Gamma gene rearrangement and expression in autoreactive helper T cells.

gamma gene rearrangements similar to those described for cytotoxic T cell lines are found in L3T4+, autoreactive, or KLH-specific cloned helper T cell lines. High levels of gamma RNA transcripts were, in addition, detected in four out of five L3T4+, class II MHC-specific, autoreactive T cell clones, and in at least one of three KLH-specific, class II MHC-restricted clones. This contrasts with previously reported (9) expression of gamma RNA in only 1 of 11 antigen-specific helper T cell lines.

T cell-specific gamma genes in C57BL/10 mice. Sequence and expression of new constant and variable region genes.

The T cell-specific gamma genes in C57BL/10 (B10) mice have been analyzed. Based on the cDNA sequences of these genes from antigen-specific MHC-restricted cytotoxic T cells, we found that the repertoire of these genes is not as limited as previously postulated (8). T cells from the B10 mice express an identical copy of V gamma J gamma C gamma (V gamma 10.8A-JC gamma 10.5) transcript previously found in T cells of BALB/c mice. In addition, a potentially functional mRNA using V gamma 10.8B and newly identified J gamma and C gamma gene segments were found. The new J gamma C gamma (JC gamma 10.8) is located 5' to the inverted V gamma 10.8B in the germline DNA of both B10 and BALB/c mice. This new C gamma is only 77 and 66% homologous to the C gamma 10.5 at the nucleotide and deduced protein sequences, respectively, thus making it a potential isotype of the C gamma genes reported previously. The V gamma 5.7, J gamma 2.3 gene segments and pseudogene C gamma 7.5 found in the germline DNA of BALB/c mice are absent in B10 mice. The loss of this gamma chain pseudogene in the B10 mouse strain, and the retention of all potentially functional V gamma, J gamma, and C gamma genes with highly conserved coding sequences supports the importance of these genes.

T cell receptor variable gene usage in a specific cytotoxic T cell response. Primary structure of the antigen-MHC receptor of four hapten-specific cytotoxic T cell clones.

The primary structure of the alpha and beta chains of the T cell antigen receptor in four cytotoxic T cell clones specific for N-iodoacetyl-sulfonic-naphthyl-ethylene-diamine (AED)-haptenated target cells displaying a particular class I MHC molecule has been determined. Two of the T cell clones, 8/10-2 and 5/10-20K, recognize AED-modified targets in association with H-2Kb, while the other two clones 5/10-20D and C9 react with AED-modified cells in the context of H-2Db. Comparison of the nucleotide sequences of both the alpha and beta chain cDNAs and their deduced protein sequences indicates that a specific variable gene segment was not used to recognize the hapten and/or class I gene products. Furthermore, there does not appear to be any conserved amino acid residues used in the AED-specific response other than the framework amino acids. However, when the two clones 8/10-2 and 5/10-20D were compared, a striking similarity was seen in the J segments. These two clones that recognize AED in the context of different MHC epitopes used identical J alpha (J alpha 810) and J beta (J beta 2.6) gene segments. C9, specific for AED-Db, shared identical V beta (V beta 6) and J beta gene segments (J beta 1.1) as those of a cytotoxic T cell that recognizes allogeneic targets expressing Db. These data indicate that a simple rule governing the usage of the variable regions of either the alpha or beta T cell receptor (TcR) genes in the recognition of antigen and MHC gene products cannot be formulated. However, subtle similarities can be detected in some situations between the primary structures of the TcR and the targets they recognize.

New type of asymmetric fission in proton-rich nuclei.

A very exotic process of ß-delayed fission of 180Tl is studied in detail by using resonant laser ionization with subsequent mass separation at ISOLDE (CERN). In contrast to common expectations, the fission-fragment mass distribution of the post-ß-decay daughter nucleus 180Hg (N/Z=1.25) is asymmetric. This asymmetry is more surprising since a mass-symmetric split of this extremely neutron-deficient nucleus would lead to two 90Zr fragments, with magic N=50 and semimagic Z=40. This is a new type of asymmetric fission, not caused by large shell effects related to fragment magic proton and neutron numbers, as observed in the actinide region. The newly measured branching ratio for ß-delayed fission of 180Tl is 3.6(7) × 10(-3)%, approximately 2 orders of magnitude larger than in an earlier study.

Proof-of-principle experiment for laser-driven cold neutron source.

The scientific and technical advances continue to support novel discoveries by allowing scientists to acquire new insights into the structure and properties of matter using new tools and sources. Notably, neutrons are among the most valuable sources in providing such a capability. At the Institute of Laser Engineering, Osaka, the first steps are taken towards the development of a table-top laser-driven neutron source, capable of producing a wide range of energies with high brightness and temporal resolution. By employing a pure hydrogen moderator, maintained at cryogenic temperature, a cold neutron ([Formula: see text]) flux of [Formula: see text]/pulse was measured at the proximity of the moderator exit surface. The beam duration of hundreds of ns to tens of [Formula: see text] is evaluated for neutron energies ranging from 100s keV down to meV via Monte-Carlo techniques. Presently, with the upcoming J-EPoCH high repetition rate laser at Osaka University, a cold neutron flux in orders of [Formula: see text] is expected to be delivered at the moderator in a compact beamline.

Predominant and developmentally regulated expression of dynamin in neurons.

We have cloned a cDNA for dynamin, a 100 kd microtubule-associated motor protein whose 5' region contains a GTP-binding motif homologous to that of the Mx proteins, from a rat brain library and analyzed its expression. Dynamin mRNA is 3.6 kb and is preferentially expressed in the brain after postnatal day 7, parallel to the developmental increase of the protein. In situ hybridization revealed high levels of dynamin transcripts in neural cells in the cerebellar cortex, hippocampus (particularly in the CA3 area), and cerebral cortex. The transcripts appeared in cerebellar granular cells only after they had ceased dividing and had migrated to the inner granular layer. We show that dynamin is expressed predominantly in neural cells after elongation of their processes, suggesting a role especially in mature neurons.

Assessing infrared intensity using the evaporation rate of liquid hydrogen inside a cryogenic integrating sphere for laser fusion targets.

Infrared (IR) heating processes have been studied to form a deuterium layer in an inertial confinement fusion target. To understand the relationship between the IR intensity and the fuel layering time constant, we have developed a new method to assess the IR intensity during irradiation. In our method, a glass flask acting as a dummy target is filled with liquid hydrogen (LH2) and is then irradiated with 2-µm light. The IR intensity is subsequently calculated from the time constant of the LH2 evaporation rate. Although LH2 evaporation is also caused by the heat inflow from the surroundings and by the background heat, the evaporation rate due to IR heating can be accurately determined by acquiring the time constant with and without irradiation. The experimentally measured IR intensity is 0.66 mW/cm2, which agrees well with a value estimated by considering the IR photon energy balance. Our results suggest that the present method can be used to measure the IR intensity inside a cryogenic system during IR irradiation of laser fusion targets.

Catalysis and energy coupling of H(+)-ATPase (ATP synthase): molecular biological approaches.

The molecular biological approach has provided important information for understanding the F0F1 H(+)-ATPase. This article focuses on our recent results on the catalytic site in the beta subunit, and the roles of alpha/beta subunit interaction and amino/carboxyl terminal interaction of the gamma subunit in energy coupling. Extensive mutagenesis of the beta subunit revealed that beta Lys-155, beta Thr-156, beta Glu-181 and beta Arg-182 are essential catalytic residues. beta Glu-185 is not absolutely essential, but a carboxyl residue may be necessary at this position. A pseudo-revertant analysis positioned beta Gly-172, beta Ser-174, beta Glu-192 and beta Val-198 in the proximity of beta Gly-149. The finding of the roles of beta Gly-149, beta Lys-155, and beta Thr-156 emphasized the importance of the glycine-rich sequence (Gly-X-X-X-X-Gly-Lys-Thr/Ser, E. coli beta residues between beta Gly-149 and beta Thr-156) conserved in many nucleotide binding proteins. The A subunits of vacuolar type ATPases may have a similar catalytic mechanism because they have conserved glycine-rich and Gly-Glu-Arg (corresponding to beta Gly-180-beta Arg-182) sequences. The results of these mutational studies are consistent with the labeling of beta Lys-155 and beta Lys-201 with AP3-PL, and of beta Glu-192 with DCCD [15]. The DCCD-binding residue of a thermophilic Bacillus corresponds to beta Glu-181, an essential catalytic residue discussed above. The defective coupling of the beta Ser-174-->Phe mutant was suppressed by the second mutation alpha Arg-296-->Cys, indicating the importance of alpha/beta interaction in energy coupling. The gamma subunit, especially its amino/carboxyl interaction, seems to be essential for energy coupling between catalysis and transport judging from studies on gamma Met-23-->Lys or Arg mutation and second-site mutations which suppressed the gamma Lys-23 mutation. Thus the conserved gamma Met-23 is not absolutely essential but is located in the important region for amino/carboxyl interaction for energy coupling.

Purification and characterization of a novel substrate for plasma kallikrein (PK-120) in human plasma.

A 120 kDa plasma protein, which is susceptible to plasma kallikrein, was purified from human plasma by polyethylene glycol fractionation followed by ion exchange chromatography using Q-Sepharose, S-Sepharose, and hydroxyapatite and gel filtration on Sephacryl S-200. The 120 kDa protein, termed PK-120 in this paper, was a single polypeptide chain containing about 20% sugar by weight and its concentration in plasma was estimated to be 80 micrograms/ml by ELISA. At least three fragments, 100, 70, and 35 kDa, were produced from PK-120 by plasma kallikrein. The N-terminal sequence and Western blot demonstrated that PK-120 was first cleaved to yield the 100 and 35 kDa fragments, then the 100 kDa fragment was cleaved into the 70 kDa fragment. N-Terminal sequence analyses of PK-120 and its fragments demonstrated that it is a novel plasma protein, distinct from high molecular weight kininogen, a natural substrate for plasma kallikrein.

Dual phasic suppression of viral replication following de novo human immunodeficiency virus type 1 (HIV-1) infection in lymphocytes of asymptomatic HIV-1 carriers.

Replication of human immunodeficiency virus type 1 (HIV-1) is suppressed in asymptomatic HIV-1 carriers (ACs). By using an in vitro experimental system, the mechanism of this suppression was investigated. Following in vitro infection of a laboratory HIV-1 strain, the peripheral blood mononuclear cells (PBMC) of ACs transiently supported a low level of viral replication, then the virus production rapidly decreased. PCR analysis revealed that HIV-1 proviral DNA integrated in the PBMC of ACs following infection gradually decreased. Such tapering consequences of in vitro HIV-1 infection in the PBMC of ACs were abrogated by depletion of CD8+ T cells from the culture. Furthermore, the viruses subsequently produced by the PBMC of an AC were less able to replicate than the virus produced by CD8+ cell-depleted PBMC of the same donor. These observations suggested that the CD8+ T cell-mediated suppression of HIV-1 replication in ACs may involve both cytocidal and cytostatic mechanisms: the former kills the cells producing viruses, and the latter inhibits viral spread by reducing viral infectivity.

The gene expression level of transforming growth factor-beta (TGF-beta) as a biological prognostic marker of hepatocellular carcinoma.

The question that transforming growth factor-beta (TGF-beta) provides a tumor-suppressive or a tumor promoting role is still unknown in hepatocellular carcinoma (HCC). In the present study, we quantitatively investigated the gene expression levels of TGF-beta in liver tissues from patients with HCC. We also evaluated the prognostic importance of TGF-beta gene in HCC patients. A total of 59 patients with primary HCC who underwent hepatectomy between 1993 and 2001 were enrolled. TGF-beta gene expression levels of tumors and of noncancerous livers were analyzed by real-time reverse transcriptase polymerase chain reaction (RT-PCR). The percentage of apoptotic cells in tumor cells (apoptotic index: AI) was evaluated with immunohistochemistry. Also the expression of survivin protein (apoptosis inhibitor) in tumors was detected by immunohistochemistry. TGF-beta gene expression levels of tumors were compared with clinicopathological findings of patients. The relative expression level of TGF-beta mRNA of 59 tumor tissues did not differ from those of 8 normal liver tissues or 59 noncancerous liver tissues. The mean AI of 29 tumors with normal expression levels of TGF-beta gene (4%) was significantly higher than that of 30 tumors with low expression levels of TGF-beta gene (2.5%, p = 0.03). Thirteen out of 30 tumors (43%) with low expression level of TGF-P gene showed survivin positive, while only 4 out of 29 tumors (14%) with preserved expression of TGF-beta gene showed survivin positive. This difference was significant (p = 0.012). The overall 5-year survival rate of 29 patients with tumors with preserved TGF-beta gene prolonged to 72% compared with that of 30 patients who had tumors with suppressed TGF-beta gene (58%, p = 0.156). In HCC, TGF-beta gene may play a defensive role against tumor progression by regulating survivin protein expression and by controlling occurrence of spontaneous apoptosis in tumors.

Quantitative and qualitative abnormalities in HIV-1-specific T cells.

OBJECTIVE: To assess the characteristics of CD4 and CD8 T cells specific for HIV-1 and cytomegalovirus (CMV) antigens in untreated and treated HIV-1-infected patients. METHODS: Antigen-specific T cell frequencies were determined by flow cytometric detection of antigen-induced intracellular cytokines. RESULTS: In untreated patients, HIV-1-specific CD4 T cell counts in peripheral blood were less than one tenth of CMV-specific CD4 T cell counts, while the number of specific CD8 T cells was approximately the same for both HIV-1 and CMV. In patients treated with highly active antiretroviral therapy (HAART) for less than 1.5 years, HIV-1-specific CD4 and CD8T cell counts were significantly lower than those in untreated patients. Perforin expression in HIV-1-specific CD8 T cells was significantly lower than that in CMV-specific CD8 T cells. CONCLUSION: These data indicate that HIV-1-specific T cells in HIV-1-infected patients have quantitative and qualitative abnormalities.

Cytomegalovirus (CMV) retinitis and CMV antigenemia as a clue to impaired adrenocortical function in patients with AIDS.

OBJECTIVE: To elucidate the relationship between the activity of CMV disease and adrenocortical function in patients with AIDS. DESIGN AND PATIENTS: CMV retinitis and CMV antigenemia assay (CMV-Ag: numbers of polymorphonuclear leukocytes positive for CMV pp65 antigen per 1.5 x 10(5) cells) are the least invasive and easily accessible examinations to assess the CMV disease activity. All HIV-infected patients with CD4+ lymphocyte counts < 50 x 10(6)/l who were admitted to the Research Hospital of the Institute of the Medical Science (University of Tokyo) between May 1995 to April 1996 were included in this study. METHODS: Fundoscopic examination on CMV retinitis and CMV-Ag were chosen as methods to assess CMV activity because of their simplicity. Adrenocortical function was evaluated by basal plasma adrenocorticotropin, plasma cortisol, plasma aldosterone, plasma renin activity, and responses of plasma cortisol and plasma aldosterone to 250 micrograms intravenous cosyntropin [rapid adrenocorticotropin test (RAT)]. RESULTS: Thirty patients were enrolled in this study with a maximum CD4+ lymphocyte count of 32 x 10(6)/l. Eleven out of 30 patients showed impaired RAT response (37%). Fourteen out of 30 patients had CMV retinitis. A significant correlation was found between the presence of CMV retinitis and subnormal cortisol response (P < 0.005). Sixteen out of the 30 patients were CMV-Ag-positive. A significant correlation was found between CMV-Ag positivity and subnormal cortisol response to RAT (P < 0.005). CMV-Ag levels in the patients with subnormal cortisol response to RAT were significantly higher than those with normal response (P < 0.001). Importantly, five patients with subnormal cortisol response but not overt adrenal insufficiency at the time of RAT developed overt disease shortly afterwards. Autopsy was performed in six patients with subnormal cortisol response and showed multiple inclusion bodies indicative of CMV adrenitis. CONCLUSION: The adrenal gland is most frequently affected by CMV in AIDS patients. Our result suggests that CMV retinitis or CMV-Ag positivity independently serve as an indication of possible adrenal dysfunction.

N-ethylmaleimide-sensitive mutant (beta Val-153-->Cys) Escherichia coli F1-ATPase: cross-linking of the mutant beta subunit with the alpha subunit.

A beta subunit mutation, beta Val-153-->Cys, in the glycine-rich sequence (phosphate-binding loop) of Escherichia coli F1 was constructed. Like vacuolar-type ATPase, the mutant enzyme was inhibited by N-ethylmaleimide (NEM) and labeled with [14C]NEM. The inhibition and labeling were prevented by ATP. m-Maleimidobenzoyl-N-hydroxysuccinimide (MBS) (3 microM) almost completely inhibited the mutant enzyme, and cross-linked one pair of alpha and beta subunits. These results suggest that the interaction of the domain near beta Val-153 with the alpha subunit is essential for catalytic cooperativity of the enzyme and that beta Val-153 is within 10 A of the alpha subunit.

Identification of multiple HIV-1 CTL epitopes presented by HLA-B*5101 molecules.

We attempted to identify and characterize HIV-1 CTL epitopes presented by HLA-B51 which is associated with a slow progression to AIDS. HLA-B*5101 stabilization assay showed that 33 out of 172 HIV-1 peptides carrying HLA-B*5101 anchor residues bound to HLA-B*5101. Seven peptides were suggested as HIV-1 CTL epitopes presented by HLA-B*5101 because the specific CTL was induced for these peptides in PBMC from three HIV-1 seropositive individuals carrying HLA-B51 by stimulation with HLA-B*5101 binding peptides. Analysis of these epitopes using the specific CTL clones confirmed that six of seven HIV-1 peptides are epitopes presented by HLA-B*5101. Three epitopes presented by HLA-B*5101 are highly conserved among the clade B strain, suggesting that the specific CTL for these epitopes might play an important role in recognition of HIV-1 infected cells. These epitopes will be useful to analyze CTL responses in HIV-1 infected individuals.

Expression of a novel Nef epitope on the surface of HIV type 1-infected cells.

We compared antibody responses to various structural and accessory gene products of HIV-1 between long-term nonprogressors and patients who have progressed to AIDS. On the basis of our results, we performed epitope mapping of the Nef protein and identified a novel epitope. This novel epitope of the Nef protein was found to be exposed on the surface of HIV-1-infected cells. The antibody response against it correlated with CD4+ cell counts among HIV-1-infected patients (r = 0.457, p < 0.001). Although further research is necessary, we suspect that antibody response against the epitope may be protective against disease progression.

Accumulation of specific amino acid substitutions in HLA-B35-restricted human immunodeficiency virus type 1 cytotoxic T lymphocyte epitopes.

HLA is one of the genetic factors that influence the clinical course of HIV-1 infection, and patients with HLA-B35 are prone to rapid disease progression. Nine viral epitopes that are recognized by cytotoxic T lymphocytes (CTLs) in an HLA-B35-restricted manner were determined. To examine how HIV-1 sequences are selected by CTLs in vivo, we sequenced the nine CTL epitopes of the virus in patient plasma. Here we show that certain amino acid substitutions at three epitopes were observed with significantly higher frequency in HLA-B35-positive patients than in HLA-B35-negative patients. By performing experiments with CTL clones established from the HLA-B35-positive patients, it was determined that one of the three substitutions was probably an escape mutation. However, concerning the other two epitopes, representative CTL clones killed target cells pulsed with mutant peptides as efficiently as those pulsed with wild-type peptides, suggesting that CTLs that can be established in vitro are not functioning properly in vivo. Amino acid sequence drift in all HLA-B35-restricted epitopes was rare during the observation period (1 year). Our results may have relevance in understanding the rapid clinical progression in HLA-B35-positive patients.

HIV type 1 V3 variation dynamics in vivo: long-term persistence of non-syncytium-inducing genotypes and transient presence of syncytium-inducing genotypes during the course of progressive AIDS.

We performed a population-based sequence analysis of the envelope V3 region of human immunodeficiency virus type 1 (HIV-1) in two infected hemophiliacs. The study was conducted over 6-9 years, extending from the asymptomatic phase to AIDS. In both patients, serial analysis showed that the V3 population at the initial stage consisted exclusively of putative non-syncytium-inducing (NSI) genotypes. Several of these clones continued to be present without change for many years until the terminal stage and often represented the dominant species in the population at each time interval. On the other hand, syncytium-inducing (SI) genotypes were initially absent but appeared shortly before severe depletion of CD4+ T cells and their proportion in the population appeared to correlate with the viral load. In sharp contrast to NSI genotypes, SI genotypes displayed a significantly shorter presence. Thus, rapid gross population changes were found in SI genotypes, which were particularly frequent in the asymptomatic phase and less frequent in the terminal stage. Furthermore, the ratio of nonsynonymous nucleotide substitutions per synonymous substitutions in the V3 region in SI genotypes was higher than the corresponding value of NSI genotypes and the phylogenetic tree analysis revealed that a longer branch length was observed in SI genotypes than in NSI genotypes. These results suggest that there might be a stronger pressure for selection on SI viruses than on NSI viruses during the high CD4 counts on the contrary to the fact that emergence of SI genotypes was well correlated with the rapid decline of CD4 count.

Elevated plasma stromal cell-derived factor 1 protein level in the progression of HIV type 1 infection/AIDS.

Stromal cell-derived factor 1 (SDF-1) is a unique chemokine involved in multiple organogenesis as well as in the regulation of HIV infection. Here we determined the plasma SDF-1 concentrations of 193 HIV-1-infected individuals and 154 normal Japanese volunteers by developing a highly sensitive measurement system based on time-resolved fluoroimmunoassay (SDF-1 TR-FIA). This system is also valid for the mouse model to quantitate circulating SDF-1 concentration in vivo and thereby its correlation with CXCR4 expression level on CD4(+) T cells. Interestingly, plasma SDF-1 concentrations in HIV-1-infected individuals were three times higher than those in a normal control group and plasma SDF-1 protein levels showed an inverse correlation with CD4(+) T cell count and a positive correlation with plasma HIV-1 RNA load. Notably, individuals with later stage HIV-1 infection, who maintained fewer than 200 CD4(+) T cells per cubic milliliter and more than 10,000 copies of HIV-1 RNA per milliliter, showed the highest plasma SDF-1 level among individuals at any stage of HIV-1 infection. These results suggest that endogenous SDF-1 is upregulated by HIV-1 infection, particularly in late-stage HIV-1 infection/AIDS.