genistein (P < 0.01) and 63 +/- 15% before and 18 +/- 4% after tyrphostin B46 (P < 0.05). In contrast, KCl-induced contractions were 82 +/- 8% and 96 +/- 9% of the control value after genistein and tyrphostin B46 treatments, respectively (P > 0.05 for both). Carbachol contractions were partially suppressed by genistein (106 +/- 8% vs. 79 +/- 8%; P < 0.05) but unaffected by tyrphostin B46 (114 +/- 10% vs. 107 +/- 12%; P > 0.05). Western blot analysis revealed a 116-kDa phosphotyrosine protein in the control muscle strips. The level of this protein was increased to 206 +/- 15% of control after SNP treatment. Both genistein and tyrphostin B46 blocked this increase. These studies show that contractions of the esophageal longitudinal muscle induced by SNP and cGMP utilize a signal transduction pathway different from that used by the depolarizing agent KCl and the muscarinic agonist carbachol. Contractions induced by SNP and cGMP involve tyrosine phosphorylation of a protein, possibly identified as a 116-kDa protein, as a key step in the signaling pathway.

Increased longevity, reduced fecundity, and delayed development in fruitfly (Zaprionus paravittiger) fed on butylated hydroxy anisole.

Oxygen free radicals are generated as a by-product during normal metabolism, and they cause damage to proteins, lipids, and DNA in organisms. The defense system of the body counteracts these highly reactive chemical moieties and neutralizes them. However, a small fraction of free radicals escapes, which causes lipid peroxidation and hence aging of the organism. It has been hypothesized that, if the free radicals are arrested/reduced, then aging can be delayed or life span could be enhanced. To test the above hypothesis, we fed butylated hydroxy anisole (BHA) to a drosophilid insect, Zaprionus paravittiger, and observed its effect on life span, fecundity, and developmental period. The insects were reared and maintained on standard corn meal agar (CMA) medium at 26 degrees +/- 2 degrees C. Various concentrations (1, 5, 10, 25, 50, and 100 mM) of BHA were mixed with CMA medium, and the cultures were reared and maintained on these mixtures to study the life span of insects. Survivor curves showed that lower concentrations (5, 10, 25 mM) of BHA increased the life span, while higher concentrations (50, 100 mM), which were rather toxic, decreased life span. The most suitable concentration was 10 mM, which increased median (LT50) (27% and 15% in male and female) and maximum (LT100) (18% and 27% in male and female) life spans of insects maximally. Females exhibited longer life spans compared with males. The cultures were fed on the optimal concentration (10 mM) of BHA to study its effect on developmental period and egg-laying rate. The total developmental period was delayed by 7.2%, and egg-laying rate was reduced by 19.7% on BHA feeding. The extension in developmental period and reduction in egg-laying capacity could be the contributory factors to the favorable effect of BHA on life span.