Insulin secretion and sensitivity before and after surgical treatment for aldosterone-producing adenoma.

AIM: Primary aldosteronism, which is usually caused by an aldosterone-producing tumour, affects glucose metabolism. The effects of this condition on insulin secretion and insulin sensitivity have remained unclear, however. To gain insight into the influence of primary aldosteronism on glucose tolerance, various parameters related to insulin secretion or insulin sensitivity in patients with an aldosterone-producing tumour were comprehensively analyzed. METHODS: To assess 14 patients with an aldosterone-producing tumour, hyperglycaemic and hyperinsulinaemic-euglycaemic clamp tests as well as oral glucose tolerance tests (OGTTs) were performed before and after tumour excision. Time between presurgical analysis and surgery was 27-559 (194±132) days, and 14-142 (51±38) days between surgery and postsurgical analysis. Various parameters related to insulin secretion or sensitivity as determined by OGTT as well as hyperglycaemic and hyperinsulinaemic-euglycaemic clamp analyses were evaluated. RESULTS: Surgical treatment of tumours ameliorated hypokalaemia and reduced plasma aldosterone levels. First and second phases of insulin secretion during the hyperglycaemic clamp, as well as the insulinogenic index and total insulin secretion measured during OGTT, were also improved after surgery. In addition, the insulin sensitivity index determined during the hyperinsulinaemic-euglycaemic clamp was reduced after surgery. CONCLUSION: Primary aldosteronism impairs insulin secretion.

Hemagglutinin-neuraminidase-independent fusion activity of simian virus 5 fusion (F) protein: difference in conformation between fusogenic and nonfusogenic F proteins on the cell surface.

The fusion (F) protein of simian virus 5 (SV5) strain W3A is known to induce cell fusion in the absence of hemagglutinin-neuraminidase (HN) protein. In contrast, the F protein of SV5 strain WR induces cell fusion only when coexpressed with the HN protein, the same as do other paramyxovirus F proteins. When Leu-22 in the subunit F2 of the WR F protein is replaced with the counterpart (Pro) in the W3A F protein, the resulting mutant L22P induces extensive cell fusion by itself. In the present study, we obtained anti-L22P monoclonal antibodies (MAbs) 21-1 and 6-7, whose epitopes were located in the middle (amino acids [aa] 227 to 320) of subunit F1. The amino-terminal region (aa 20 to 47) of subunit F2 was also involved in the formation of MAb 21-1 epitope. Flow cytometric analysis revealed that both the MAbs reacted very faintly with native WR F protein that was expressed on the cell surface whereas they reacted efficiently with native L22P irrespective of whether it is cleaved into F1 and F2. However, by heating the cells at 47 degrees C after mild formaldehyde fixation, the epitopes for MAb 6-7 and mAb 21-1 in the WR F protein were exposed and the reactivity of the MAbs with the WR F protein became comparable to their reactivity with L22P. Thus, the two MAbs seem to distinguish the difference in native conformation between fusogenic mutant L22P and its parental nonfusogenic WR F protein. The native conformation of L22P may represent an intermediate between native and postfusion conformations of a typical paramyxovirus F protein.

High resistance of human parainfluenza type 2 virus protein-expressing cells to the antiviral and anti-cell proliferative activities of alpha/beta interferons: cysteine-rich V-specific domain is required for high resistance to the interferons.

Human parainfluenza type 2 virus (hPIV-2)-infected HeLa (HeLa-CA) cells and hPIV-2 V-expressing HeLa (HeLa-V) cells show high resistance to alpha/beta interferons (IFN-alpha/beta) irrespective of whether vesicular stomatitis virus or Sindbis virus is used as a challenge virus. When Sindbis virus is used, these cells show high susceptibility to human IFN-gamma. Furthermore, the multiplication of HeLa-V cells is not inhibited by IFN-alpha/beta. HeLa cells expressing the N-terminally truncated V protein show resistance to IFN-alpha/beta, showing that the IFN resistance determinant maps to the cysteine-rich V-specific domain. A complete defect of Stat2 is found in HeLa-CA and HeLa-V cells, whereas the levels of Stat1 expression are not significantly different among HeLa, HeLa-CA, HeLa-P, and HeLa-V cells, indicating that IFN-alpha/beta resistance of HeLa-CA and HeLa-V cells is due to a defect of Stat2. HeLa-SV41V cells show high resistance to all IFNs, and no expression of Stat1 can be detected. Stat2 mRNA is fully detected in HeLa-V cells. Stat2 was scarcely pulse-labeled in the HeLa-V cells, indicating that synthesis of Stat2 is suppressed or Stat2 is very rapidly degraded in HeLa-V cells. The V protein suppresses the in vitro translation of Stat2 mRNA more extensively than that of Stat1 mRNA. An extremely small amount of Stat2 can be detected in HeLa-V cells treated with proteasome inhibitors. The half-life of Stat2 is approximately 3.5 and 2 h in uninfected and hPIV-2-infected HeLa cells, respectively. This study shows that synthesis of Stat2 may be suppressed and Stat2 degradation is also enhanced in hPIV-2-infected HeLa and HeLa-V cells.

Recovery of infectious human parainfluenza type 2 virus from cDNA clones and properties of the defective virus without V-specific cysteine-rich domain.

A full-length cDNA clone was constructed from the genome of the human parainfluenza type 2 virus (hPIV2). First, Vero cells were infected with recombinant vaccinia virus expressing T7 RNA polymerase, and then the plasmid encoding the antigenome sequence was transfected into Vero cells together with polymerase unit plasmids, NP, P, and L, which were under control of the T7 polymerase promoter. Subsequently, the transfected cells were cocultured with fresh Vero cells. Rescue of recombinant hPIV2 (rPIV2) from cDNA clone was demonstrated by finding the introduced genetic tag. As an application of reverse genetics, we introduced one nucleotide change (UCU to ACU) to immediate downstream of the RNA-editing site of the V gene in the full-length hPIV2 cDNA and were able to obtain infectious viruses [rPIV2V(-)] from the cDNA. The rPIV2V(-) possessed a defective V protein that did not have the unique cysteine-rich domain in its carboxyl terminus (the V-protein-specific domain). The rPIV2V(-) showed no growth in CV-1 and FL cells. Replication of the rPIV2V(-) in these cells, however, was partially recovered by adding anti-interferon (IFN)-beta antibody into the culture medium, showing that the rPIV2V(-) is highly sensitive against IFN and that no growth of rPIV2V(-) in CV-1 and FL cells is mainly due to its hypersensitivity to endogenously produced IFN. These findings indicate that the V-protein-specific domain of hPIV2 is related to IFN resistance. On the other hand, the rPIV2V(-) efficiently replicated in Vero cells, which are known as a IFN-non-producers. However, the virus yields of rPIV2V(-) in Vero cells were 10- to100-fold lower than those of control rPIV2, although syntheses of the viral-specific proteins and their mRNAs in rPIV2V(-)-infected Vero cells were augmented up to 48 p.i. in comparison with those of rPIV2. Furthermore, the rPIV2V(-) virions showed anomalous in size as compared with rPIV2 virions. These results suggest that the V protein plays an important role in the hPIV2 assembly, maturation, and morphogenesis.

Ruptured dissecting aneurysm in bilateral iliac arteries caused by Ehlers-Danlos syndrome type IV: report of a case.

Ehlers-Danlos syndrome (EDS) is an inherited disorder of connective tissue characterized by hyperextensible skin, hypermobile joints, and abnormalities of the cardiovascular system. Ten types and several subtypes of EDS have so far been recognized based on genetic, clinical, and biochemical characteristics. The spectrum of the disorder varies from mild to life-threatening vascular complications. EDS type IV is a particularly dangerous form with a lethal spontaneous rupture of the major arteries and aneurysmal formation. We present herein a case of a ruptured dissecting aneurysm in the bilateral iliac arteries caused by EDS type IV. A previously healthy 33-year-old man without any physical features of this connective tissue disorder experienced a metachronous vascular rupture two times. Successful synthetic bypass grafting was performed with great difficulty. The diagnosis of EDS type IV was made afterwards based on an electrophoresis analysis of a skin biopsy specimen which revealed a lack of type III collagen. Surgical intervention in cases of arterial complications in EDS type IV patients have been reported to be both difficult and frequently unsuccessful. The early clinical recognition of this syndrome is therefore of great importance due to the hazards of such surgical therapies.

N-glycosylation contributes to the limited cross-reactivity between hemagglutinin neuraminidase proteins of human parainfluenza virus type 4A and 4B.

cDNAs encoding human parainfluenza virus type 4B (hPIV-4B) hemagglutinin neuraminidase (HN) protein were cloned and the nucleotide sequences were determined. A high degree of identity (81.4%) was observed between the nucleotide sequences of hPIV-4A and -4B HN proteins, and an 87.3% identity was found between the deduced amino acid sequences. This degree of identity is considered to be greater than immunological similarity between hPIV-4A and -4B HN proteins determined using monoclonal antibodies. To elucidate the causes of the antigenic difference between HN proteins of hPIV-4A and -4B, we constructed three cDNAs of hPIV-4B HN whose potential N-glycosylation sites were partially or completely the same as in hPIV-4A HN cDNA. We compared the antigenicity of the expressed wild-type and mutant proteins, and found that the antigenicities of the mutant hPIV-4B HN proteins were more similar to the hPIV-4A HN protein than to the non-mutant hPIV-4B HN protein. This study indicated that the antigenic diversity between hPIV-4A and -4B was partly caused by deletion or creation of glycosylation sites, showing that the point mutations resulting in deletion or creation of glycosylation sites is one of the initial steps leading to the division of virus into subtypes.

Incomplete replication of human parainfluenza virus type 4 in LLC-MK2 cells and in L929 cells.

Human parainfluenza virus type 4A (hPIV-4A) and type 4B (hPIV-4B) were tested for their ability to replicate in the monkey kidney LLC-MK2 cell line (MK2 cells) and the murine L929 cell line (L929 cells). These cells are normally non-permissive for replication of hPIV-4; however, treatment with acetylated trypsin led to virus replication in MK2 cells, but was less effective for L929 cells. Endogenously produced interferon (IFN) played no role in virus replication in L929 cells. Synthesis of virus-specific polypeptides was suppressed in L929 cells. Whereas NP-mRNA and HN-mRNA were detected in MK2 cells, no HN-mRNA was detected in L929 cells. These results indicate that hPIV-4 can infect both MK2 cells and L929 cells. In MK2 cells, when protease exists in the extracellular medium, hPIV-4 exhibits multistep growth. In L929 cells, however, the cause of incomplete replication might be lack of other unknown factors.

An amino acid in the heptad repeat 1 domain is important for the haemagglutinin-neuraminidase-independent fusing activity of simian virus 5 fusion protein.

A canine isolate (strain T1) of simian virus 5 (SV-5) performed multiple replication in BHK cells but did not induce cell fusion for up to 3 days. In contrast, a prototype strain (WR) provoked extensive cell fusion within 2 days during the course of its replication. Accordingly, the fusion (F) protein of the T1 strain did not cause cell fusion even when co-expressed with the SV-5 haemagglutinin-neuraminidase (HN) protein, whereas the WR F protein induced cell fusion in the presence of the HN protein. Differences in the predicted amino acid sequences of the T1 and WR F proteins were found at 12 positions and it was proved that the T1 F protein had a longer cytoplasmic tail than the WR F protein. By reducing the length of the cytoplasmic tail or by replacing the tail with the WR F counterpart, the T1 F protein partly restored its HN-dependent fusing activity. Chimeric and mutational analyses between the T1 F protein and the mutant F protein (L22P) suggested that Glu-132 in the heptad repeat 1 domain was involved in the HN-independent fusing activity in addition to the previously identified Pro-22 at the F(2) N terminus. It was also shown that Ala-290 in the heptad repeat 3 domain contributed to the HN-independent fusing activity to some extent.

Suppression of FRP-1/CD98-mediated multinucleated giant cell and osteoclast formation by an anti-FRP-1/CD98 mAb, HBJ 127, that inhibits c-src expression.

When anti-CD98 mAb 6-1-13, 4-5-1, or 38-2-2 was added to the culture fluids of monocytes, extensive cell aggregation and polykaryocyte formation were induced. These multinucleated giant cells were tartrate-resistant acid phosphatase (TRAP) positive. On the other hand, when monocytes were incubated with another anti-CD98 mAb, HBJ 127, polykaryocyte formation was not detected, although extensive cell aggregation was induced. When HBJ 127 and 6-1-13 were simultaneously added to the culture fluids, anti-CD98 mAb-induced cell fusion was inhibited almost completely. HBJ 127 suppressed formation of 6-1-13-induced cell fusion in a dose-dependent manner. If, however, HBJ 127 was added after incubation of monocytes with mAb 6-1-13 for 6 h, an appreciable degree of TRAP-positive polykaryocyte formation was found. The bindings of 6-1-13 and HBJ 127 were not mutually competed. When monocytes were incubated with 6-1-13 or HBJ 127, 6-1-13 induced c-src mRNA, while HBJ 127 did not. Furthermore, when monocytes were incubated with both 6-1-13 and HBJ 127, c-src mRNA could not be detected, showing that HBJ 127 suppresses c-src expression. Therefore, CD98-mediated osteoclast formation can be regulated by modification of CD98 system.

Isolation and characterization of monoclonal antibodies directed against murine FRP-1/CD98/4F2 heavy chain: murine FRP-1 is an alloantigen and amino acid change at 129 (P<-->R) is related to the alloantigenicity.

Nineteen mAb directed against murine fusion regulatory protein-1 (mFRP-1)/4F2/CD98 were isolated and their biological properties were analysed. Intriguingly, mFRP-1 was found to be an alloantigen, namely, FRP-1.1 (DBA/2 and CBA mice type) and FRP-1.2 (BALB/c, C57BL/6 and C3H/He mice type). The nucleotide sequences of FRP-1.1 and FRP-1.2 were determined, demonstrating that amino acid change at 129 (P<-->R) is related to the alloantigenicity. mFRP-1 is expressed on thymocytes, on spleen cells, on peripheral lymphocytes and on blood monocytes, suggesting that the physiological role in vivo of murine FRP-1 is different from that of human FRP-1. The biological activities of antimFRP-1 mAbs showed by the present study are: (i) enhancement of Newcastle disease virus-induced cell fusion; (ii) suppression of HIVgp160-mediated cell fusion; and (iii) induction of aggregation and multinucleated giant cells of monocytes/macrophages.

Cutting edge: primary structure of the light chain of fusion regulatory protein-1/CD98/4F2 predicts a protein with multiple transmembrane domains that is almost identical to the amino acid transporter E16.

The CD98 light chain (CD98LC) was copurified from HeLa S3 cells by an affinity chromatography using a mAb specific for the fusion regulatory protein-1 (FRP-1) which is identical to the CD98 heavy chain. On the basis of the N-terminal sequence (63 amino acids) of purified CD98LC polypeptide, we have cloned a PCR fragment (155 bp) from a HeLa S3 cDNA library and finally obtained a full cDNA clone encoding the CD98LC. Fluorescence in situ hybridization analysis using the cDNA assigned the CD98LC gene to the long arm of human chromosome 16 (16q24). The predicted amino acid sequence suggested that CD98LC is a protein with multiple transmembrane domains and is almost identical to the amino acid transporter E16. Resting monocytes and lymphocytes expressed CD98LC as analyzed by a newly isolated anti-CD98LC mAb, which showed cross-reactivity with insect Sf9 cells as well as with various mammalian cell lines.

Isolation of monoclonal antibodies directed against the V protein of human parainfluenza virus type 2 and localization of the V protein in virus-infected cells.

Two monoclonal antibodies (mAbs) specific for the human parainfluenza virus type 2 (hPIV-2) V protein were obtained by immunizing mice with the V protein recombinantly expressed in Escherichia coli. Both mAbs were found to react with the V protein in ELISA and in Western blot analysis. Using these mAbs and previously obtained mAbs specific for hPIV-2 nucleoprotein (NP) or hPIV-2 phospho-(P) protein, we examined the intracellular distributions of the V, P and NP proteins in hPIV-2-infected cells by indirect immunofluorescence analyses. The P and NP proteins were organized in numerous granules in the cytoplasm of hPIV-2 infected cells. In contrast, the V protein showed diffuse nuclear and cytoplasm distributions.

Identification of regions on the fusion protein of human parainfluenza virus type 2 which are required for haemagglutinin-neuraminidase proteins to promote cell fusion.

Using a plasmid expression system in HeLa cells, we have previously shown that the fusion (F) protein of simian virus 41 (SV-41) induces cell fusion when coexpressed with the haemagglutinin-neuraminidase (HN) protein of human parainfluenza virus type 2 (PIV-2), while the PIV-2 F protein does not induce cell fusion with the SV-41 HN protein. In the present study, we found that the PIV-2 F protein induced extensive cell fusion with the HN protein of mumps virus (MuV), whereas the SV-41 F protein did not. Chimaeric analyses of the F proteins of PIV-2 and SV-41 identified two regions (designated M1 and M2) on the PIV-2 F protein, either of which was necessary for chimaeric F proteins to show fusogenic activity with the MuV HN protein. Subsequently, two additional regions (P1 and P2) were identified on the PIV-2 F protein, both of which were necessary for chimaeric F proteins to prevent induction of cell fusion with the SV-41 HN protein. Consequently, it was proved that a given chimaeric F protein, harbouring regions P1 and P2 together with either of region M1 or M2, induced cell fusion specifically with HN proteins of PIV-2 and MuV, the same as the PIV-2 F protein. Region M2 was located at the membrane proximal end of the PIV-2 F1 ectodomain, while regions P1, M1 and P2 clustered together in the middle of the ectodomain. These regions on the PIV-2 F protein may be involved in a putative functional interaction with HN proteins, which is considered to be a prerequisite for cell fusion.

Enhancement of human parainfluenza virus-induced cell fusion by pradimicin, a low molecular weight mannose-binding antibiotic.

Oligosaccharides, especially mannose residues, expressed on the cell surface, are thought to be important for virus-induced membrane fusion. We examined the effect of mannose-binding compounds, pradimicin derivative BMY-28,864 (PRM) and concanavalin A (Con A), on cell fusion of human parainfluenza type 2 virus (hPIV2)-infected HeLa cells. Syncytium formation of hPIV2-infected HeLa cells was suppressed in the presence of Con A. On the other hand, PRM enhanced cell fusion of hPIV2-infected HeLa cells. These effects were blocked by addition of mannose-rich mannan. However, PRM shows little effect on virus growth and the expression of viral glycoproteins on the cell surface in hPIV2-infected HeLa cells. Fluorescein-isothiocyanate-labeled pradimicin and Con A bound to both uninfected and hPIV2-infected mononuclear cells, indicating that these compounds have an affinity to several cellular component(s). In contrast to Con A, PRM had little affinity to the viral glycoproteins. It is inferred from these results that the enhancement of hPIV2-induced cell fusion is probably due to the interaction between PRM and cellular component(s).

Protein tyrosine kinase activation provides an early and obligatory signal in anti-FRP-1/CD98/4F2 monoclonal antibody induced cell fusion mediated by HIV gp160.

The mechanism by which anti-fusion regulatory protein-1 (FRP-1) monoclonal antibody (mAb) induced cell fusion was investigated using U2ME-7 cells that are CD4+U937 cells transfected with the HIV gp160 gene. Protein kinase inhibitors (H-7, H-89, herbimycin A and genistein) suppressed cell fusion of Cd+U2ME-7 cells induced by anti-FRP-1 mAb. H-7 and H-89 also inhibited the cell aggregation, but herbimycin A and genistein did not. Intriguingly, only when herbimycin A was added either before or simultaneously with addition of anti-FRP-1 mAb, was cell fusion suppressed, suggesting that tyrosine kinase is related with the initial step of polykaryocyte formation. Anti-FRP-1 mAb induced the rapid tyrosine phosphorylation of multiple cellular proteins. These effects occurred within 1 min and returned to near baseline by 60 min. The rapid tyrosine phosphorylation was suppressed by herbimycin A and genistein. Although it remains to be determined which protein tyrosine kinase(s) is involved in this response, pp130 tyrosine phosphorylation appears to be a specific and early signal transmitted after the interaction of FRP-1 with a specific antibody. pp130 was present in the cytosol fraction and was distinct from pp125FAK, p130CAS, vinculin, and beta 1-integrin. Thus, our study may present evidence for a novel pathway of protein tyrosine kinases that phosphorylate specific, still unknown protein substrates during polykaryocyte formation.

Role of a single amino acid at the amino terminus of the simian virus 5 F2 subunit in syncytium formation.

The fusion (F) protein of simian virus 5 (strain W3A) induced extensive cell fusion in BHK cells when expressed alone, while that of strain WR did not. Mutational analysis demonstrated that the fusing activity can be transferred to the WR F protein by a proline residue at position 22 of subunit W3A F2.

Human parainfluenza virus type 2 phosphoprotein: mapping of monoclonal antibody epitopes and location of the multimerization domain.

The epitopes recognized by 42 monoclonal antibodies directed against the human parainfluenza virus type 2 (hPIV-2) phosphoprotein (P) were mapped on the primary structure of the P protein by testing their reactivities with deletion mutants. By Western Immunoblotting with these monoclonal antibodies and P protein deletion mutants the region essential for P-P interactions was determined. The P protein region encompassing amino acids 211-248 was required for proper folding and oligomerization which is mediated by predicted coiled-coils in this region. The oligomer was shown to be a homotrimer by chemical cross-linking experiments.

Paramyxovirus-induced syncytium cell formation is suppressed by a dominant negative fusion regulatory protein-1 (FRP-1)/CD98 mutated construct: an important role of FRP-1 in virus-induced cell fusion.

Syncytium formation and subsequent generalized cell fusion have been reported as potentially important mechanisms of virus-induced cytotoxic effects. We tried to clarify the roles of fusion regulatory factor-1 (FRP-1) in virus-induced cell fusion. Two mutated human FRP-1/CD98 proteins [FRP-1/HN, in which the cytoplasmic domain was replaced with the cytoplasmic domain of human parainfluenza virus type 2 (HPIV-2) haemagglutinin-neuraminidase (HN), and FRP-1/330 (serine), in which a cysteine at amino acid 330 was mutated to serine], when expressed stably in L929 cells, were lacking in cell-fusion-enhancing activity stimulated by anti-FRP-1 antibodies. Anti-FRP-1 antibodies enhanced Newcastle disease virus (NDV)-mediated polykaryocyte formation in parent HeLa cells, while anti-FRP-1 antibodies showed no/low effect on polykaryocyte formation in NDV-infected HeLa cells constitutively expressing FRP-1/HN (HeLa-FRP-1/HN cells), indicating that the FRP-1/HN molecule is capable of acting as a dominant negative inhibitor. Furthermore, when HeLa-FRP-1/HN cells were infected with various rubulaviruses (HPIV-2, mumps virus, simian viruses 5 and 41), virus-induced cell fusion was also suppressed, although virus replication was not inhibited in these cells, showing that FRP-1 molecules are required for virus-induced cell fusion. Therefore, FRP-1 is considered to be related to the pathogenesis of paramyxoviruses.