Identification and characterization of dominant helper T-cell epitopes in the nucleocapsid protein of severe acute respiratory syndrome coronavirus

By using a series of overlapping synthetic peptides covering 98% of the amino acid sequence of the nucleocapsid protein (NP) of severe acute respiratory syndrome coronavirus (SARS-CoV), four helper T-cell (Th) epitopes (NP11, residues 11 to 25; NP51, residues 51 to 65; NP61, residues 61 to 75; and NP111, residues 111 to 125) in C57BL mice (H-2(b)), four (NP21, residues 21 to 35; NP91, residues 91 to 105; NP331, residues 331 to 345; and NP351, residues 351 to 365) in C3H mice (H-2(k)), and two (NP81, residues 81 to 95; and NP351, residues 351 to 365) in BALB/c mice (H-2(d)) have been identified. All of these peptides were able to stimulate the proliferation of NP-specific T-cell lines or freshly isolated lymph node cells from mice immunized with recombinant NP. Immunization of mice with synthetic peptides containing appropriate Th epitopes elicited strong cellular immunity in vivo, as evidenced by delayed-type hypersensitivity. Priming with the helper peptides (e.g., NP111 and NP351) significantly accelerated the immune response induced by recombinant NP, as determined by the production of NP-specific antibodies. When fused with a conserved neutralizing epitope (SP1143-1157) from the spike protein of SARS-CoV, NP111 and NP351 assisted in the production of high-titer neutralizing antibodies in vivo. These data provide useful insights regarding immunity against SARS-CoV and have the potential to help guide the design of peptide-based vaccines.     

Localization of rotavirus VP4 neutralization epitopes involved in antibody-induced conformational changes of virus structure.

We previously characterized three neutralization-positive epitopes (NP1 [1a and 1b], NP2, and NP3) and three neutralization-negative epitopes on the simian rotavirus SA11 VP4 with 13 monoclonal antibodies (MAbs). Conformational changes occurred as a result of the binding of NP1 MAbs to the SA11 spike VP4, and enhanced binding of all neutralization-negative MAbs was observed when NP1 MAbs bound VP4 in a competitive MAb capture enzyme-linked immunosorbent assay. To further understand the structure and function of VP4, we have continued studies with these MAbs. Electron microscopic and sucrose gradient analyses of SA11-MAb complexes showed that triple-layered viral particles disassembled following treatment with NP1b MAbs 10G6 and 7G6 but not following treatment with NP1a MAb 9F6, NP2 MAb 2G4, and NP3 MAb 23. Virus infectivity was reduced approximately 3 to 5 logs by the NP1b MAbs. These results suggest that NP1b MAb neutralization occurs by a novel mechanism. We selected four neutralization escape mutants of SA11 with these VP4 MAbs and characterized them by using plaque reduction neutralization assays, hemagglutination inhibition assays, and an antigen capture enzyme-linked immunosorbent assay. These analyses support the previous assignment of the NP1a, NP1b, NP2, and NP3 MAbs into separate epitopes and confirmed that the viruses were truly neutralization escape mutants. Nucleotide sequence analyses found 1 amino acid (aa) substitution in VP8* of VP4 at (i) aa 136 for NP1a MAb mutant 9F6R, (ii) aa 180 and 183 for NP1b MAb mutants 7G6R and 10G6R, respectively, and (iii) aa 194 for NP3 MAb mutant 23R. The NP1b MAb mutants showed an unexpected enhanced binding with heterologous nonneutralization MAb to VP7 compared with parental SA11 and the other mutants. Taken together, these results suggest that the NP1b epitope is a critical site for VP4 and VP7 interactions and for virus stability.     

The crystal structure of nitrophorin 2. A trifunctional antihemostatic protein from the saliva of Rhodnius prolixus

Nitrophorin 2 (NP2) (also known as prolixin-S) is a salivary protein that transports nitric oxide, binds histamine, and acts as an anticoagulant during blood feeding by the insect Rhodnius prolixus. The 2.0-A crystal structure of NP2 reveals an eight-stranded antiparallel beta-barrel containing a ferric heme coordinated through His(57), similar to the structures of NP1 and NP4. All four Rhodnius nitrophorins transport NO and sequester histamine through heme binding, but only NP2 acts as an anticoagulant. Here, we demonstrate that recombinant NP2, but not recombinant NP1 or NP4, is a potent anticoagulant; recombinant NP3 also displays minor activity. Comparison of the nitrophorin structures suggests that a surface region near the C terminus and the loops between beta strands B-C and E-F is responsible for the anticoagulant activity. NP2 also displays larger NO association rates and smaller dissociation rates than NP1 and NP4, which may result from a more open and more hydrophobic distal pocket, allowing more rapid solvent reorganization on ligand binding. The NP2 protein core differs from NP1 and NP4 in that buried Glu(53), which allows for larger NO release rates when deprotonated, hydrogen bonds to invariant Tyr(81). Surprisingly, this tyrosine lies on the protein surface in NP1 and NP4.     

A Family of Novel DNA-Binding Nuclear Proteins Having Polypyrimidine Tract-Binding Motif and Arginine/Serine-Rich Motif

NP220 is a DNA-binding nuclear protein originally cloned from human cell lines. Human NP220 (hNP220) has an arginine/serine-rich motif found in non-small nuclear RNP splicing factors (SR proteins) and shares three domains (MH1, MH2 and MH3) with an acidic nuclear matrix protein, matrin 3. The MH2 domain repeats three times and has homology to the polypyrimidine tract-binding motif of heterogeneous nuclear RNP I/L. NP220 also has a DNA-binding domain and nine repeats of the sequence LVTVDEVIEEEDL (acidic repeat). We have now isolated mouse equivalents of NP220 (mNP220s) and found that NP220s form a family of proteins with four members produced by alternative splicing of a common pre-mRNA. Two longer forms (NP220α and NP220β) have all functional domains mentioned above while two shorter forms (NP220γ and NP220δ) lack the DNA-binding domain and the acidic repeat. The structural aspects of NP220s are distinct from that of the SR proteins but rather resemble U2AF and Tra2 which activate a specific 3′-splicing site of specific genes in response to differentiation-dependent signals.     

Rubredoxin refolding on nanostructured hydrophobic surfaces: Evidence for a new type of biomimetic chaperones

Rubredoxins (Rds) are small proteins containing a tetrahedral Fe(SCys)₄ site. Folded forms of metal free Rds (apoRds) show greatly impaired ability to incorporate iron compared with chaotropically unfolded apoRds. In this study, formation of the Rd holoprotein (holoRd) on addition of iron to a structured, but iron‐uptake incompetent apoRd was investigated in the presence of polystyrene nanoparticles (NP). In our rationale, hydrophobic contacts between apoRd and the NP surface would expose protein regions (including ligand cysteines) buried in the structured apoRd, allowing iron incorporation and folding to the native holoRd. Burial of the hydrophobic regions in the folded holoRd would allow its detachment from the NP surface. We found that both rate and yield of holoRd formation increased significantly in the presence of NP and were influenced by the NP concentration and size. Rates and yields had an optimum at “catalytic” NP concentrations (0.2 g/L NP) when using relatively small NP (46 nm diameter). At these optimal conditions, only a fraction of the apoRd was bound to the NP, consistent with the occurrence of turnover events on the NP surface. Lower rates and yields at higher NP concentrations or when using larger NP (200 nm) suggest that steric effects and molecular crowding on the NP surface favor specific “iron‐uptake‐competent” conformations of apoRd on the NP surface. This bio‐mimetic chaperone system may be applicable to other proteins requiring an unfolding step before cofactor‐triggered refolding, particularly when over‐expressed under limited cofactor accessibility. Proteins 2014; 82:3154–3162. © 2014 Wiley Periodicals, Inc.     

NMR studies of nitrophorin distal pocket side chain effects on the heme orientation and seating of NP2 as compared to NP1

The nitrophorins (NP) of the adult blood-sucking insect Rhodnius prolixus fall into two pairs based on sequence identity (NP1,4 (90%) and NP2,3 (79%)), which differ significantly in the size of side chains of residues which contact the heme. These residues include those in the distal pocket of NP2 (I120) and NP1 (T121) and the “belt” that surrounds the heme of NP2 (S40, F42), and NP1(A42, L44). To determine the importance of these residues and others conserved or very similar for the two pairs, including L122(123), L132(133), appropriate mutants of NP2 and NP1 have been prepared and studied by ¹H NMR spectroscopy. Wild-type NP2 has heme orientation ratio (A:B) of 1:8 at equilibrium, while wild-type NP1 has A:B ~ 1:1 at equilibrium. Another difference between NP2 and NP1 is in the heme seating with regard to His57(59). It is found that among the distal pocket residues investigated, the residue most responsible for heme orientation and seating is I120(T121). F42(L44) and L106(F107) may also be important, but must be investigated in greater detail.     

Identification of an RNA binding region within the N-terminal third of the influenza A virus nucleoprotein.

The influenza A virus nucleoprotein (NP) has been examined with regard to its RNA-binding characteristics. NP, purified from virions and devoid of RNA, bound synthetic RNAs in vitro and interacted with the ribonucleotide homopolymers poly(A), poly(G), poly(U), and poly(C) in a salt-dependent manner, showing higher binding affinity for polypyrimidine homopolymers. To map the NP regions involved in RNA binding, a series of deleted forms of the NP were prepared, and these truncated polypeptides were tested for their ability to bind poly(U) and poly(C) homopolymers linked to agarose beads. Proteins containing deletions at the N terminus of the NP molecule showed reduced RNA-binding activity, indicating that this part of the protein was required to bind RNA. To identify the NP region or regions which directly interact with RNA, proteins having the maltose-binding protein fused with various NP fragments were obtained and tested for binding to radioactively labeled RNAs in three different assays: (i) nitrocellulose filter binding assays, (ii) gel shift assays, and (iii) UV light-induced cross-linking experiments. A maltose-binding protein fusion containing the N-terminal 180 amino acids of NP behaved as an RNA-binding protein in the three assays, demonstrating that the N terminus of NP can directly interact with RNA. This NP region could be further subdivided into two smaller regions (amino acids 1 to 77 and 79 to 180) that also retained RNA-binding activity.     

Metabolic pathway of xenoestrogenic short ethoxy chain-nonylphenol to nonylphenol by aerobic bacteria, Ensifer sp. strain AS08 and Pseudomonas sp. strain AS90

Ensifer sp. strain AS08 and Pseudomonas sp. strain AS90 degrading short ethoxy (EO) chain-nonylphenol (NP) [NPEO_av2.0 containing NP mono- ∼ tetraethoxylates (NP1EO ∼ NP4EO); average 2.0 EO units] were isolated by enrichment cultures. Both strains grew on NP but not on octyl- and nonylphenol polyethoxylates (NPEOs) (average 10 EO units). Growth and degradation of NPEO_av2.0 was increased with increased concentrations of yeast extract (0.02–0.5%) in a culture medium. Culture supernatants of both strains grown on NPEO_av2.0 were analyzed by high-performance liquid chromatography, showing degradation of NP4EO–NP1EO. The metabolites from nonylphenol diethoxylate (NP2EO) by resting cells of both strains were identified by gas chromatography–mass spectrometry as nonylphenoxyethoxyacetic acid, NP1EO, nonylphenoxyacetic acid (NP1EC), and NP, while those from NP1EO were identified as NP1EC and NP. Cell-free extracts from strain AS08 grown on NPEO_av2.0 dehydrogenated NPEOs, NPEO_av2.0, NP2EO, NP1EO, and PEG 400, but the extracts were inactive toward di- ∼ tetraethylene glycol. Aldehydes were formed in the reaction mixture of each substrate with cell-free extracts. From these results, the aerobic metabolic pathway for short EO chain-NP is proposed: A terminal alcohol group of the EO chain is oxidized to a carboxylic acid via an aldehyde, and then one EO unit is removed. This process is repeated until NP is produced.English edition: The paper was edited by a native speaker through KN international ()     

Variation in microfilariae and infective stages of two types of Wuchereria bancrofti from the Thai-Myanmar border

Comparative morphometric and morphological studies of microfilariae and infective stages were undertaken in nocturnally periodic and subperiodic Wuchereria bancrofti. For microfilariae, the body dimensions of nocturnally periodic (NP) were significantly smaller than nocturnally subperiodic (NSP), i.e., body length 268.03+/-14.75 microm (NP), 307.61+/-11.52 microm (NSP); cephalic space length 4.21+/-0.62 microm (NP), 5.32+/-0.79 microm (NSP); head to nerve ring 49.39+/-5.43 microm (NP), 57.40+/-4.46 microm (NSP); innenk?rper length 33.05+/-5.89 microm (NP), 44.02+/-8.71 microm (NSP); cephalic space width 4.28+/-0.59 microm (NP), 6.04+/-0.68 microm (NSP); body width at nerve ring 5.01+/-0.57 microm (NP), 7.45+/-0.75 microm (NSP). The number of nuclei between the cephalic space and nerve ring of NP (66.67+/-5.19) was also significantly less than in NSP (94.74+/-6.95). For infective stages, the body dimensions of NP were significantly smaller than NSP, i.e., body length 1632.50+/-131.48 microm (NP), 2002.63+/-222.60 microm (NSP); head to nerve ring 103.09+/-7.47 microm (NP), 122.44+/-9.62 microm (NSP); head to oesophago-intestinal junction 567.69+/-94.84 microm (NP), 666.75+/-110.08 microm (NSP); body width at oesophago-intestinal junction 23.15+/-1.55 microm (NP), 26.78+/-1.62 microm (NSP). It is too early to infer the NP type as an additional sibling species of W. bancrofti but it is reasonable to treat it as a new variety and additional work is needed to clarify its status.     

Assignment of the 1H NMR resonances of protein residues in close proximity to the heme of the nitrophorins: similarities and differences among the four proteins from the saliva of the adult blood-sucking insect Rhodnius prolixus

The nuclear Overhauser effects (NOEs) observed between heme substituent protons and a small number of nearby protein side chain protons in the water-elimination Fourier transform NOE spectroscopy (WEFT-NOESY) spectra of high- and low-spin wild-type nitrophorin (NP) 2 and its ligand complexes have been analyzed and compared with those observed for the same complexes of wild-type NP3. These assignments were made on naturally abundant isotope samples, with the most useful protein side chains being those of Ile120, Leu122, and Leu132 for NP2 and NP3, and Thr121, Leu123, and Leu133 for NP1 and NP4. It is found that the NOEs observed are identical, with extremely similar protein side chain proton chemical shifts. This is strong evidence that the structure of NP3, for which no X-ray crystal structures are available, is essentially identical to that of NP2, at least near the heme binding pocket. Similarly, the NOEs observed between heme substituents and protein side chains for NP1 and NP4 also indicate that the structures of the protein having both A and B heme orientations are very similar to each other, as well as to the proteins with major B heme orientation of NP2 and NP3. These A and B connectivities can be seen, even though the two heme orientations have similar populations in NP1 and NP4, which complicates the analysis of the NOESY spectra. The histamine complex of wild-type NP2 shows significant shifts of the Leu132 side chain protons relative to all other ligand complexes of NP1-NP4 because of the perturbation of the structure near Leu132 caused by the histamine's side chain ammonium hydrogen bond to the Asp29 side chain carboxylate.     

CD24 is expressed specifically in the nucleus pulposus of intervertebral discs

Intervertebral disc (IVD) consists of a soft gelatinous material in its center, the nucleus pulposus (NP), bounded peripherally by fibrocartilage, annulus fibrosus (AF). Despite the number of patients with IVD degeneration, gene expression analysis has not been undertaken in NP and therefore little is known about the molecular markers expressed in NP. Here, we undertook a microarray screen in NP with the other nine tissues to identify the specific cell surface markers for NP. Five membrane associating molecules out of 10,490 genes were identified as highly expressing genes in NP compared with the other tissues. Among them, we identified CD24, a glycosylphosphatidylinositol (GPI) anchor protein as a cell surface marker for NP. CD24 expression was also detected in the herniated NP and chordoma, a malignant primary tumor derived from notochordal cells, while it was absent in chondrosarcoma. Therefore, CD24 is a molecular marker for NP as well as the diseases of IVD.     

Process for degradation of nitrobenzene: combining electron beam irradiation with biotransformation

Electron beam irradiations of aqueous solutions containing 15-30 mg/L of nitrobenzene at 60 kGy dose removed 78% of the contaminant. Three mononitrophenols were detected as by-products of electron beam treatment of nitrobenzene. A mixed culture enriched on a mixture of 2-, 3-, and 4-nitrophenol degraded both the residual nitrobenzene and the nitrophenol products. Percentage removal of nitrobenzene increased with increasing electron beam dose. This observation led to the conceptual design of a two-stage electron beam microbial process for degradation of nitrobenzene. Three groups of pure isolates were characterized from the mixed culture based on their abilities to grow on cor- responding nitrophenol substrates: Group A, 2NP(-)3NP(-)4NP(+); Group B, 2NP(+)3NP(+)4NP(-); and Group C, 2NP(-)3NP(+)4NP(-). Bacteria that grew on 3-NP transformed nitrobenzene into ammonia in the electron beam-treated nitrobenzene samples.     

Expression of pro-angiogenic cytokines and their inhibition by dexamethasone in an ex vivo model of nasal polyps

We used an organ culture of nasal polyps (NP) to provide ex vivo model to study the expression of pro-angiogenic cytokines and the effect of glucocorticoids in the pathogenesis of NP. Glucocorticoids are the drugs of choice for clinical treatment of NP; however, their mechanism of action is not fully understood. The cell-cell and cell-matrix integrity is well maintained in cultured NP. Expression of IL-6, IL-8, bFGF, GRO, and MCP-1 was increased in cultured NP compared to pre-cultured NP. Expression levels of IL-6, bFGF, and GRO in cultured NP were downregulated by dexamethasone (DEX) treatment, while MCP-1 expression was not suppressed. Further, RT-PCR and immunohistochemical analysis showed that HIF-1α and VEGF were suppressed in DEX-treated NP compared to untreated NP. Taken together, these results suggest that ex vivo organ culture can be considered a useful model to study the pathogenesis and regulation of pro-angiogenic cytokines in nasal polypogenesis.     

Alterations of bovine nucleus pulposus cells with aging

Aging is one of the major etiological factors driving intervertebral disc (IVD) degeneration, the main cause of low back pain. The nucleus pulposus (NP) includes a heterogeneous cell population, which is still poorly characterized. Here, we aimed to uncover main alterations in NP cells with aging. For that, bovine coccygeal discs from young (12 months) and old (10–16 years old) animals were dissected and primary NP cells were isolated. Gene expression and proteomics of fresh NP cells were performed. NP cells were labelled with propidium iodide and analysed by flow cytometry for the expression of CD29, CD44, CD45, CD146, GD2, Tie2, CD34 and Stro-1. Morphological cell features were also dissected by imaging flow cytometry. Elder NP cells (up-regulated bIL-6 and bMMP1 gene expression) presented lower percentages of CD29+, CD44+, CD45+ and Tie2+ cells compared with young NP cells (upregulated bIL-8, bCOL2A1 and bACAN gene expression), while GD2, CD146, Stro-1 and CD34 expression were maintained with age. NP cellulome showed an upregulation of proteins related to endoplasmic reticulum (ER) and melanosome independently of age, whereas proteins upregulated in elder NP cells were also associated with glycosylation and disulfide bonds. Flow cytometry analysis of NP cells disclosed the existence of 4 subpopulations with distinct auto-fluorescence and size with different dynamics along aging. Regarding cell morphology, aging increases NP cell area, diameter and vesicles. These results contribute to a better understanding of NP cells aging and highlighting potential anti-aging targets that can help to mitigate age-related disc disease.     

Grafting of a calcium-binding loop of thermolysin to Bacillus subtilis neutral protease

The surface loop which in the Bacillus subtilis neutral protease (NP) extends from amino acid residue 188 to residue 194 was replaced, by site-directed mutagenesis, with the 10-residue segment which in the homologous polypeptide chain of thermolysin (TLN) binds calcium-4 [Matthews, B. W., Weaver, L. H., & Kester, W. R. (1974) J. Biol. Chem. 249, 8030-8044]. The mutant NP was isolated to homogeneity, and its structural, functional, calcium-binding, and stability properties were investigated. Proteolytic fragmentation with Staphylococcus aureus V8 protease of mutant NP was used to isolate and analyze the protein fragment encompassing the site of mutation, unambiguously establishing the effective insertion of the new 10-residue segment. Atomic absorption measurements allowed us to demonstrate that mutant NP binds three calcium ions instead of the two ions bound to wild-type NP, showing that indeed the chain segment grafted from TLN to NP maintains its calcium-binding properties. The mutant NP showed kinetic parameters essentially similar to those of the wild-type NP with Z-Phe-Leu-Ala-OH as substrate. The enzyme inactivation of mutant vs wild-type NP was studied as a function of free [Ca2+]. It was found that mutant NP was much less stable than the wild-type NP when enzyme solutions were dialyzed at neutral pH in the presence of [Ca2+] below 10(-3) M. On the other hand, the kinetic thermal stability to irreversible inactivation of mutant NP, when measured in the presence of 0.1 M CaCl2, was found to be increased about 2-fold over that of the wild-type NP. Thus, modulation of enzyme stability by free [Ca2+] in mutant NP correlates with similar findings previously reported for thermolysin. Overall, the results obtained indicate that protein engineering experiments can be used to prepare hybrid proteins on the basis of sequence and function analysis of homologous protein molecules and show the feasibility of engineering metal ion binding sites into proteins.     

Determination of 4-nonylphenol, nonylphenol monoethoxylate, nonylphenol diethoxylate and other alkylphenols in fish and shellfish by high-performance liquid chromatography with fluorescence detection

A simple and highly sensitive method is described for the HPLC determination of 4-nonylphenol (NP), 4-nonylphenol mono- (NP1EO) and diethoxylates (NP2EO) in fish and shellfish together with bisphenol A (BPA), 4-tert.-butylphenol (BP) and 4-tert.-octylphenol (OP). The NP, NP1EO, NP2EO and other alkylphenols in the samples are extracted with acetonitrile and the lipid in the sample extract is eliminated by partitioning between hexane and acetonitrile. After Florisil PR clean-up the sample extract is analyzed by HPLC with a fluorescence detection. Recoveries in Japanese smelt, carp and corbicura are 81.8–84.3% for NP, 83.5–84.3% for NP1EO, 90.5–96.2% for NP2EO, 70.7–72.9% for BPA, 71.0–73.4% for BP and 77.1–83.2% for OP spiked at 0.5 μg each chemical per 5 g of the fish and shellfish samples. The detection limits are 2 ng/g for NP, NP1EO and NP2EO, and 1ng/g for BPA, BP and OP.     

Nuclear import and export of influenza virus nucleoprotein.

Influenza virus nucleoprotein (NP) shuttles between the nucleus and the cytoplasm. A nuclear localization signal (NLS) has been identified in NP at amino acids 327 to 345 (J. Davey et al., Cell 40:667-675, 1985). However, some NP mutants that lack this region still localize to the nucleus, suggesting an additional NLS in NP. We therefore investigated the nucleocytoplasmic transport of NP from influenza virus A/WSN/33 (H1N1). NP deletion constructs lacking the 38 N-terminal amino acids, as well as those lacking the 38 N-terminal amino acids and the previously identified NLS, localized to both the cytoplasm and the nucleus. Nuclear localization of a protein containing amino acids 1 to 38 of NP fused to LacZ proved that these 38 amino acids function as an NLS. Within this region, we identified two basic amino acids, Lys7 and Arg8, that are crucial for NP nuclear import. After being imported into the nucleus, the wild-type NP and the NP-LacZ fusion construct containing amino acids 1 to 38 of NP were both transported back to the cytoplasm, where they accumulated. These data indicate that NP has intrinsic structural features that allow nuclear import, nuclear export, and cytoplasmic accumulation in the absence of any other viral proteins. Further, the information required for nuclear import and export is located in the 38 N-terminal amino acids of NP, although other NP nuclear export signals may exist. Treatment of cells with a protein kinase C inhibitor increased the amounts of nuclear NP, whereas treatment of cells with a phosphorylation stimulator increased the amounts of cytoplasmic NP. These findings suggest a role of phosphorylation in nucleocytoplasmic transport of NP.     

Hypertension in response to CD4(+) T cells from reduced uterine perfusion pregnant rats is associated with activation of the endothelin-1 system

We have shown that adoptive transfer of CD4(+) T cells from placental ischemia (reduction in uteroplacental perfusion, RUPP) rats causes hypertension and elevated inflammatory cytokines during pregnancy. In this study we tested the hypothesis that adoptive transfer of RUPP CD4(+) T cells was associated with endothelin-1 activation as a mechanism to increase blood pressure during pregnancy. CD4(+) T cells from RUPP or normal pregnant (NP) rats were adoptively transferred into NP rats on gestational day 13. Mean arterial pressure (MAP) was analyzed on gestational day 19, and tissues were collected for endothelin-1 analysis. MAP increased in placental ischemic RUPP rats versus NP rats (124.1 ± 3 vs. 96.2 ± 3 mmHg; P = 0.0001) and increased in NP recipients of RUPP CD4(+) T cells (117.8 ± 2 mmHg; P = 0.001 compared with NP). Adoptive transfer of RUPP CD4(+) T cells increased placental preproendothelin-1 mRNA 2.1-fold compared with NP CD4(+) T cell rats and 1.7-fold compared with NP. Endothelin-1 secretion from endothelial cells exposed to NP rat serum was 52.2 ± 1.9 pg·mg(-1)·ml(-1), 77.5 ± 4.3 pg·mg(-1)·ml(-1) with RUPP rat serum (P = 0.0003); 47.2 ± .16 pg·mg(-1)·ml(-1) with NP+NP CD4(+) T cell serum, and 62.2 ± 2.1 pg·mg(-1)·ml(-1) with NP+RUPP CD4(+) T cell serum (P = 0.002). To test the role of endothelin-1 in RUPP CD4(+) T cell-induced hypertension, pregnant rats were treated with an endothelin A (ET(A)) receptor antagonist (ABT-627, 5 mg/kg) via drinking water. MAP was 92 ± 2 mmHg in NP+ET(A) blockade and 108 ± 3 mmHg in RUPP+ET(A) blockade; 95 ± 5 mmHg in NP+NP CD4(+) T cells+ET(A) blockade and 102 ± 2 mmHg in NP+RUPP CD4(+) T cells+ET(A) blockade. These data indicate the importance of endothelin-1 activation to cause hypertension via chronic exposure to activated CD4(+) T cells in response to placental ischemia.     

Primary structures of sperm-specific basic nuclear proteins and gene expression in Japanese newt,Cynops pyrrhogaster

Electrophoretic analyses of acid extracts from mature sperm of newt, Cynops pyrrhogaster, on acid/urea/Triton X-100 polyacrylamide gel showed the exclusive occurrence of sperm-specific nuclear basic proteins (SBPs), which moved faster than somatic histones on the gel. These SBPs were eluted separately by reversed phase-high-performance liquid chromatography as two large peaks and a few small peaks. Of these, only the small peaks disappeared with treatment of the acid extracts with alkaline phosphatase before they were injected into the column, so that there were only two distinct components: NP1 and NP2. Determination of amino acid sequences by the Edman method as well as by sequencing of cDNA for both components indicated that each protein consisted of 43 (NP1) or 48 (NP2) amino acid residues, rich in arginine residues (53.5% in NP1; 47.9% in NP2), forming the clusters. They had molecular masses of 5,386 Da (NP1) and 5,748 Da (NP2), respectively. Northern blot analysis using cDNAs as probes indicated that mRNAs for both NP1 and NP2 occurred not in primary spermatocytes but in round spermatids. In situ hybridization analyses using antisense RNA for NP1 as a probe clearly showed the first appearance of NP1 mRNA at the late stage of round spermatid. Mol. Reprod. Dev. 46:243–251, 1997. © 1997 Wiley-Liss, Inc.     

Intracellular proteolytic cleavage of the influenza virus protein NP as a sign of the epidemicity of virus strains?

The main nucleocapsid protein (NP) of human epidemic viruses was found to be cleaved via NP56----HP53 mol. wt. reduction in infected cells, while the NP of animal influenza viruses was refractory to analogous intracellular modification. Like animal influenza viruses, the strain A/Baku/799/82(H1N3) isolated from a sick child has been observed to exhibit the intracellular resistance of NP to intracellular proteolysis. The similar NP resistance has been revealed for A/New Jersey/8/76(H1N1) and A/seal/Massachusetts/81 (H7N7) viruses, which are able to induce only a sporadic human influenza viral infection. Thus, the results reveal a correlation between the viral strains epidemicity and intracellular cleavability of their NPs. The influenza viral strains epidemic for humans are characterized by cleavable NP, whereas the strains, which are known to induce the sporadic influenza human infection are found to exhibit the resistance of NP to intracellular proteolysis. It is reasonable to consider the phenomenon of NP56----NP53 proteolytic modification as a sign of viral strain epidemicity for humans.