Measles viruses with altered envelope protein cytoplasmic tails gain cell fusion competence

The cytoplasmic tail of the measles virus (MV) fusion (F) protein is often altered in viruses which spread through the brain of patients suffering from subacute sclerosing panencephalitis (SSPE). We transferred the coding regions of F tails from SSPE viruses in an MV genomic cDNA. Similarly, we constructed and transferred mutated tail-encoding regions of the other viral glycoprotein hemagglutinin (H) gene. From the mutated genomic cDNAs, we achieved rescue of viruses that harbor different alterations of the F tail, deletions in the membrane-distal half of the H tail, and combinations of these mutations. Viruses with alterations in any of the tails spread rapidly through the monolayer via enhanced cell-cell fusion. Double-tail mutants had even higher fusion competence but slightly decreased infectivity. Analysis of the protein composition of released mutant viral particles indicated that the tails are necessary for accurate virus envelope assembly and suggested a direct F tail-matrix (M) protein interaction. Since even tail-altered glycoproteins colocalized with M protein in intracellular patches, additional interactions may exist. We conclude that in MV infections, including SSPE, the glycoprotein tails are involved not only in virus envelope assembly but also in the control of virus-induced cell fusion.     

Recombinant measles viruses with mutations in the C, V, or F gene have altered growth phenotypes in vivo

An understanding of the determinants of measles virus (MV) virulence has been hampered by the lack of an experimental model of infection. We have previously demonstrated that virulence phenotypes in human infections are faithfully reproduced by infection of human thymus/liver (thy/liv) implants engrafted into SCID mice, where the virus grows primarily in stromal cells but induces thymocyte apoptosis (P. G. Auwaerter et al., J. Virol. 70:3734-3740, 1996). To begin to elucidate the roles of the C protein, V protein, and the 5' untranslated region of the F gene (F 5'UTR) in MV infection in vivo, the replication of strains bearing mutations of these genes was compared to that of the parent sequence-tagged Edmonston strain (EdTag). Growth curves show that mutants fall into two phenotypic classes. One class of mutants demonstrated kinetics of growth similar to that of EdTag, with decreased peak titers. The second class of mutants manifested peak titers similar to that of EdTag but had different replication kinetics. Abrogation of V expression led to delayed and markedly prolonged replication. Additionally, thymocyte survival was prolonged and implant architecture was preserved throughout the course of infection. In contrast, massive bystander thymocyte death occurred after infection with EdTag and all other mutants. A mutant which overexpressed V in Vero cells (V+) had the opposite phenotype of the A mutant not expressing V (V-). V+ grew more rapidly than EdTag with 100-fold-greater levels of virus production 3 days after infection. These results suggest that C, V, and the F 5'UTR are accessory factors required for efficient virus replication in vivo. In addition, thymocyte survival after V- infection suggests this protein may play multiple roles in pathogenesis of MV infection of thymus. Since these recombinant mutant viruses grew identically to the parent virus in Vero cells, the data show that thy/liv implants are an excellent model for investigating the determinants of MV virulence.     

A role for the Sendai virus P protein trimer in RNA synthesis

The SeV P protein is found as a homotrimer (P3) when it is expressed in mammalian cells, and trimerization is mediated by a predicted coiled-coil motif which maps within amino acids (aa) 344 to 411 (the BoxA region). The bacterially expressed protein also appears to be trimeric, apparently precluding a role for phosphorylation in the association of the P monomers. I have examined the role of P trimerization both in the protein's interaction with the nucleocapsid (N:RNA) template and in the protein's function on the template during RNA synthesis. As with the results of earlier experiments (32), I found that both the BoxA and BoxC (aa 479 to 568) regions were required for stable binding of P to the N:RNA. Binding was also observed with P proteins containing less than three BoxC regions, suggesting that trimerization may be required to permit contacts between multiple BoxC regions and the N:RNA. However, these heterologous trimers failed to function in viral RNA synthesis, indicating that the third C-terminal leg of the trimer plays an essential role in P function on the template. We speculate that this function may involve the movement of P (and possibly the polymerase complex) on the template and the maintenance of processivity.     

Importance of the cysteine-rich carboxyl-terminal half of V protein for Sendai virus pathogenesis

The Sendai virus V protein is a nonstructural trans-frame protein whose cysteine-rich C-terminal half is fused to the acidic N-terminal half of the P protein via mRNA editing. We recently created a mutant by disrupting the editing motif, which is devoid of mRNA editing and hence unable to produce the V protein, and demonstrated that this V(-) virus replicated normally or even faster with augmented gene expression and cytopathogenicity in cells in vitro, but was strongly attenuated in pathogenicity for mice (A. Kato, K. Kiyotani, Y. Sakai, T. Yoshida, and Y. Nagai, EMBO J. 16:578-587, 1997). Thus, although categorized as a nonessential protein, the V protein appeared to encode a luxury function required for the viral in vivo pathogenesis. Here, we created another version of a V-deficient mutant, VdeltaC, encoding only the N-terminal half but not the V-specific C-terminal half, by introducing a stop codon in the trans-V frame, and then we compared its in vitro and in vivo phenotypes with those of the V(-) and wild-type viruses. The VdeltaC virus was found to be similar to the wild-type virus in vitro with no augmented gene expression and cytopathogenicity, but in vivo, it resembled the V(-) virus, displaying a similarly attenuated phenotype. Thus, the pathogenicity determinant in the V protein was mapped to the C-terminal half. The N-terminal half was likely sufficient to confer normal (wild-type) in vitro phenotypes.     

The relationship of non-Hodgkin's lymphoma with P16 protein expression

The expression of P16 protein was examined in 45 cases of non-Hodgkin's lymphoma (NHL), 4 cases of reactive hyperplasia of lymph node (RHLN) and 6 cases of normal spleen with immunohistochemical staining. Positive reaction of P16 protein was observed in some lymphocytes of RHLN and splenic nodule and around splenic sinus. The positive rate of P16 protein expression in NHL was 37.8% (17/45). The positive rates of P16 protein expression for the low intermadiate- and high-grade malignancy groups were 66.7% (10/15), 37.5% (6/16) and 7.1% (1/14) respectively. There was a significant difference between the low malignancy NHL and the high malignancy NHL (P < 0.005) in expression rate of P16 protein. It suggests that P16 protein expression may be one of the important parameters in estimating the prognosis of patients with NHL.     

Vascular remodeling and altered protein expression during growth of coronary collateral arteries

The cellular mechanism of growth of coronary collateral vessels (adaptive arteriogenesis) is still poorly understood. To define a possible role of an altered expression pattern of cellular and matrix proteins in this process we implanted a constricting device around the left circumflex artery in 25 canine hearts and sacrificed the animals at the time of initiation (3 weeks), high activity (6 weeks) and discontinuation (8 weeks) of vessel growth. Methods were electron microscopy, labeling with Ki-67, the TUNEL method and immunofluorescence with confocal laser microscopy. As described earlier, the collateral vessels increased in wall thickness by the formation of a neointima without luminal narrowing. We report here for the first time that extensive vascular remodeling including migration, proliferation and apoptosis in all cell types takes place during the growth phase but not in more mature vessels. The most obvious difference with normal vessels is the reiteration of an embryonal expression pattern in smooth muscle cells of the neointima which includes a significant reduction of desmin and alpha-smooth muscle actin, calponin and vinculin. Fibronectin as a promoter of migration and adhesion was abundant, its antagonist tenascin and chondroitin sulfate showed patchy localization. A completely new finding in arteriogenesis is the involvement of mast cells releasing histamine and serotonin and probably cytokines. Vascular protein expression returned to almost normal at 8 weeks indicating cessation of remodeling. We conclude that in collateral vessel development an altered cellular and matrix protein expression is involved in a drastic case of positive vascular remodeling finally resulting in mature vessels 20-fold increased in size which are capable of maintaining the functional and structural integrity of the myocardium at risk.     

Selectable viruses and altered susceptibility mutants in Arabidopsis thaliana

The genetic basis for susceptibility or nonsusceptibility of plants to viruses is understood poorly. Two selectable tobacco etch virus (TEV) strains were developed for identification of Arabidopsis thaliana mutants with either gain-of-susceptibility or loss-of-susceptibility phenotypes. These strains conferred a conditional-survival phenotype to Arabidopsis based on systemic expression of herbicide resistance or proherbicide sensitivity genes, thereby facilitating mass selections and screens for Arabidopsis mutants that enhance or suppress TEV replication, cell-to-cell movement, or long-distance movement. A multicomponent mechanism that restricts systemic invasion of TEV was identified through isolation of gain-of-susceptibility mutants with alterations at two loci.     

Influenza A and Sendai viruses preferentially bind to fucosylated gangliosides with linear poly-N-acetyllactosaminyl chains from human granulocytes

Seven solitary fibrous tumors (SFTs) of the meninges are presented and their clinicopathologic features are compared with those of 64 fibrous meningiomas (FM). Patients with SFT included 5 females and 2 males age 47 to 73 years. The dura-based tumors involved the parasagittal region (1), tentorium (2), cerebellopontine angle (2), and spinal region (2). One each showed invasion of brain and of a spinal nerve root. Of four SFTs with at least 1-year follow-up, one subtotally resected example recurred. No tumors metastasized. All consisted of spindle cells disposed in fascicles between prominent, eosinophilic bands of collagen. Whorls and storiform cell arrangements were lacking. Mitoses ranged from 1 to 7/10 400 x fields. MIB-1 labeling indices ranged from 1% to 18% (mean 4%). All were PAS negative and showed strong immunoreactivity for vimentin and CD34. Of cases studied, half were estrogen and all were progesterone receptor immunopositive. The majority (72%) of FMs occurred in females and most (72%) were supratentorial. Recurrence was noted in 15%. Mitotic activity varied from 0 to 3 mitoses per 10 400 x fields (mean < 1). MIB-1 labeling indices ranged from 1% to 5% (mean 1.5%). Unlike SFT, FMs were glycogen-containing and variously exhibited a storiform pattern (13 of 20), psammoma body formation (9 of 20), and calcification of collagen (4 of 20). Immunoreactivities included vimentin (100%), focal to patchy EMA (80%), S-100 protein (80%), collagen IV (25%), and patchy, mild-to-moderate CD34 staining (60%). Of cases studied, nearly half were estrogen and all were progesterone receptor staining positive. Meningeal SFTs represent a distinct morphologic entity, the morphologic and immunohistochemical features of which differ from those of FM and suggest a histogenetic relationship to pleural SFT. Although a minority histologically appear to be low grade malignant, our limited experience suggests that they behave in a benign fashion. The classification of mesenchymal tumors affecting the central nervous system must be expanded to include SFT.     

P53 protein immunohistochemical expression in colonic adenomas with and without associated carcinoma

On the basis of the positive outcome of animal experiments, several large placebo-controlled trials are underway and aiming for the first time at the prevention of an immune-mediated disease, type 1 diabetes. The first of these trials, The Deutsche Nicotinamide Intervention Study (DENIS), evaluated the clinical efficacy of high doses of nicotinamide in children at high risk for IDDM. Nicotinamide has been shown to protect beta-cells from inflammatory insults and to improve residual beta-cell function in patients after onset of IDDM. Individuals at high risk for developing IDDM within 3 years were identified by screening the siblings (age 3-12 years) of patients with IDDM for the presence of high titer (> or =20 Juvenile Diabetes Foundation [JDF] U) islet cell antibodies. Probands (n = 55) were randomized into placebo and nicotinamide (slow release, 1.2 g x m(-2) x day(-1)) receiving groups and followed prospectively in a controlled clinical trial using a sequential design. Rates of diabetes onset were similar in both groups throughout the observation period (maximum 3.8 years, median 2.1 years). This sequential design provides a 10% probability of a type II error against a reduction of the cumulative diabetes incidence at 3 years from 30 to 6% by nicotinamide. The trial was terminated when the second sequential interim analysis after the eleventh case of diabetes showed that the trial had failed to detect a reduction of the cumulative diabetes incidence at 3 years from 30 to 6% (P = 0.97). The group receiving nicotinamide exhibited decreased first-phase insulin secretion in response to intravenous glucose (P = 0.03). No other side effects were observed. We conclude that in this subgroup of diabetes-prone individuals at very high risk and with an assumed rapid disease progression, nicotinamide treatment did not cause a major decrease or delay of diabetes development. However, the data do not exclude the possibility of a less strong, but potentially meaningful, risk reduction in this cohort, or a major clinical effect of nicotinamide in individuals with less risk of progression to IDDM than studied here.     

Altered retinoblastoma protein expression in nonsmall cell lung cancer: its synergistic effects with altered ras and p53 protein status on prognosis

BACKGROUND: Inactivation of the retinoblastoma (Rb) gene has been documented in various types of cancer, including lung cancer. Alterations of the p53 and ras genes are also common features in the molecular biology of lung carcinoma, and the authors of this article have reported previously on the prognostic significance of both of them. In the present study, the authors evaluated the prognostic significance of the loss of Rb protein expression alone, then performed a combined analysis of Rb protein and ras p21 status (Rb/ras) as well as an analysis of Rb and p53 protein status (Rb/p53) in patients with nonsmall cell lung cancer (NSCLC). METHODS: Ninety-one patients with NSCLC underwent potentially curative resection between 1977 and 1988, 65 of whom received postoperative combination chemotherapy. Tumor specimens were analyzed for Rb protein expression by immunohistochemistry. Univariate and multivariate analyses were performed to assess the association between Rb protein expression and survival. RESULTS: Nineteen (21%) of the 91 NSCLCs showed negative Rb protein expression. Positive or negative Rb protein expression (Rb+ or Rb-) as an individual factor was not statistically correlated with survival or prognosis in this cohort of NSCLC patients, although a tendency among Rb- patients to do worse was observed. The authors then combined the Rb protein status with previously studied results of ras p21 and p53 protein expression in the same tumor specimens, and compared the prognosis between the individuals with theoretically the best pattern of gene expression in their tumors and those with theoretically the worst pattern of expression, i.e., Rb+/ras- versus Rb-/ras+ and Rb+/p53- versus Rb-/p53+. In patients with adenocarcinoma, those with Rb-/ras+ tumors survived for a significantly shorter period after surgery (13% 5-year survival) than those with Rb+/ras- tumors (82% 5-year survival) (P = 0.01). Similarly, patients with Rb-/p53+ tumors survived for a significantly shorter period (20% 5-year survival) compared with those who had Rb+/p53- tumors (73% 5-year survival) (P = 0.008). Rb/ras status was a significant prognostic factor (P = 0.02 by univariate analysis, P = 0.048 by multivariate analysis), and Rb/p53 status tended to be significant as a prognostic factor (P = 0.04 by univariate analysis, P = 0.08 by multivariate analysis). In patients with squamous cell carcinoma, neither Rb/ras nor Rb/p53 status was a significant prognostic factor in this cohort. CONCLUSIONS: These results suggest that combined immunohistochemical analyses of Rb and ras p21 proteins and of Rb and p53 proteins may indicate their potentially synergistic effects on survival and prognosis. These analyses may also be useful for stratifying patients with adenocarcinoma of the lung into different prognostic groups and identifying populations with different risks of recurrence. Larger prospective studies with Stage I NSCLC patients are necessary to confirm the current findings.     

A comparison of hepatic cytochrome P450 protein expression between infancy and postinfancy

We immunochemically measured the contents of 9 different cytochrome P450 (CYP) isoenzymes expressed in the liver and compared them between two groups: one group of 6 infant and 4 perinatal patients and one group of 10 patients after infancy (over 1 year old). CYP protein expressed in human liver can be divided into three groups on the basis of expression pattern: (a) CYP2A6, 2C9, 2D6, 2E1, and 3A were present in all samples and no difference was observed between the two groups; (b) CYP1A2, 2B6, and 2C8 were expressed more after infancy than during infancy; and (c) CYP3A7, which has been considered a major CYP enzyme in fetal liver microsomes, was expressed in all infants as well as the four perinatal patients, whereas it was detected in only 2 patients after infancy. These results implied that CYP2A6, 2C9, 2D6, 2E1, and 3A are already expressed during perinatal and infant period, while CYP1A2, 2B6, and 2C8 are expressed highly in subjects over 1 year old, and CYP3A7 disappeared after infancy.     

Syd, a SecY-interacting protein, excludes SecA from the SecYE complex with an altered SecY24 subunit

Syd is an Escherichia coli cytosolic protein that interacts with SecY. Overproduction of this protein causes a number of protein translocation-related phenotypes, including the strong toxicity against the secY24 mutant cells. Previously, this mutation was shown to impair the interaction between SecY and SecE, the two fundamental subunits of the membrane-embedded part of protein translocase. We have now studied in vitro the mechanisms of the Syd-directed inhibition of protein translocation. Pro-OmpA translocation into inverted membrane vesicles (IMVs) prepared from the secY24 mutant cells as well as the accompanied translocation ATPase activity of SecA were rapidly inhibited by purified Syd protein. In the course of protein translocation, high affinity binding of preprotein-bearing SecA to the translocase on the IMV is followed by ATP-driven insertion of the 30-kDa SecA segment into the membrane. Our experiments using 125I-labeled SecA and the secY24 mutant IMV showed that Syd abolished both the high affinity SecA binding and the SecA insertion. Syd was even able to release the inserted form of SecA that had been stabilized by a nonhydrolyzable ATP analog. Syd affected markedly the proteolytic digestion pattern of the IMV-integrated SecY24 protein, suggesting that Syd exerts its inhibitory effect by interacting directly with the SecY24 protein. In accordance with this notion, a SecY24 variant with a second site mutation (secY249) resisted the Syd action both in vivo and in vitro. Thus, Syd acts against the SecY24 form of translocase, in which SecY-SecE interaction has been compromised, to exclude the SecA motor protein from the SecYE channel complex.     

Nongenetic variation, genetic-environmental interactions and altered gene expression. III. Posttranslational modifications

The use of protein electrophoretic data for determining the relationships among species or populations is widespread and generally accepted. However, posttranslational modifications have been discovered in many of the commonly analyzed proteins and enzymes. Posttranslational modifications often alter the electrophoretic mobility of the modified enzyme or protein. Because posttranslational modifications may affect only a fraction of the total enzyme or protein, an additional staining band often appears on gels as a result, and this may confound interpretations. Deamidation, acteylation, proteolytic modification, and oxidation of sulfhydryl groups are modifications that often result in an electrophoretic mobility shift. Sialic acid-induced heterogeneity has been documented for many enzymes, but neuraminidase treatment can often remove sialic acids and produce gel patterns that are easier to interpret. In some cases, ontogenetic and tissue-specific expression may be due to posttranslational modifications rather than gene control and restricted expression, respectively. Methods of preventing, detecting and eliminating posttranslational modifications are discussed. Some posttranslational modifications may be useful for detecting cryptic genetic polymorphisms.     

P53 protein expression and DNA ploidy in common epithelial tumors of the ovary

OBJECTIVE: To investigate p53 protein expression and DNA content in imprints from surgical biopsies of common epithelial tumors of the ovary. STUDY DESIGN: The study was based on 60 cases of epithelial tumors of the ovary (15 benign, 3 border-line and 42 malignant). For the demonstration of p53 protein, immunocytochemical staining with the avidin-extravidin technique was performed using monoclonal antibody p53 DO-7. DNA content was measured by image cytometry after Feulgen staining. RESULTS: There was a strong correlation between p53 expression and aneuploidy, with the difference between diploid and aneuploid tumors statistically significant (P < .001). A correlation was found between DNA ploidy, histologic grade and clinical stage (P < .001 and P < .05), respectively. There was no correlation between DNA ploidy and histologic type (P = .89). No correlation was observed between p53 protein expression and grade or clinical stage of the tumors. Nevertheless, a correlation of p53 expression between early (I, II) and advanced stages (III, IV) (P < .05) was observed. All benign and borderline tumors were diploid and did not express p53 protein. CONCLUSION: The results of the present study and the data in the literature stress the value of p53 expression and DNA ploidy in assessing the malignant potential of common epithelial ovarian cancers. However, the clinical application of these data requires further study.