Ir(iii) complex-based oxygen imaging of living cells and ocular fundus with a gated ICCD camera.

Phosphorescence lifetime imaging methods using oxygen-sensitive probes are very useful for visualizing the oxygen status of living cells and tissues with high spatial resolution. We aim to develop a useful oxygen detection technique combining a phosphorescent oxygen probe and an optimal detection method. Herein we present a biological oxygen imaging method using a microscope equipped with a gated intensified charge-coupled device (ICCD) camera as a detector and an Ir(iii) complex as a phosphorescent oxygen probe. Microscopic luminescence images of monolayer HT-29 cells (human colorectal adenocarcinoma cells) obtained using the cell-penetrating Ir(iii) complex BTPDM1 and an inverted microscope demonstrated that this method allowed visualization of the oxygen gradient produced in a monolayer of cultured cells when the monolayer is covered with a thin coverslip. Furthermore, combining the IR-emitting Ir(iii) complex DTTPH-PEG24 with a macrozoom microscope equipped with a gated ICCD camera enabled both the visualization of retinal vessels near the optic disc and the monitoring of oxygen level changes in a rabbit retina upon changing the inhaled oxygen content.

Cytological criteria of endometrial lesions with emphasis on stromal and epithelial cell clusters: result of 8 years of experience with intrauterine sampling.

OBJECTIVE: There are a number of unresolved issues in endometrial cytology. They include the significance of nuclear atypia for the diagnosis of grade1 adenocarcinoma (G1AC) and atypical endometrial hyperplasia (AEH), cytological criteria of endometrial hyperplasia without atypia, and recognition of stromal cell cluster (SC) and its distinction from epithelial cell cluster (EC). METHODS: We examined nuclear atypia, SC and EC in typical cases of five categories: normal endometrium (NEM), simple endometrial hyperplasia without atypia (SEH), complex endometrial hyperplasia without atypia (CEH), G1AC and grade2 adenocarcinoma (G2AC). We classified EC into four types: simple EC (SPEC), large regular EC (LREC), large irregular EC (LIEC) and small irregular EC (SIEC). Based on the results, we developed criteria of endometrial cytology and have evaluated 13 639 cases over 8 years. RESULTS: Nuclear atypia was significantly more frequent in G2AC than in any of the other four categories (P < 0.001). SC was significantly more frequent in NEM and SEH than in the other three categories (P < 0.001). G1AC and G2AC showed significantly higher frequency of LIEC than the other three categories (P < 0.001). CEH exhibited significantly higher frequency of LREC than the four categories (P < 0.001). The sensitivity and the specificity was 88.8% and 99.0% respectively. CONCLUSIONS: We could diagnose G1AC, G2AC and CEH with high accuracy using the established criteria mainly based on SC and EC. We think that the criteria may facilitate an effective screening and an objective interpretation of endometrial samples.

Monoclonal antibody against human gallbladder carcinoma-associated antigen.

Monoclonal antibody HI-531 of immunoglobulin G2b subclass was produced against a human gallbladder carcinoma cell line. HI-531 was investigated for reactivity with a panel comprising ten types of different origin in fluorescence-activated cell sorter analysis. The antibody reacted with the gallbladder carcinoma cell line G-415 used for immunization and with four unrelated tumors. HI-531 was further shown, with the use of the avidin-biotin complex-immunoperoxidase technique and surgically resected tissues, to be strongly reactive with carcinoma of the gallbladder, pancreas, bile duct, and gastrointestinal tract. The antibody was reacted with several types of normal epithelial cells but often more weakly expressed than on corresponding tumors. One of six fetal lung tissues was weakly stained. All other fetal organ tissues tested showed negative staining reactions. These observations suggest that HI-531 may be of value in identifying the tumor-associated antigen expressed in gallbladder carcinoma. HI-531 immunoprecipitated the Mr 43,000 molecule from extracts of Na125I- or [35S]methionine-labeled tumor cells, but not from those of [3H]glucosamine-labeled tumor cells. In addition, cytofluorometric analysis showed that cells treated with trypsin or protease greatly decreased a reactivity to the antibody. The findings suggest that the antibody recognizes a Mr 43,000 protein molecule. Sequential immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis studies and analyses by nonequilibrium pH gradient and polyacrylamide gel electrophoreses showed that the Mr 43,000 molecule defined by HI-531 was not a Mr 43,000 heavy chain of HLA-A,B,C antigens detected by monoclonal antibody W6/32.

Sequence and functional properties of African green monkey CD4 silencer.

We reported previously that in African green monkey (AGM) CD4 lymphocytes, CD4 mRNA expression undergoes a decrease following in vitro activation, and CD4 cells are therefore subject to loss of CD4 expression on the cell surface. To examine the transcriptional regulation of the CD4 gene in this species. we analyzed the CD4 silencer, which has been identified as a regulatory element responsible for the down regulation of CD4 transcription in CD8 cell lineage cells. Sequence analysis indicated that the CD4 silencer of the AGM was highly homologous to that of humans. However, two nucleotide substitutions were present in one of the nuclear protein binding sites, which was characterized as the FP II site having a strong enhancing effect on transgene expression in CD4 cells. By performing transient transfection assays. we found that the enhancing activities of the CD4 silencer or FP II-containing fragment of the AGM were greatly reduced in a human CD4 cell line as compared to those of human materials. The CD4 mRNA level was significantly decreased in the human CD4 cell line when synthetic oligonucleotide corresponding to the human FP II sequence was added to the culture. These observations imply that FP II-protein interaction might be required for the maintenance of sufficient expression of the CD4 gene, and the enhancing activity mediated by the above interaction might be decreased in the AGM CD4 silencer, due probably to the nucleotide changes occurring at the FP II site.

Molecular characterization of three HLA class II molecules on DR4 and DRw9 haplotypes: serologic and structural relationships at the polypeptides level.

By using alloantisera, three distinct HLA-D/DR region-encoded class II molecules were identified from cells carrying the HLA-DR4 and DRw9 haplotypes. Both DRw-53 and DQw3 molecules that bear the "supertypic" specificity were isolated independently from the DR antigen. The light chains of the DR4 antigens from different HLA-D types were distinct from one another, whereas the DRw53 molecules had identical charge and molecular weight in both heavy and light chains. On the other hand, the DQw3 molecules from the DR4 cell lines (Dw4 and Dw 10) were apparently identical but were polymorphic at least in the light chains among the DR4, DR5, and DRw9 haplotypes. In addition, monoclonal antibodies which specifically precipitate DR4 and DQw3 molecules have been isolated. The variable extent of homogeneity and diversity of three class II molecules may aid in our understanding of the role of class II antigens in the human immune regulation.

Simian T cell leukemia virus type I-induced malignant adult T cell leukemia-like disease in a naturally infected African green monkey: implication of CD8+ T cell leukemia.

Spontaneous T cell leukemia was found in an African green monkey (Cercopithecus aethiops, AGM) naturally infected with simian T cell leukemia virus type I (STLV-I). The hematological features and the evidence for monoclonal integration of provirus DNA in the leukemic cells revealed that the leukemia was an ATL-like disease. The expression of surface markers on the leukemic cells indicated that they were defined as an activated CD8+ T cell subset. Together with the finding that seven in vitro spontaneously STLV-I-transformed cell lines were CD4-CD8+, it is likely that CD8+ T cells are transformed by STLV-I in AGMs, in contrast with human ATL. Finally, we assessed characteristics of the CD8 chains on these transformed cells. The result indicated that the leukemic cells expressed only the alpha chains but not the beta chains. However, in the case of in vitro-transformed cell lines the expression pattern of the CD8 chains varied in individual monkeys. Thus, STLV-I may preferentially transform CD8+ (both alphaalpha+ and alphabeta+) T cells in AGMs.

Effects of cerulein on the normal pancreas and on experimental pancreatic carcinoma in the Syrian golden hamster.

The effects of cerulein on normal pancreas and on N-nitrobis (2-hydroxypropyl) amine (BHP)-induced experimental pancreatic carcinoma in Syrian golden hamsters were studied. Twenty hamsters received a subcutaneous injection of cerulein (20 micrograms/kg). The results showed that when cerulein was injected subcutaneously for 10 days, pancreatic weight and amylase increased. DNA and the pancreatic weight/DNA ratio were also increased significantly in treated hamsters compared with controls (p less than 0.02 versus p less than 0.01). These results indicated that chronic cerulein injection had hypertrophic and hyperplastic effects. DNA synthesis, as measured by histoautoradiography of tritiated thymidine-labeled tissue, increased in pancreatic acinar cells (p less than 0.01) and increased slightly in islet cells and in ductal cells. Tritiated thymidine uptake in the pancreas of the treated group indicated a rather selective exocrine gland incorporation by acinar rather than ductal cells. Sixty hamsters received a subcutaneous injection of BHP (500 mg/kg) once a week, while 63 hamsters received BHP (500 mg/kg) plus cerulein (20 micrograms/kg). Twenty-seven hamsters received cerulein (20 micrograms/kg) alone. All animals were killed from 8 to 27 weeks later, and no cancer-bearing hamsters were observed during the eighth and ninth week following administration. From the 10th to 14th weeks after administration of BHP and cerulein, 87.9% (13 of 15) had tumors compared with 18.7% (3 of 16) after BHP alone (p less than 0.01). One of three and two of 13 tumors were adenoma.(ABSTRACT TRUNCATED AT 250 WORDS)

Antiviral effects of 6-chloro-2',3'-dideoxyguanosine in rhesus monkeys acutely infected with simian immunodeficiency virus.

A lipophilic dideoxynucleoside analogue, 6-chloro-2',3'-dideoxyguanosine (6-Cl-ddG), was expected to be effective against AIDS-related dementia. In this study, we tested the effect of 6-Cl-ddG on simian immunodeficiency virus (SIVmac239) replication in vitro and on acute infection of six rhesus monkeys (Macaca mulatta) with SIVmac239. This compound inhibited SIV-induced cytopathic effect in CEM x 174 cells and SIV replication in vitro with an ED50 value of 2.5 microM. A dose of 25 mg/kg 6-Cl-ddG was administered to three monkeys every 8 h for 10 days and an untreated group of three monkeys was injected with the solvent without drug. Although 6-Cl-ddG was not detected in the plasma, the metabolite ddG was maintained at a concentration of more than 3 microM for 8 h after administration. In the cerebrospinal fluid, the ddG concentration was 2 microM at 2 h after administration. SIV antigen (p27) and antibody appearance in the plasma were delayed for 5-8 days compared with the mock-treated group. The occurrence of lymphadenopathy in treated monkeys was delayed for 6 days compared with the mock-treated group. Signs of 6-Cl-ddG toxicity were minimal after the treatment. The results of this study provide further evidence that 6-Cl-ddG may act as a potent anti-human immunodeficiency virus agent in vivo.

Adaptive acquisition and tracking for deep space array feed antennas.

The use of radial basis function (RBF) networks and least squares algorithms for acquisition and fine tracking of NASA's 70-m-deep space network antennas is described and evaluated. We demonstrate that such a network, trained using the computationally efficient orthogonal least squares algorithm and working in conjunction with an array feed compensation system, can point a 70-m-deep space antenna with root mean square (rms) errors of 0.1-0.5 millidegrees (mdeg) under a wide range of signal-to-noise ratios and antenna elevations. This pointing accuracy is significantly better than the 0.8 mdeg benchmark for communications at Ka-band frequencies (32 GHz). Continuous adaptation strategies for the RBF network were also implemented to compensate for antenna aging, thermal gradients, and other factors leading to time-varying changes in the antenna structure, resulting in dramatic improvements in system performance. The systems described here are currently in testing phases at NASA's Goldstone Deep Space Network (DSN) and were evaluated using Ka-band telemetry from the Cassini spacecraft.

Detection of B virus infection in cynomolgus monkeys by ELISA using simian agent 8 as alternative antigen.

The use of simian agent 8 (SA8) as an antigen for B virus (BV) antibody detection was evaluated in cynomolgus monkeys. Seventy-two sera judged as positive using BV antigen were all positive when the SA8 antigen was used. Out of 28 BV-negative sera 2 were positive against the SA8 antigen and one serum was classified as indeterminate. The present data indicates that detection of BV antibody can be achieved accurately and safely by enzyme-linked immunosorbent assay (ELISA) using SA8 antigen.

Efficacy of 6-chloro-2',3'-dideoxyguanosine (6-Cl-ddG) on an ARC/AIDS rhesus macaque (Macaca mulatta) infected with simian immunodeficiency virus.

The efficacy of 6-chloro-2',3'-dideoxyguanosine (6-Cl-ddG) was investigated in vivo by using a male ARC/AIDS rhesus macaque infected with simian immunodeficiency virus (SIVmac251/32H). He was administered subcutaneously 6-Cl-ddG (50 mg/kg B.W.) every 8 hr for 14 days when he showed clinical features of recurrent weight loss, severe diarrhea and neuropathy. The number of CD4+, CD8+ cells and total T cells increased rapidly after administration of 6-Cl-ddG and a high level was maintained for 2 months, but the B cell count decreased during the treatment. The antibody titer to SIV did not change significantly during or after the treatment, but the virus load in the plasma measured by RT-PCR dropped to one-third at the start of the 6-Cl-ddG treatment. Within 3 days after the start of 6-Cl-ddG administration, he began to show recovery in clinical signs including weight increase, and disappearance of diarrhea and neuropathy. These findings suggested that 6-Cl-ddG was effective at the stage of ARC/AIDS in a rhesus monkey infected with SIV.

Effects of 6-chloro-2',3'-dideoxyguanosine (6-Cl-ddG) in surface lymph nodes of rhesus monkeys (Macaca mulatta) chronically infected with simian immunodeficiency virus (SIVmac239).

We studied the effects of 6-chloro-2',3'-dideoxyguanosine (6-Cl-ddG), an antiretroviral drug, in surface lymph nodes of rhesus monkeys (Macaca mulatta) chronically infected with simian immunodeficiency virus (SIV). The rhesus monkeys were treated with 25 mg/kg of 6-Cl-ddG every 8 hr for 2 weeks. We performed sequential biopsies of the surface lymph nodes three times: before, during, and after the drug treatment. The 6-Cl-ddG dramatically decreased the number of infectious virus (measured by limiting dilution assay) in lymph node mononuclear cells. This decrease was consistent with the decrease in the number of viral RNA-positive cells in lymph nodes (analyzed by in situ hybridization). Histopathological analysis revealed that hyperplastic lymphoid follicles were reduced in size, especially, enlarged areas of centroblasts in lymphoid follicles (the so-called dark areas of germinal centers) were declined. Our results demonstrated that 6-Cl-ddG decreased the viral burden concomitantly with reduced hyper-activation of germinal centers in lymphoid follicles of SIV-infected rhesus monkeys.

[Trophic effect of caerulein on the normal pancreas and enhancement in experimental pancreatic cancer by caerulein in the Syrian golden hamster].

The author investigated the hyperplastic and hypertrophic effect of caerulein in the pancreas of normal syrian golden hamsters, and the promoting effect of experimental pancreatic cancer in hamster induced by N-nitroso-bis (2-hydroxypropyl) amine (BHP). The results are as follow: Repeated subcutaneous injections of caerulein in every 12 hours for 10 days elicited a marked trophic effect on the pancreas, characterized by increased pancreatic weight and pancreatic weight/DNA ratios with an enhanced content of DNA and amylase in the pancreas in treated hamsters. DNA synthesis, as measured by histoautoradiography of tritiated thymidine labeled tissues, was increased in pancreatic acinar but little in islet nor in ductal cells. Weekly subcutaneous administration of BHP with caerulein brought pancreatic carcinomas earlier and in higher incidence than BHP alone. The majority of induced carcinomas were well differentiated adenocarcinomas, and acinar cell carcinoma was seen in neither groups. In addition, a further investigation was performed in search target cells of both BHP and caerulein. Repeated injections of caerulein in every 12 hours for 5 days before one shot of subcutaneous BHP administration led to increase both mitotic and labeling index using tritiated thymidine in most acinar cells. These results suggested that caerulein has a trophic action on the pancreas, and acts as a promotor in experimental pancreatic carcinoma.

HTLV-1 HBZ positively regulates the mTOR signaling pathway via inhibition of GADD34 activity in the cytoplasm.

Human T-cell leukemia virus type-1 (HTLV-1) infection causes adult T-cell leukemia (ATL). Modulation of the transcriptional control of cellular genes by HTLV-1 is thought to be associated with the development of ATL. The viral protein HTLV-1 basic leucine-zipper factor (HBZ) has been shown to dysregulate the activity of cellular transcription factors. Here, we demonstrate that HBZ is exported from the nucleus to the cytoplasm, where it activates the mammalian target of rapamycin (mTOR) signaling pathway through an association with growth arrest and DNA damage gene 34 (GADD34). The N-terminal region of HBZ interacts with the C-terminal region of GADD34. HBZ contains a functional nuclear export signal (NES) sequence within its N-terminal region and it is exported from the nucleus via the CRM1-dependent pathway. Nuclear export of HBZ is essential for its interaction with GADD34 and increased phosphorylation of S6 kinase, which is an established downstream target of the mTOR pathway. Starvation-induced autophagy is significantly suppressed by the overexpression of HBZ. These findings indicate that HBZ is actively exported to the cytoplasm, where it dysregulates the function of cellular factors.

In vitro system that synthesizes circular viral DNA of bacteriophage phi X 174.

An extract prepared from Escherichia coli cells infected with phi chi 174 bacteriophage was capable of incorporating dTTP into phage-specific DNAs in vitro. The synthesized DNAs were associated with proteins and sedimented with S values of 20, 50, and 90 in a sucrose gradient sedimentation. DNA isolated from 20S material was open circular replicative form (RF), DNA in 50S material was replicative-form DNA with an extended single-stranded viral DNA that ranged up to one genome in length, and DNA in 90S material consisted of circular and linear single-stranded viral DNA of full genome length and single-stranded viral DNA shorter than full genome length. Pulse and pulse-chase experiments indicated that 90S material derived from 50S material.

Detection of B virus antibody in monkey sera using glycoprotein D expressed in mammalian cells.

The gene encoding glycoprotein D (gD) of the monkey B virus (Cercopithecine herpesvirus 1) was cloned into a mammalian expression vector, pcDNA3.1(-), and the recombinant plasmid DNA was transfected into COS7 cells. The expression of gD in transfected COS7 cells was detected by indirect immunofluorescence assay or radioimmunoprecipitation analysis (RIPA). Although the expressed gD protein was revealed to react well with sera from monkeys naturally infected with B virus by RIPA, some sera showed reduced reactivity when analyzed by the Western blotting (WB) method. Some sera also showed relatively high background when the WB was performed using gD expressed from recombinant plasmid. The mutant gD protein lacking the transmembrane domain (TM) and cytoplasmic tail (CT) was next expressed in COS7 cells. The mutant protein was secreted into culture medium without apparent loss of the antigenicity. Using the secretory form of the gD protein as antigen in dot blot analysis, sera from B virus-infected monkeys were shown to react with the mutant protein without nonspecific reaction. Since the recombinant gD or its derivative lacking TM and CT could be expressed in mammalian cells with proper antigenicity, these antigens appeared to be useful for serological detection of B virus infection in monkeys.

Role of DNA gyrase subunits in synthesis of bacteriophage phi X174 viral DNA.

The role of Escherichia coli DNA gyrase subunit A and subunit B during phi X174 viral DNA synthesis was investigated. Addition of nalidixic acid (an inhibitor of gyrase subunit A) and novobiocin (an inhibitor of gyrase subunit B) to an in vitro system capable of synthesizing phi X174 viral DNA inhibited DNA synthesis. The inhibition caused by novobiocin, however, was not due specifically to an inhibition of gyrase subunit B because DNA synthesis in an in vitro system composed of an extract containing novobiocin-resistant gyrase subunit B was also inhibited by novobiocin. The requirement for gyrase subunit A and the dispensability of gyrase subunit B during viral strand synthesis was confirmed in vivo by examining phi X174 viral DNA synthesis in host bacteria containing temperature-sensitive gyrase subunits.

An African green monkey lacking peripheral CD4 lymphocytes that retains helper T cell activity and coexists with SIVagm.

Natural infection with simian immunodeficiency virus (SIV) is known to occur in the African green monkey (AGM). The actual onset of the disease has not been recognized in SIVagm infected AGM, and the precise reason for such apathogenicity in the AGM remains unclear. We reported previously that AGM peripheral CD4 lymphocytes underwent a peculiar differentiation from CD4+ to CD4- cells after in vitro activation, and we inferred that the AGM does not fall into a fatal immunodeficient state because of the generation of CD4- helper T cells in vivo. To evaluate this possibility, we examined the relationship between CD4 expression and helper T cell activity in the naturally infected AGM. We identified a healthy monkey almost lacking CD4 T cells in the periphery. This AGM showed no signs and symptoms of immunodeficiency and retained a helper T cell activity in antibody production comparable to those of CD4+ AGMs. In addition, SIVagm could be isolated from CD8+ lymphocytes in the CD4- AGM. These observations suggest that a unique host-virus adaptation has developed in the AGM, and may be helpful in explaining the fundamental reason for the apathogenicity occurring in this monkey.

Isolation and identification of bacteriophage phi X174 prohead.

The morphogenesis of bacteriophage phi X174 has been investigated by using an in vitro DNA synthesizing system. An extract of a B-mutant-infected cell is capable of synthesizing infectious phage in vitro when the B gene function is provided by the addition of an ammonium sulfate fraction of a C-mutant-infected-cell extract. This fraction contains the omega complex, a complex of phage-coded proteins with S = 108; the B-mutant extract does not. The purified omega complex, isolated from the C-mutant extract, caused the synthesis and encapsidation of viral DNA when added to B-mutant extract. The omega complex contains the B protein but it is the intact omega complex that functioned in the in vitro complementation of the B-mutant extract because other fractions containing B protein but no omega complex had little or no complementing activity. The results indicate that the omega complex is the phi X174 prohead. The B protein is not found in either the 132S or 114S phage particles but is an essential component of the prohead. This suggests that it may have a scaffolding function.

Purification of phi X174 gene C protein.

The product of gene C of bacteriophage phi X174 is required for the replication of phiX174 single-stranded DNA in Escherichia coli cells infected with phi X174. The protein has been purified to homogeneity using an in vitro complementation system. The protein exhibits a molecular weight of 5800 under denaturing conditions. The NH2-terminal amino acid sequence of the protein is (NH2)-Met-Arg-Lys, which is consistent with the sequence predicted from the nucleotide sequence of gene C. The protein has an affinity for single-stranded DNA but less for double-stranded DNA.