Highly increased plasma concentrations of the nicked form of beta(2) glycoprotein I in patients with leukemia and with lupus anticoagulant: measurement with a monoclonal antibody specific for a nicked form of domain V

beta(2)-Glycoprotein I (beta(2)GPI) consists of five tandem repeated domains (I, II, III, IV, and V). The nicked form of beta(2)GPI (N-beta(2)GPI ) which was cleaved by plasmin in vitro at Lys 317-Thr 318 in domain V, showed reduced affinity for the negatively charged phospholipids, especially cardiolipin (CL). Recently, the N-beta(2)GPI was detected in the plasma of patients with disseminated intravascular coagulation syndrome (DIC) by an immunological method. In the present study, we prepared monoclonal antibodies for the nicked form, and demonstrated that the concentrations of this form of beta(2)GPI, which were analyzed by a sandwich ELISA using two specially prepared monoclonal antibodies, were significantly increased in the plasma of patients with leukemia (n = 51, mean +/- SD: 162.0 +/- 118.3 ng/ml) and with lupus anticoagulant (LA) (n =40, mean +/- SD: 3,041.5 +/- 16,579.7 ng/ml), compared to the normals (n = 33, mean +/- SD: 1.04 +/- 1.54 ng/ml). We found a significant correlation between the concentrations of N-beta(2)GPI and those of typical molecular markers for a fibrinolytic state such as plasmin-alpha(2) plasmin inhibitor complex (PIC) and D-dimer in patients with leukemia, but not in patients with LA. These results suggested that the generation of N-beta(2)GPI was caused by plasmin in the patients with leukemia, and by unknown proteases in the patients with LA. In the patients with LA, the levels of N-beta(2)GPI tended to be higher in those without thrombosis than in those with thrombosis.     

IL-18 prevents the development of chronic graft-versus-host disease in mice

The development of chronic graft-versus-host disease (GVHD), which is induced by the transfer of DBA/2 spleen cells into (C57BL/6 x DBA/2)F1 (BDF1) mice, is closely related to diminished donor anti-host CTL activity and host B cell hyperactivation. Therefore, an approach which activates donor CD8+ T cells or suppresses donor CD4+ T cell-host B cell interaction may have clinical utility in the treatment of chronic GVHD. We have previously demonstrated that IL-18 induces the development of naive CD8+ T cells into type I effector cells in DBA/2 anti-BDF1 MLC. In this paper we examined the effect of IL-18 administration on the development of chronic GVHD in mice. The treatment was started before or after the onset of clinical evidence of the disease. Regardless of the treatment schedule, IL-18 significantly decreased immunological parameters indicative of chronic GVHD, such as elevated serum IgG antinuclear Abs, IgG1, and IgE levels, and host B cell numbers and their activation. Importantly, IL-18-treated mice did not show the same acute GVHD-like symptoms reported for IL-12 treatment, because there was no weight loss, death, or severe immunodeficiency as indicated by a decrease in IL-2 and IFN-gamma production by Con A-stimulated spleen cells. In contrast, IL-18 treatment partially but significantly restored the production of these cytokines. Data further suggested that these IL-18-mediated therapeutic effects may be due to the induction of donor CD8+ CTL, the decrease in donor CD4+ T cell numbers, and a down-regulation of host B cell MHC class II expression. Thus, our results suggest that IL-18 has beneficial effects in the prevention and treatment of chronic GVHD.     

Postextrasystolic contractile decay always contains exponential and alternans components in canine heart

In isolated, blood-perfused canine hearts, postextrasystolic potentiation (PESP) decays monotonically after a noncompensatory pause following a spontaneous extrasystole (ES). The monotonic PESP decay yields myocardial internal Ca(2+) recirculation fraction (RF). We have found that after a compensatory pause (CP), PESP decays in alternans, consisting of an exponential and a sinusoidal decay component. We have proposed that this exponential component also yields RF. In the present study, we examined the reliability of this alternative method by widely changing the ES coupling interval (ESI), CP, and heart rate in the canine excised, cross-circulated left ventricle. We found that all PESP decays consisted of the sum of an exponential and a sinusoidal decay component of variable magnitudes whether a CP existed or not. Their decay constants as well as the calculated RF were independent of the ESI and CP. This confirmed the utility of our alternative RF determination method regardless of the ESI, CP, and heart rate. Direct experimental evidence of Ca(2+) dynamics supportive of this alternative method, however, remains to be obtained.     

Identification of a novel mammalian endoplasmic reticulum-resident KDEL protein using an EST database motif search

Several endoplasmic reticulum (ER)-resident proteins contain a unique C-terminal sequence (KDEL) which is required for the retention of these proteins in the ER. By searching a mouse EST database for records containing the nucleotide sequence encoding the KDEL motif, we extracted cDNAs encoding putative novel ER-resident proteins in addition to all of the known ER proteins bearing the KDEL motif. Using the sequence information obtained by this database search, we cloned the cDNA encoding a novel KDEL motif-bearing protein, ER protein 58 (EP58), sharing no significant homology to any of the known ER-resident proteins. Subcellular localization of EP58 in the ER was confirmed by cytoimmunofluorescence studies using epitope-tagged EP58. The EP58 gene was primarily expressed in embryo, placenta, and adult heart. Neither heat shock nor ER stress as tested here was sufficient to induce expression of the EP58 gene. A putative role of the N-terminal half of EP58 in protein-protein interaction is suggested by its similarity to the filamin rod domain. Similarity of the EP58 sequence with bacterial and fungus proteins suggests a possible role for EP58 in polysaccharide biosynthesis.     

Concerted Evolution of a Highly Repetitive DNA Family in Eptatretidae (Cyclostomata, Agnatha) Implies Specifically Differential Homogenization and Amplification Events in Their Germ Cells

In eight hagfish species, it is known that chromosome elimination occurs during early embryogenesis, and some highly repetitive DNA families, restricted to germ cells, have been isolated. One of these families, ``EEEo2,' has been isolated as DNA fragments by restriction enzyme analyses from Eptatretus okinoseanus and E. cirrhatus. In this study, EEEo2 sequences were isolated from germline DNA in E. burgeri, Paramyxine sheni, and P. atami using PCR methods. Sequence analysis revealed that these sequences are intraspecifically homogeneous, except in E. burgeri, and are interspecifically conserved with heterogeneity. The intraspecific sequence variability tends to decrease as the copy number increases. These results indicate that EEEo2 has evolved in a concerted manner. Moreover, an ancestral repeating motif consisting of triplicate subrepeats was deduced. These results suggest that EEEo2 arose as an initial amplification of this subrepeat and has evolved by saltatory replication. Phylogenetic analyses suggested the possibility that EEEo2 in E. okinoseanus and E. cirrhatus has been subjected to strong homogenizing forces for concerted evolution, whereas the force is weak in E. burgeri. In addition, EEEo2 in P. sheni and P. atami appear to have been incompletely subjected to these forces. Chromosomal in situ hybridization experiments revealed that EEEo2 sequences were located along almost their entire length of several heterochromatic chromosomes that are restricted to germ cells. These chromosomes are disposed to form a secondary association during the first meiotic metaphases, except in P. sheni. This chromosomal distribution may promote a concerted mode of sequence evolution in both nonhomologous chromosomes and homologous chromosomes and reflect the differential driving forces between species. Received: 17 April 1999 / Accepted: 10 September 1999     

Highly repetitive DNA sequences that are restricted to the germ line in the hagfish Eptatretus cirrhatus: a mosaic of eliminated elements

The New Zealand hagfish, Eptatretus cirrhatus, is known to eliminate parts of its chromosomes during embryogenesis from presumptive somatic cells. Electrophoresis of germ line and somatic DNAs of this species, after treatment with the restriction endonucleases DraI and EcoRI, revealed three fragments of DNA that were restricted to the germ line. DNA filter hybridization experiments demonstrated that these fragments were present almost exclusively in the germ line DNA of E. cirrhatus and that they were highly and tandemly repeated. Thus, these DNA fragments appeared to be eliminated during embryogenesis. Moreover, one fragment (a DraI fragment) cross-hybridized with the germ line DNA from other species of hagfish, namely, Eptatretus okinoseanus and Paramyxine atami. Molecular cloning and sequence analysis revealed that the DraI fragment was composed mainly of closely related sequences of 85 bp in length and that this sequence was about 75% homologous to the sequence of EEEo2 (eliminated element of E. okinoseanus 2) which is a germ line-restricted and highly repetitive sequence that was isolated previously from E. okinoseanus. The other two fragments were composed of three families of closely related sequences that were 172 bp long (designated EEEc1), 61 bp long (EEEc2) and 54 bp long (EEEc3). Fluorescence in situ hybridization experiments revealed that each eliminated element was distributed on several chromosomes that are limited to germ cells. EEEo2 was dispersed on 12 C-band-positive chromosomes. EEEc1 and EEEc3 were dispersed on all C-band-positive and several C-band-negative chromosomes. By contrast, EEEc2 was located to terminal regions of several C-band-negative chromosomes. These results suggest that the eliminated chromosomes in hagfish are mosaics of highly repeated, germ line-restricted families of DNA sequences. Received: ██; in revised form: 25 October 1997 / Accepted: ██     

Effectiveness of weekly subcutaneous recombinant human erythropoietin administration for chemotherapy-induced anemia

The effects of weekly subcutaneousrecombinant human erythropoietin (r-hEPO)administration on anemia during chemotherapy includingcisplatin and 5-fluorouracil in patients with head andneck carcinomas were examined. Weekly subcutaneousr-hEPO administration in cancer patients has not beeninvestigated previously. Patients were treated withr-hEPO 100 IU/kg (2 patients), 200 IU/kg (6 patients),or 400 IU/kg (5 patients), or placebo, andeffectiveness was evaluated by monitoring hemoglobinconcentration changes after administration for 8weeks. Hemoglobin concentrations in all 3 r-hEPOdosage groups were higher than that in the controlgroup during chemotherapy. All r-hEPO doses producedimprovements in the anemia induced by chemotherapy;however, the 400 IU/kg dose was most effective. Therequirement for blood transfusions decreased inpatients receiving r-hEPO therapy, and no significantside-effects were associated with r-hEPOadministration. These results suggest thatchemotherapy-induced anemia can be prevented by weeklysubcutaneous r-hEPO administration.     

Novel frame-shift mutation in Slc5a2 encoding SGLT2 in a strain of senescence-accelerated mouse SAMP10

The senescence-accelerated mouse prone10 (SAMP10) strain, a model of aging, exhibits cognitive impairments and cerebral atrophy. We noticed that SAMP10/TaSlc mice, a SAMP10 substrain, have developed persistent glucosuria over the past few years. In the present study, we characterized SAMP10/TaSlc mice and further identified a spontaneous mutation in the Slc5a2 gene encoding sodium-glucose co-transporter (SGLT) 2. The mean concentration of urine glucose was high in SAMP10/TaSlc mice and increased further with advancing age, whereas other strains of senescence-accelerated mice, including SAMP1/SkuSlc, SAMP6/TaSlc and SAMP8/TaSlc or normal aging control SAMR1/TaSlc mice, exhibited no detectable glucose in urine. SAMP10/TaSlc mice consumed increasing amounts of food and water compared to SAMR1/TaSlc mice, suggesting the compensation of polyuria and the loss of glucose. Oral glucose tolerance tests showed decreased glucose reabsorption in the kidney of SAMP10/TaSlc mice. In addition, blood glucose levels decreased in an age-dependent fashion. The kidney was innately larger than that of control mice with no histological alterations. We examined the expression levels of glucose transporters in the kidney. Among SGLT1, SGLT2, glucose transporter (GLUT) 1 and GLUT2, we found a significant decrease only in the level of SGLT2. DNA sequencing of SGLT2 in SAMP10/TaSlc mice revealed a single nucleotide deletion of guanine at 1236, which resulted in a frameshift mutation that produced a truncated protein. We designate this strain as SAMP10/TaSlc-Slc5a2^slc (SAMP10-ΔSglt2). Recently, SGLT2 inhibitors have been demonstrated to be effective for the treatment of patients with type 2 diabetes (T2D). SAMP10-ΔSglt2 mice may serve as a unique preclinical model to study the link between aging-related neurodegenerative disorders and T2D.