Synthesis of antitumor-active conjugates of adriamycin or daunomycin with the copolymer of divinyl ether and maleic anhydride

Polymeric conjugates of adriamycin (ADR) ( 2 ) or daunomycin (DM) ( 3 ) were synthesized by reaction of the drugs with the copolymer of divinyl ether and maleic anyhdride (DIVEMA) ( 1 ). The content of ADR moieties in the DIVEMA conjugate ( 4 ) could be varied depending on the reaction conditions up to 35,8 wt.-%. Considering the low toxicity and the high possibility of renal excretion, DIVEMA with M?_w of 7000 and M?_w/M?_n = 1,6 was used for the conjugation. The rate of drug release from the conjugate was determined under physiological conditions by reversed phase HPLC. Within 14 days only 15% of the attached ADR was released from conjugate 4 . The antitumor activity of the conjugates was tested in vitro and in vivo against mouse P388 leukemia. Conjugate 4 proved to be 28 times less active than ADR in vitro, which could be explained from the slow drug-release. On the contrary 50% of the leukemic mice treated by 4 survived more than 60 days, whereas no mice given ADR alone or the admixture of ADR and DIVEMA survived 30 days. An antitumor activity of the polymeric conjugate better than that of the free drug was also observed in vivo with DM. Such a polymeric effect can be attributed either to the change in body distribution, the difference in pharmacokinetics, or the slow drugrelease.     

Plasma brain natriuretic peptide and the evaluation of volume overload in infants and children with congenital heart disease

This study was designed to explore whether it was possible to evaluate the severity of VSD, PDA, and ASD by measuring brain natriuretic peptide (BNP) levels. We also investigated normal BNP levels in children to provide a baseline for our study. We measured BNP levels in 253 normal children, including 11 normal neonates, and in 91 VSD patients, 29 PDA patients, and 34 ASD patients. BNP levels showed no age-related differences in normal children (the mean value: 5.3 +/- 3.8 pg/ml). In the healthy neonates, BNP levels rose from 10.4 +/- 11.9 pg/ml in cord blood to 118.8 +/- 83.2 pg/ml on day 0, then fell to 15.3 +/- 7.8 pg/ml by day 7. In VSD and PDA patients, BNP levels correlated significantly with Qp/Qs, LVEDV, and peak RVP/LVP. In ASD patients, BNP levels correlated with Qp/Qs and RVEDV. Especially, in VSD patients, as an index corresponding to 1.5-2.0 of the Qp/Qs ratio, BNP levels of 20-35 pg/ml were found to be best with regard to both sensitivity and specificity. In the healthy neonates, BNP levels changed rapidly after birth. In VSD, PDA, and ASD patients, BNP levels were well-correlated with the severity of the disease. Especially, in VSD patients, it that appears BNP levels may be useful in evaluating surgical indications, with 20-35 pg/ml levels being the appropriate cut-off value.     

Germ cell transplantation: a review and progress report on ICSI from spermatozoa generated in xenogeneic testes

Results from the transplantation of donor male germ cells into xenogeneic recipient seminiferous tubules indicate that donor spermatogonia are capable of differentiating to form spermatozoa morphologically characteristic of the donor species. Germ cell transplantation procedures combined with developments in freezing, culturing or enriching germ cell populations have applications of paramount importance in medicine, basic sciences and animal reproduction. Additionally, these techniques can serve as an alternative approach for gonadal protection and fertility preservation in patients with cancer. This article is a chronological critical review of the technological advances that followed the initial successful transplantation of mouse germ cells into recipient mice. Furthermore, the factors responsible for the immunological privilege properties of the testis and the parameters influencing the potential of mammalian germ cells to undergo mitosis and meiosis within a xenogeneic testis are described. Finally, the role of human germ cell transplantation procedures in the therapeutic management of non-obstructive azoospermia is discussed.     

Identification and characterization of temperature-sensitive mild mutations in three Japanese patients with nonsevere forms of very-long-chain acyl-CoA dehydrogenase deficiency

Very-long-chain acyl-CoA dehydrogenase (VLCAD) deficiency is clinically classified into severe, intermediate, and myopathic forms. We identified mutations in three unrelated Japanese patients with VLCAD deficiency: two with the myopathic form and one with the intermediate form, all compound heterozygotes of K264E/M437V, A416T/1798delA, and P89S/IVS16-3delAA, respectively. We characterized four missense mutations, K264E, M437V, A416T, and P89S, by transisent expression analysis, using SV40-transformed fibroblasts derived from a VLCAD-null patient, as recipient cells. In transient expression of the wild-type VLCAD cDNA, VLCAD activity at 30 degrees C was higher than at 37 degrees C. Moreover, this temperature-sensitive character is more evident in all the mutant proteins tested than in wild type. Based on characterization of the five missense mutations identified in four Japanese patients, including data on one patient with the myopathic form previously reported, patients with the nonsevere forms (intermediate or myopathic forms) have missense mutations with residual activities in at least one allele. Expression analysis at 30 degrees C may be more useful for evaluating these missense mutations, compared with that at 37 degrees C.     

An Experimental Comparison of Flat-Panel Detector Performance for Direct and Indirect Systems (Initial Experiences and Physical Evaluation)

The purpose of this work was to compare direct and indirect detectors in terms of their system linearity, presampled modulation transfer function (MTF), Wiener spectrum (WS), noise equivalent quanta (NEQ), and power spectrum. Measurements were made on two flat-panel detectors, GE Revolution XR/d (indirect) and Shimadzu Safire (direct) radiographic techniques. The system linearity of the systems was measured using a time-scale method. The MTF of the systems was measured using an edge method. The WS of the systems was determined for a variable range of exposure levels by two-dimensional Fourier analysis. The NEQ was assessed from the measured MTF, WS, and estimated ideal signal-to-noise ratios. Power spectrum analyzed the chest phantom within artificial lesions. System linearity was excellent for the direct systems. For the direct system, the MTF was found to be significantly higher than that for the indirect systems. For the direct system, the WS was relatively uniform across all frequencies. In comparison, the indirect system exhibited a drop in the WS at high frequencies. At lower frequencies, the NEQ for the indirect system was noticeably higher than for the direct system. Power spectrum for the direct system was relatively flat and similar to that for white noise. The indirect system exhibited significant reduction at high spatial frequencies. In general, the direct systems exhibit improved image quality over indirect systems at comparable exposure dose.     

Neural fibrolipoma of the superficial peroneal nerve in the ankle: a case report with immunohistochemical analysis

This report presents a case of neural fibrolipoma arising from the superficial peroneal nerve in the ankle. A 28-year-old woman was referred with a soft tissue mass in the anterior aspect of the right ankle, which had been gradually enlarging for the past 10 years. Magnetic resonance imaging showed a mass lesion, measuring approximately 8 x 3 x 2 cm, with high to partially low signal intensity on both T1- and T2-weighted images. A band of low signal intensity within the lesion, which is indicative of coexistence with the tumor and the superficial peroneal nerve, could be detected on both T1- and T2-weighted images. The patient underwent an excisional biopsy. The specimen microscopically consisted of nerve bundles and fibro-fatty proliferation with abundant collagen fibers. Immunoreactivity for CD34 antigen antibody was detected in fibrous spindle cells. This is the first report to present an immunohistochemical profile of neural fibrolipoma. Neural fibrolipoma should be considered as a differential diagnosis when a lipomatous lesion is encountered in the foot or ankle as well as in the upper extremities.     

Neuronal protein NP25 interacts with F-actin

Neuronal protein NP25 is a neuron-specific protein present in highly differentiated neural cells, but its functional properties have not been well characterized. NP25 shows high amino acid sequence homology with the smooth muscle cell cytoskeleton-associated proteins, SM22, mp20, and calponin. To gain an insight into the biological functions of NP25, we first examined its subcellular localization in the human neuroblastoma cell line, SK-N-SH. NP25 diffusely distributed in the cytoplasm and fiber-like staining was also observed. It showed that NP25 co-localized with F-actin on stress fibers. A co-sedimentation assay demonstrated that NP25 bound to filamentous actin. Further investigations using fluorescence resonance energy transfer (FRET) technique revealed intracellular binding of NP25 and actin. The significance of the interaction between NP25 and F-actin is discussed.     

B Lymphocyte signaling established by the CD19/CD22 loop regulates autoimmunity in the tight-skin mouse

Systemic sclerosis (SSc) is characterized by fibrosis and autoimmmunity. Peripheral blood B cells from SSc patients specifically overexpress CD19, a critical cell-surface signal transduction molecule in B cells. CD19 deficiency in B cells also attenuates skin fibrosis in the tight-skin (TSK/+) mouse, a genetic model for SSc. Herein we analyzed two transgenic mouse lines that overexpress CD19. Remarkably, 20% increase of CD19 expression in mice spontaneously induced SSc-specific anti-DNA topoisomerase I (topo I) antibody (Ab) production, which was further augmented by 200% overexpression. In TSK/+ mice overexpressing CD19, skin thickness did not increase, although anti-topo I Ab levels were significantly augmented, indicating that abnormal CD19 signaling influences autoimmunity in TSK/+ mice and also that anti-topo I Ab does not have a pathogenic role. The molecular mechanisms for abnormal CD19 signaling were further assessed. B-cell antigen receptor crosslinking induced exaggerated calcium responses and augmented activation of extracellular signal-regulated kinase in TSK/+ B cells. CD22 function was specifically impaired in TSK/+ B cells. Consistently, CD19, a major target of CD22-negative regulation, was hyperphosphorylated in TSK/+ B cells. These findings indicate that reduced inhibitory signal provided by CD22 results in abnormal activation of signaling pathways including CD19 in TSK/+ mice and also suggest that this disrupted B cell signaling contribute to specific autoantibody production.     

Effects of L-arginine on spontaneous contraction of the rat portal vein

The effects of L-arginine on spontaneous contraction of endothelium-denuded longitudinal preparations of the rat portal vein were studied. L-arginine increased the frequency of spontaneous contraction concentration-dependently between 10 microM and 1 mM. Changes in contraction amplitude and duration were not remarkable. D-arginine had a negligible effect on spontaneous contraction. N(omega)-nitro-L-arginine (1 mM) did not affect spontaneous contraction or the response to L-arginine. Addition of N(G)-monomethyl-L-arginine (1 mM), l-lysine (1 mM) or N-ethymaleimide (0.1 mM) increased the frequency of spontaneous contractions and inhibited the effect of L-arginine. Glibenclamide (10 microM) did not affect spontaneous contraction or the response to L-arginine. Spontaneous increase in concentration of intracellular Ca2+, estimated as the ratio of Fura-PE3 fluorescence occurred synchronously with spontaneous contraction. Spontaneous increase in concentration of intracellular Ca2+ occurred more frequently in the presence of L-arginine (1 mM). L-arginine (1 mM) also increased the number of action potential bursts/min in the longitudinal smooth muscle layer. L-arginine (1 mM) also depolarized cell membranes. This study indicates that L-arginine increases the frequency of spontaneous contraction of longitudinal muscle in the rat portal vein by membrane depolarization through mechanisms that do not involve nitric oxide or the inhibition of ATP-sensitive K+ channels.     

Cartilage engineering from ovine umbilical cord blood mesenchymal progenitor cells

We aimed to determine whether three-dimensional (3D) cartilage could be engineered from umbilical cord blood (CB) cells and compare it with both engineered fetal cartilage and native tissue. Ovine mesenchymal progenitor cells were isolated from CB samples (n=4) harvested at 80-120 days of gestation by low-density fractionation, expanded, and seeded onto polyglycolic acid scaffolds. Constructs (n=28) were maintained in a rotating bioreactor with serum-free medium supplemented with transforming growth factor-beta1 for 4-12 weeks. Similar constructs seeded with fetal chondrocytes (n=13) were cultured in parallel for 8 weeks. All specimens were analyzed and compared with native fetal cartilage samples (n=10). Statistical analysis was by analysis of variance and Student's t-test (p<.01). At 12 weeks, CB constructs exhibited chondrogenic differentiation by both standard and matrix-specific staining. In the CB constructs, there was a significant time-dependent increase in extracellular matrix levels of glycosaminoglycans (GAGs) and type-II collagen (C-II) but not of elastin (EL). Fetal chondrocyte and CB constructs had similar GAG and C-II contents, but CB constructs had less EL. Compared with both hyaline and elastic native fetal cartilage, C-II and EL levels were, respectively, similar and lower in the CB constructs, which had correspondingly lower and similar GAG levels than native hyaline and elastic fetal cartilage. We conclude that CB mesenchymal progenitor cells can be successfully used for the engineering of 3D cartilaginous tissue in vitro, displaying select histological and functional properties of both native and engineered fetal cartilage. Cartilage engineered from CB may prove useful for the treatment of select congenital anomalies.