Diffusion Tensor Imaging MRI With Spin-Echo Sequence and Long-Duration Measurement for Evaluation of Renal Fibrosis in a Rat Fibrosis Model.

BACKGROUND: Renal fibrosis (RF) is a well-known marker for chronic kidney disease (CKD) progression, including chronic renal injury after renal transplantation. However, invasive biopsy is an available examination for evaluation of RF. Diffusion MRI was once recognized as a promising option for RF. However, it is now controversial for RF evaluation in a unilateral ureteral obstruction (UUO) model. METHODS: To seek an optimal imaging method applicable for RF in UUO model kidneys, we attempted a series of MRI methods, including proton density-weighted imaging, T1-weighted imaging, T2-weighted imaging, T2*-weighted imaging, diffusion-weighted imaging, and diffusion tensor imaging (DTI). RESULTS: We identified DTI MRI by spin-echo sequence plus a special kidney attachment as the best option for evaluation of renal UUO fibrosis, compared with normal kidney on the opposite side. To confirm these results, we applied this technique to a rat UUO therapeutic model with the anti-fibrotic reagent Fasudil. Fractional anisotropy values calculated from DTI MRI showed statistically significant linear correlation with the RF area measured by use of Sirius red or Masson trichrome staining of the positive area [cortex (r = 0.6397, P = .0283) and outer stripe of the outer medulla (r = 0.7810, P = .0039)]. CONCLUSIONS: By use of the DTI MRI with spin-echo sequence, it may be possible to accurately evaluate RF in CKD.

Studies of Pig Complement: Measurement of Pig CH50, ACH50, and Components.

BACKGROUND: On the basis of a comparison of the hemolytic complement titer in pigs with that in humans, the complement system of pigs was investigated. The response of innate immunity, such as the natural antibodies, against humans was also examined. METHODS: Hemolytic complement activity of pig serum was measured with the use of a microtitration technique. CH50 was determined according to the method of Mayer. ACH50 was assayed according to the methods of Platts-Milles and Ishizaka. Hemolytic activities of C1, C4, C2, C3, C5, C8, and C9 were estimated through the use of intermediate cells and reagents, as described previously. In addition, the pig natural anti-human antibody was studied with the use of human peripheral blood mononuclear cells (PBMCs). Human PBMCs were stained with 5% pig serum, followed by staining with fluorescein isothiocyanate-labeled goat anti-pig IgG and IgM. The resulting stained cells were quantified by use of a FACScalibur system. The alternative pathway of pig complement was also measured with the use of human erythrocytes and normal pooled pig serum with or without Mg(++)EGTA. RESULTS: Both the CH50 and ACH50 titers were lower than those of humans. Concerning the components, except for C3, each component, that is, C1, C4, C2, C5, C8, and C9, was also lower than that of humans, based on measured values for human complement components. Pig serum clearly contains natural antibodies, IgG and IgM, to human PBMCs. The alternative pathway of pig complement reacted with human erythrocytes. CONCLUSIONS: As a whole, pig innate immunity, the complement system and natural antibody, recognizes the surfaces of human cells.

Functional cross-talk of HIV-1 Tat with p53 through its C-terminal domain.

Human immunodeficiency virus type 1 (HIV-1) Tat repressed the p53-dependent gene expression through its C-terminal domain of Tat (amino acid residues 73-86) independent of the involvement of NF-kappaB and coactivator CBP/p300. Although Tat did not directly bind to p53, this repression required the N-terminal domain of p53. In contrast, Tat and p53 cooperated in the activation of HIV-1 gene expression. Thus, the cross-talk between Tat and p53 may be linked with cellular transformation by HIV-1 infection or activation of HIV-1 replication.

The cyclic AMP/PKA signal pathway is required for initiation of spore germination in Schizosaccharomyces pombe.

Spore germination, a transition from the quiescent G0 phase to the proliferation cycle, is triggered by glucose in Schizosaccharomyces pombe. The role of cAMP/protein kinase A (PKA) signalling in germination is investigated. Gene disruption of cyr1+, pka1+ and gpa2+ encoding adenylate cyclase, PKA and the alpha-subunit of a trimeric GTP-binding protein, respectively, reduced the colony-forming efficiency of spores in minimal medium. Isolated spores of these null mutants did not germinate in minimal medium for up to 12 h, at which time wild-type spores had completed germination and formed germ projections. In wild-type spores, cortical actin patches randomly distributed in the early stage of outgrowth and then localized to one side of spores before the formation of projections. In contrast, the mutant spores exhibited no actin patches, but the cell surface was predominantly stained, like ungerminated spores of wild-type. Flow fluorocytometric analysis of propidium iodide-stained spores revealed a distinct 1C DNA peak after germination was completed. The fluorescent profile of the mutant spores, however, did not change during 12 h incubation in the minimal medium. These observations indicate that spores harbouring either cyr1Delta, pka1Delta or gpa2Delta are hardly triggered to germination. When wild-type spores were exposed to glucose, the intracellular cAMP level transiently increased in a few minutes, but gpa2Delta spores did not respond to glucose. We conclude that S. pombe spores initiate germination in response to glucose through the cyclic AMP-PKA pathway.

My4+/LeuM3- molecule and CD19 antigen are down-modulate by low affinity Fc gamma receptor II (CD32) stimulation on CD56-positive B-lymphoma cells.

My4+/LeuM3- molecule is recognized by My4, but not by LeuM3, both well known mAbs to CD14. In a previous study we showed that the My4+/LeuM3- molecule on a human monoblastic cell line, U937, is not CD14, but another cell surface antigen. The roles and functions of the My4+/LeuM3- molecule remained unknown. We now report that specific stimulation of Fc gammaR with aggregated IgG or anti-Fc gammaRII antibody down-modulated the My4+/LeuM3- molecules, as well as CD19, in a case of CD56-positive B cell lymphoma. Stimulation of Fc gammaR with anti-mu antibody, which induced concomitant stimulation of sIg, did not induce down-modulation of either molecule. Stimulation of CR2 (CD21), a protein which is functionally or physically associated with CD19, with anti-CR2 (CD21) mAbs also had no effect. The modulation occurred specifically on CD56-positive B-lymphoma cells, since My4+/LeuM3(-)-positive, CD56-negative B-lymphoma cells did not respond to the stimulation. These results suggest that CD19 and My4+/LeuM3- molecules are functionally or physically associated with Fc gammaR II on CD56 positive B-lymphoma cells defined as being at a terminal B cell differentiation stage.

Molecular remodelling of human CD46 for xenotransplantation: designing a potent complement regulator without measles virus receptor activity.

In pig-to-human discordant xenotransplantation, human complement (C) is a major barrier to long survival of xenografts. The current idea on how to cope with this barrier is that human complement regulatory proteins are forcibly expressed on xenografts to serve as safeguards against host C-induced hyperacute rejection of xenografts. Co-expression of decay-accelerating factor (DAF) (CD55) and membrane cofactor protein (MCP) (CD46) would be the first choice for this trial, because most of the human cells are protected from C-mediated damage by two different modes with these two kinds of C-regulators. Many problems have arisen, however, for MCP expression on grafts. (i) MCP acts as a measles virus receptor, which may function to render donor pigs measles virus (MV) sensitive. (ii) MCP signals immune suppression which causes devastation of the recipient's immune responses. (iii) MCP exerts relatively low self-protective activity against C compared with other cofactors; development of more efficient forms is desirable. (iv) Grafts with a high expression level of MCP are difficult to produce. In this study, we made a number of cDNA constructs of MCP, expressed them on swine endothelial cell lines, and tested cell-protective potency and MV susceptibility. The short consensus repeat 1 (SCR1)-deleted MCP with glycosyl phosphatidylinositol (GPI)-anchored form (Delta1MCP-PI) of MCP was found to be most suitable for the purpose of overcoming these problems. However, it was also found that MV induces two modes of cytopathic effect (CPE) on swine endothelial cells, either MCP-dependent or -independent. Here, we discuss these two points which will be raised through study of MCP-transgenic animals.

Suppression of the poly(ADP-ribose) polymerase activity by DNA-dependent protein kinase in vitro.

It has been suggested that DNA-dependent protein kinase (DNA-PK) is a central component of DNA double-strand-break repair. The mechanism of DNA-PK action, however, has not been fully understood. Poly(ADP-ribose) polymerase (PARP) is another nuclear enzyme which has high affinity to DNA ends. In this study, we analysed the interaction between these two enzymes. First, DNA-PK was found to suppress the PARP activity and alters the pattern of poly(ADP-ribosyl)ation. Although DNA-PK phosphorylates PARP in a DNA-dependent manner, this modification is unlikely to be responsible for the suppression of PARP activity, since this suppression occurs even in the absence of ATP. Conversely, PARP was found to ADP-ribosylate DNA-PK in vitro. However, the auto-phosphorylation activity of DNA-PK was not influenced by this modification. In a competitive electrophoretic mobility shift assay, Ku 70/80 complex, the DNA binding component of DNA-PK, was found to have higher affinity to a short fragment of DNA than does PARP. Furthermore, co-immunoprecipitation analysis suggested direct or close association between Ku and PARP. Thus, DNA-PK suppresses PARP activity, probably through direct binding and/or sequestration of DNA-ends which serve as an important stimulator for both enzymes.

The human T cell leukemia virus type I-tax gene is responsible for the development of both inflammatory polyarthropathy resembling rheumatoid arthritis and noninflammatory ankylotic arthropathy in transgenic mice.

We previously reported that inflammatory arthropathy resembling rheumatoid arthritis (RA) develops among transgenic mice carrying the long terminal repeat (LTR)-env-pX-LTR region of human T cell leukemia virus type I (LTR-pX-Tg mice). Because four genes are encoded in this region, we produced transgenic mice that only express the tax gene to examine its role in the development of arthritis. Transgenic mice were produced by constructing DNAs that express the tax gene alone under the control of either its own LTR or CD4 enhancer/promoter and by microinjecting them into C3H/HeN-fertilized ova. We produced seven transgenic mice carrying the LTR-tax gene and nine mice carrying the CD4-tax and found that one of the LTR-tax-Tg mice and five of CD4-tax-Tg mice developed RA-like inflammatory arthropathy similar to LTR-pX-Tg mice, indicating that the tax gene is arthritogenic. On the other hand, the other two LTR-tax-Tg mice had ankylotic changes caused by new bone formation without inflammation. In these ankylotic mice, tax mRNA, inflammatory cytokine mRNA, and autoantibody levels except for TGF-beta1 level were lower than those in LTR-pX- or CD4-tax-Tg mice. These results show that Tax is responsible for the development of inflammatory arthropathy resembling RA and that this protein also causes ankylotic arthropathy.

Ability of teicoplanin and vancomycin to induce contraction of, and histamine release from, pulmonary tissue of humans, monkeys and guinea pigs.

To assess the safety of teicoplanin and vancomycin with respect to airway tissue, we evaluated whether these two antibiotics induce pulmonary tissue contraction and histamine release in human, monkey and guinea pig specimens in vitro. The effects of these drugs on the release of histamine from monkey blood leucocytes and mouse bone marrow-derived mast cells (BMMC) were also studied. Neither teicoplanin nor vancomycin (10(-6)-10(-3) g/mL) induced contractions of guinea pig trachea or lung parenchyma. Similarly, these drugs induced no appreciable change in the resting tonus of cynomolgus monkey bronchus or lung parenchyma. The tonus of monkey trachea was not influenced by teicoplanin, whereas 10(-3) g/mL vancomycin caused contraction. The spontaneous tonus of human lung parenchyma was not altered by teicoplanin or vancomycin, and that of the bronchus was not influenced by teicoplanin; however, 10(-3) g/mL vancomycin elicited obvious contraction of the bronchus. Neither drug promoted the release of significant amounts of histamine from these pulmonary tissues or from monkey blood leucocytes and BMMC. These results suggest that, compared with vancomycin, teicoplanin may be associated with a lower risk of inducing bronchospasm when used for inhalation therapy.

Insertion of two independent enhancers in the long terminal repeat of a self-inactivating vector results in high-titer retroviral vectors with tissue-specific expression.

The use of retroviral vectors (RVs) derived from the murine oncoretroviruses for gene therapy is associated with the risk of malignant transformation of infected cells and ectopic expression of the proteins of interest. Targeting retroviral vectors to specific tissues would increase their safety and clinical applicability. To explore the potential of targeting vector expression to skeletal muscle, the murine leukemia virus broad transcriptional tropism was modified by substituting the viral promoter and/or enhancer with a transcriptional cassette containing the human T cell leukemia virus type I Tax-responsive element and the minimal muscle creatine kinase enhancer and promoter. The resulting retroviral vectors could be transcriptionally trans-activated by tax. In the absence of Tax, however, the viruses showed muscle-specific expression. Trans-complementing packaging and indicator cells stably expressing Tax were used to isolate high-titer producer cell clones (10(6) CFU/ml). In vitro, the levels of expression of these RVs in Tax-expressing fibroblasts were 10,000-fold higher than in normal fibroblasts and 1000-fold higher in C2C12 myotubes than in C2C12 myoblasts. Expression of the vectors and the endogenous muscle creatine kinase gene was similarly dependent on the maturity of the muscle cultures. One vector with modified LTRs was also tested in vivo in regenerating muscle and showed a delayed pattern of expression in myofibers compared with the vector containing the wild-type LTRs. These vectors can be easily modified to contain different tissue-specific enhancer and promoter elements and the availability of complementing packaging and indicator cells expressing Tax should allow their application in a variety of gene therapy settings.

In vivo phosphorylation of poly(ADP-ribose) polymerase is independent of its activation.

Poly(ADP-ribose) polymerase (PARP) is a nuclear enzyme, which is activated by DNA strand breaks. Although PARP is known to be cleaved by the cysteine protease, caspase-3/CPP32, during apoptosis, signal cascade which regulates the PARP activity has not been fully understood. In this study, we investigated post-translational modification of PARP. We found that PARP was phosphorylated by a serine kinase in vivo. PARP was activated temporarily and extensive auto-modification occurred on PARP, possibly by the fragmented DNA during apoptosis induced by etoposide in Jurkat cells. However, the phosphorylation level was not changed for up to 6 h, after PARP cleavage began in apoptosis by the treatment with etoposide. Furthermore, we showed the presence of a PARP-associated kinase in nuclear extracts of the HTLV-I infected T-cell lines but not in uninfected T-cell lines, whereas this kinase did not inhibit the PARP activity even in the presence of ATP. Taken together, in vivo phosphorylation of PARP might be independent of the activation or cleavage of PARP.

Characterization of the internal promoter of human T-cell leukemia virus type I.

The HTLV-I provirus contains two different promoters: the classical retroviral promoter in the 5' long terminal repeat (LTR) and our previously identified second promoter in the pol gene just upstream of the ATG codon of the tax gene. Here, we demonstrated that the internal promoter expresses the gene for Tax but not Rex. As the deletion of upstream of the transcriptional initiation site (nt 5130) caused down-regulation of the promoter activity, we termed the region HTLV-I internal regulatory element (HIRE). We found a cellular sequence-specific DNA binding protein which binds to HIRE. Furthermore, we demonstrated that the 3' LTR regulates Tax expression from the internal promoter. These findings may shed light on a novel mechanism for gene expression in complex retroviruses of the HTLV family.

Modulation of HTLV-I gene expression by HIV-1 Rev through an alternative RxRE-independent pathway mediated by the RU5 portion of the 5'-LTR.

The 5'-RU5 portion of human T-lymphocyte virus type I (HTLV-I) long terminal repeat (LTR) had been reported to contain cis-acting elements for the controlled viral gene expression by the rex gene product. In this study, the human immunodeficiency virus type I (HIV-1) Rev protein was found to enhance gene expression, acting through the 5'-RU5 portion of HTLV-I, while the Rex-responsive element (RxRE)-mediated activation by Rev was reconfirmed to be negative. This positive action of HIV-1 Rev on HTLV-I gene expression seemed to be distinct from the widely accepted Rex or Rev function to facilitate the nuclear export of RxRE-containing unspliced viral mRNAs, since a trans-dominant, nuclear export-deficient mutant (RevM10) still retained the RU5-mediated effector function. Analyses of the functional aspects of Tat/Rev fusion proteins on the HTLV-I RU5 suggested a specific interaction of Rev and RU5, but lacked evidence for the binding of Rev to the RU5 at the RNA level. These results suggest an answer to the controversy regarding a Rex-like function occasionally observed with HIV-1 Rev and its related proteins. It may also be suggested that particular care should be taken when such a trans-dominant Rev mutant is considered to be used as a genetic therapy against HIV-I infection, in individuals infected with both HIV-I and HTLV-1.

Expression of caveolin-1 in human T cell leukemia cell lines.

Caveolae are plasma membrane invaginations that function as a center for signal transduction. Recent studies indicate that caveolins, the main proteins in caveolae, serve as scaffolding proteins onto which many classes of signaling molecules are assembled. There are multiple forms of caveolins: caveolin-1 and caveolin-2 are expressed as stable heterooligomeric complexes within most cell types, while caveolin-3 is restricted to striated muscle cells. However, neither caveolin proteins nor caveolae structures are detected in peripheral blood cells or blood cell lines. We identified caveolin-1 in one T cell leukemia cell line, a subline of Jurkat cells, by immunostaining and Western blotting. The cells showed enlarged cell bodies similar to activated T cells. This led us to investigate caveolin expression in adult T cell leukemia (ATL) cell lines, which are known to be constitutively activated. Two of five ATL cell lines expressed caveolin-1. The phenotype of caveolin-1-positive cells expressed not only high levels of the T cell activation markers, as with CD25 or HLA-DR, but also CD54 at extremely high levels. These findings demonstrate for the first time that hematological cells express caveolin-1 in certain states of cell activation. In addition, the caveolin-1 expression may be a useful marker for the diagnosis of ATL malignancy.

A new nonamyloid transthyretin variant, G101S, detected by electrospray ionization/mass spectrometry. Mutations in brief no. 201. Online.

Familial amyloidotic polyneuropathy is caused by transthyretin (TTR) variants. The identification of new variants with and without amyloidosis may help to clarify the mechanism of amyloid fibril formation. We detected several variant TTRs from patients with and without symptoms of amyloidosis using mass spectrometry (MS). TTR was isolated by mixing test serum with anti-transthyretin antiserum, and the generated immunoprecipitate was analyzed by high performanced liquid chromatography/electrospray ionization (HPLC/ESI) MS. Variant TTRs showed extra peaks in addition to normal TTR peaks. A variant found in nonamyloid group was sequenced by HPLC/ESI tandem MS using peptides obtained by protelytic digestion of TTR and by DNA analysis. The structure was new, [G101S], and was found in a 74 years old Japanese male. This mutation results from substitution in a CpG hot spot. The substitution in the surface loop, 98-102, between F and G b-strands may not cause amyloid formation.

A novel protein that participates in nonself discrimination of malignant cells by homologous complement.

The human complement (C) system protects an individual against substances of nonself origin, including xenografts and microbial pathogens. Human cells express C-regulatory proteins, CD46 and CD55, thereby circumventing attack by C3, a major effector of C. Nevertheless, certain malignant cells, particularly those undergoing apoptotic stress, can activate homologous C, overcoming the regulatory actions of CD46 and/or CD55. The molecular mechanisms whereby malignant cells are tagged by homologous C3 remain largely unknown. We identified a novel gene product that converts human cells into targets for homologous complement. Only malignant cells and cell lines exposed to Fas or X-irradiation stimuli produced this protein, designated M161Ag, which was an unglycosylated 43-kDa protein. Analysis of cloned cDNAs indicated that this molecule was a secretory protein containing five amino acids encoded by TGA codons. Its functions were unique in that once secreted from the tumor cells, it bound back to the surface of these cells and activated homologous complement (C3) via the alternative pathway, allowing for C3 deposition on the membrane. This molecule may offer new insight into innate immunity; surveillance of tumor cells by complement is a common feature in the human immune system.

HIV-1 capsid mutants inhibit the replication of wild-type virus at both early and late infection phases.

In-frame mutations were introduced into various portions of the human immunodeficiency virus type 1 (HIV-1) gag gene, and potentials of the mutants to suppress the replication of wild-type HIV-1 were monitored. In contrast to results obtained with matrix and nucleocapsid mutants, almost all capsid mutants blocked HIV-1 replication completely in single-round replication assays. A capsid mutant designated C6b was demonstrated to be one of the most efficient inhibitors for HIV-1 reported to date, and to be effective at both early and late viral replication phases. T-cells, which are engineered to express the C6b Gag in response to HIV-1 infection, were perfectly resistant to HIV-1.

Crystal structure of calcium bound domain VI of calpain at 1.9 A resolution and its role in enzyme assembly, regulation, and inhibitor binding.

The three dimensional structure of calcium-bound domain VI of porcine calpain has been determined to 1.9 A resolution. The crystal structure reveals five EF-hands, one more than previously suggested. There are two EF-hand pairs, one pair (EF1-EF2) displays an 'open' conformation and the other (EF3-EF4) a 'closed' conformation. Unusually, a calcium atom is found at the C-terminal end of the calcium binding loop of EF4. With two additional residues in the calcium binding loop, the fifth EF-hand (EF5) is in a 'closed' conformation. EF5 pairs up with the corresponding fifth EF-hand of a non-crystallographically related molecule. Considering the EF5's role in a homodimer formation of domain VI, we suggest a model for the assembly of heterodimeric calpain. The crystal structure of a Ca2+ bound domain VI-inhibitor (PD150606) complex has been refined to 2.1 A resolution. A possible mode for calpain inhibition is discussed.

HTLV-I env-pX transgenic rats: prototype animal model for collagen vascular diseases.

To evaluate the function of HTLV-I env-pX gene in vivo, we developed two lines of transgenic rats (env-pX rats) that expressed env-pX gene products, under control of own LTR promotor. In various tissues of the rats, env and pX mRNAs were constitutively expressed, irrespective of age. At age 5 weeks, swelling of the bilateral ankle joints histologically showing synovial lining hyperplasia, severe chronic inflammation, erosion of the joint cartilage, and bone destruction with pannus formation began to develop in these env-pX rats. These histologic features resemble those of rheumatoid arthritis (RA) in man. High titered rheumatoid factors and low anti-dsDNA antibodies and hyper-gamma globulinemia were detected. Necrotizing arteritis resembling polyarteritis nodosa, polymyositis, myocarditis and Sjögren syndrome-like sialoadenitis developed, together with RA-like arthritis even in one individual animal. Thymic atrophy with low body weight was also observed. The evidence indicates that env-pX rats appear to be suitable animal models for elucidating pathogenetic mechanisms involved in not only HTLV-I related diseases but also various collegen vascular and autoimmune diseases of unknown etiology in man.