Heat shock protein 70 regulates degradation of the mumps virus phosphoprotein via the ubiquitin-proteasome pathway.

UNLABELLED: Mumps virus (MuV) infection induces formation of cytoplasmic inclusion bodies (IBs). Growing evidence indicates that IBs are the sites where RNA viruses synthesize their viral RNA. However, in the case of MuV infection, little is known about the viral and cellular compositions and biological functions of the IBs. In this study, pulldown purification and N-terminal amino acid sequencing revealed that stress-inducible heat shock protein 70 (Hsp72) was a binding partner of MuV phosphoprotein (P protein), which was an essential component of the IB formation. Immunofluorescence and immunoblotting analyses revealed that Hsp72 was colocalized with the P protein in the IBs, and its expression was increased during MuV infection. Knockdown of Hsp72 using small interfering RNAs (siRNAs) had little, if any, effect on viral propagation in cultured cells. Knockdown of Hsp72 caused accumulation of ubiquitinated P protein and delayed P protein degradation. These results show that Hsp72 is recruited to IBs and regulates the degradation of MuV P protein through the ubiquitin-proteasome pathway. IMPORTANCE: Formation of cytoplasmic inclusion bodies (IBs) is a common characteristic feature in mononegavirus infections. IBs are considered to be the sites of viral RNA replication and transcription. However, there have been few studies focused on host factors recruited to the IBs and their biological functions. Here, we identified stress-inducible heat shock protein 70 (Hsp72) as the first cellular partner of mumps virus (MuV) phosphoprotein (P protein), which is an essential component of the IBs and is involved in viral RNA replication/transcription. We found that the Hsp72 mobilized to the IBs promoted degradation of the MuV P protein through the ubiquitin-proteasome pathway. Our data provide new insight into the role played by IBs in mononegavirus infection.

LDH isoenzymes in semen of infertile men.

LDH isoenzymes (LDH1, LDH2, LDH3, LDH4, LDH5, and LDHx) were evaluated in 68 infertile men and in a control of 10 fertile men. No statistically significant difference was found in the value of the 5 isoenzymes (LDH 1-5) in the different subgroups of infertile men and in the comparison of the infertile men with the control group. LDHx was detected in the infertile men, regardless of the cause of infertility, as long as the sperm concentration was greater than or equal to 16 million/ml, LDHx was not detected not only in all azoospermic men but, also, in those infertile men whose sperm count was less than or equal to 15 million/ml. There is a positive relationship (p less than 0.001) between LDHx and sperm concentration, but there is no such relationship between LDHx and % motility.

Amylase inhibitor from wheat: its action and clinical application.

The electrophoretic and column chromatographic characteristics of an amylase inhibitor of wheat origin were investigated. Further, the clinical usefulness of this inhibitor for determining the ratio of pancreatic to salivary isoamylase activity in serum was evaluated. Amylase inhibitor inhibits the action of salivary alpha-amylase by making an amylase-inhibitor complex, which is easily separated into its individual component during electrophoresis with full recovery of amylase activity. Using the specific inhibitory effect of this inhibitor on salivary alpha-amylase activity, the ratio of pancreatic to salivary isoamylase activity (P/S) in serum was determined. There was a good correlation in P/S ratio in serum between the results obtained with the inhibitor method and those with electrophoretic method. The P/S ratio in sera from patients with acute pancreatitis was over 8.0, whereas that in sera from patients with salivary-type hyperamylasemia such as mumps, pulmonary diseases and following surgery was less than 0.1. However, hyperamylasemia due to macroamylase or renal failure could not be identified by the inhibitor method.

Inhibition of human serum alpha-amylase activity.

Amylase activity was measured in normal sera and serum of patients with acute pancreatitis and mumps. A significant decrease of enzyme activity in patients with acute pancreatitis was observed after incubation with specific antibody against hog pancreas amylase. A minimal effect of the antibody on normal serum and on that from mumps patients was noted. A comparable effect was observed in the presence of glyceraldehyde used as an amylase differentiating factor.

Serum alpha-amylase isozymes in mumps: estimation of salivary and pancreatic isozymes by isoelectric focusing.

Serum alpha-amylase isozymes were separated into three major isozymes by thin-layer gel isoelectric focusing and detected by a starch-iodine zymogram procedure. Of the three groups of isozymes, one (S isozyme) corresponded to a salivary specific form, one (P isozyme) to a pancreatic specific form and the third (SP isozyme) to isozymes of similar isoelectric point common to both secretions. The levels of total alpha-amylase and of these three isozymes were estimated in the sera of 54 patients with mumps. Total alpha-amylase and salivary isozyme concentrations were greatly increased in the sera of all patients compared with controls. Pancreatic isozyme concentrations however, were only slightly increased and did not correlate with clinical pancreatitis. Indeed, in patients with mumps associated with pancreatitis, meningoencephalitis or orchitis, levels of total serum amylase, although higher than controls, were lower than levels in patients who presented solely with mumps sialadenitis.

The relative values of serum immuno-reactive trypsin concentration and total amylase activity in the diagnosis of mumps, chronic renal failure, and pancreatic disease.

Fasting serum concentrations of trypsin and amylase activity have been compared in 107 subjects, including 18 controls and patients with mumps, acute pancreatitis, chronic pancreatitis, cancer of the pancreas, and chronic renal failure. There was no significant correlation between amylase activity and trypsin concentrations in any of these groups. In all 12 patients with acute pancreatitis and all 16 with chronic renal failure the serum immuno-reactive trypsin concentrations were elevated. Amylase activity was increased in 87% (20 out of 23) of patients with mumps, but only 13% (3 out of 23) had hypertrypsinaemia suggesting subclinical pancreatitis. In 18 patients with chronic pancreatitis low levels of serum trypsin were measured in 11 (61%), reflecting a decrease in pancreatic acinar mass. In contrast, serum amylase was normal or raised in all 18. Subnormal values of the trypsin to amylase ratio was obtained in 15 (83%). Trypsin levels in 20 patients with carcinoma of the pancreas were abnormal in 11 (55%). Six (30%) had abnormal amylase levels. It is concluded that it is more useful to measure the serum trypsin concentration than the amylase activity in the diagnosis of both mumps-pancreatitis and chronic pancreatic disease and that the trypsin to amylase ratio is more sensitive than either enzyme alone in the diagnosis of chronic pancreatitis.

Amylase isoenzymes in mumps.

The amylase isoenzymes activities in serum and urine from 75 children treated for complications of mumps were evaluated. The clinical observations, especially in cases with suspected pancreatitis, were compared with the P- and S-type amylase activities. Increased total activity of serum amylase in 79% of patients was due to increased S-type amylase. However, in 39% of patients there was also a markedly increased P-type serum amylase. Amylase isoenzymes may offer a new insights into the relationship between the pancreas and the parotid glands in mumps.

Cerebrospinal fluid lysozyme in bacterial and viral meningitis.

The concentration of lysozyme (LZM) in cerebrospinal fluid was determined in 25 patients with bacterial meningitis, in 18 patients with viral meningitis and in 25 control patients who had other fibrile illnesses. The concentration of LZM was less than 1.5 microgram/ml in all control patients, and slightly to markedly raised in 10 patients with viral meningitis and in 11 out of 13 patients with untreated bacterial meningitis. The concentration of LZM was significantly different in the viral and bacterial meningitis patients (p less than 0.001). Most raised concentrations of cerebrospinal fluid LZM persisted for at least one week after the start of antibiotic treatment. The concentrations of LZM correlated well with concentrations of lactic dehydrogenase. These results show that the determination of cerebrospinal fluid LZM is a useful tool in the differential diagnosis of meningitis, particularly when the prehospital treatment with antibiotics may be responsible for a diagnostically misleading negative bacterial culture of the cerebrospinal fluid and altered cerebrospinal fluid cytology.

Amylase in the lung.

Although elevated amylase levels in serum, pleural fluid, and extracts of tumor tissue in primary lung cancer have been reported, electrophoretic and column-chromatographic studies have not revealed the ectopic production of amylase but have merely shown an increase of amylase activity of chiefly the salivary type in these materials. The present study was designed to make clear the nature of the amylase or amylase-like substance in the serum, pleural fluid and tumor extracts, and to determine whether amylase might be produced ectopically in tumor tissues. Our data not only confirmed that the hyperamylasemia in some cases of primary lung cancer was due to an increase in salivary type isoamylases, but also showed that the same isoamylase pattern occurs in serum, pleural fluids, and diseased lung tissue of patients with pneumonia. However, the elution pattern of amylase in these materials in column-chromatography on Sephadex G-75 Superfine was different from that of salivary amylase. On the basis of our observations, it seems reasonable to conclude that the salivary type hyperamylasemia in some cases of primary lung cancer may be due to an increase in the amylase contained in normal lung tissues, resulting from activation and release into the blood stream by some inflammatory process. However, ectopic production of amylase was demonstrated in one particular case of primary lung cancer in which a high amylase content and a peculiar isoamylase were found both in the primary and metastatic lesions.

Amylase thermolability in body fluids.

The thermolability of amylase was measured in saliva, pancreatic juice, urine, adult and neonatal sera. The mean percentage thermolability from these fluids was 100%, 99%, 87%, 44% and 23% respectively. In patients with acute pancreatitis and mumps the amylase was 84% and 83% thermolabile during the acute phase. On resolution of the pancreatitis this dropped towards normal. Patients with a pancreatic pseudocyst showed a high mean percentage thermolability (82%). These results could suggest that a component of amylase in human serum is not of pancreatic or salivary origin. In addition, this simple technique may be helpful in the diagnosis of pancreatic pseudocyst.

Cerebrospinal fluid fructose-biphosphate aldolase (aldolase) activity in infectious diseases of the central nervous system.

Aldolase levels were estimated in the cerebrospinal fluid of patients with infectious diseases of the central nervous system. A significant rise was found in bacterial and cryptococcal meningitis but the investigation failed to elucidate the source and clinical significance of the elevated activity.

An evaluation of the usefulness of amylase isozyme differentiation in patients with hyperamylasemia.

Amylase assays measure total activity without differentiating the relative contributions of pancreatic- and salivary-type amylase isozymes. Since polyacrylamide electrophoresis allows identification of salivary-and pancreatic-type isoxymes and their respective variants, serum and urine specimens from patients with the clinical diagnoses of mumps (4), pancreatitis (16), or undiagnosed hyperamylasemias (5) were compared with specimens from control subjects. Patients with mumps had elevations of salivary-type isozymes, while those with pancreatitis had elevations of pancreatic-type isozymes. Elevation of salivary-type isozymes was identified in the five patients who had undiagnosed hyperamylasemias; among these, the isozymes of two originated in neoplastic ovarian tissue and those of three, probably in the salivary glands. Amylase isozyme differentiation cannot unamibiguously identify the tissue source of hyperamylasemia. However, in patients whose hyperamylasemia is of unknown etiology or who respond atypically to therapy, amylase electrophoresis provides identification of the elevated isozyme type, thus providing the basis for the rational selection of further diagnostic procedures.