Mutation in saposin D domain of sphingolipid activator protein gene causes urinary system defects and cerebellar Purkinje cell degeneration with accumulation of hydroxy fatty acid-containing ceramide in mouse

The sphingolipid activator proteins (saposins A, B, C and D) are small homologous glycoproteins that are encoded by a single gene in tandem within a large precursor protein (prosaposin) and are required for in vivo degradation of some sphingolipids with relatively short carbohydrate chains. Human patients with prosaposin or specific saposin B or C deficiency are known, and prosaposin- and saposin A-deficient mouse lines have been generated. Experimental evidence suggests that saposin D may be a lysosomal acid ceramidase activator. However, no specific saposin D deficiency state is known in any mammalian species. We have generated a specific saposin D(-/-) mouse by introducing a mutation (C509S) into the saposin D domain of the mouse prosaposin gene. Saposin D(-/-) mice developed progressive polyuria at around 2 months and ataxia at around 4 months. Pathologically, the kidney of saposin D(-/-) mice showed renal tubular degeneration and eventual hydronephrosis. In the nervous system, progressive and selective loss of the cerebellar Purkinje cells in a striped pattern was conspicuous, and almost all Purkinje cells disappeared by 12 months. Biochemically, ceramides, particularly those containing hydroxy fatty acids accumulated in the kidney and the brain, most prominently in the cerebellum. These results not only indicate the role of saposin D in in vivo ceramide metabolism, but also suggest possible cytotoxicity of ceramide underlying the cerebellar Purkinje cell and renal tubular cell degeneration.     

Mechanisms responsible for delayed and immediate hemolytic transfusion reactions in a patient with anti-E + Jk(b)+ Di(b) and anti-HLA alloantibodies

Immediate hemolytic transfusion reactions (IHTR) occurred in the course of delayed hemolytic transfusion reactions (DHTR). An 84-year-old man had received a blood transfusion 20 years ago. Progressive anemia developed, because of continuous bleeding from a bladder tumor. He was transfused with concentrated red blood cells (CRC) which were Rh-E antigen negative, because he had anti-E antibodies (day 0). He received CRC on day 3, and underwent resection of bladder tumor on day 6. Although crossmatch-compatible CRCs were prepared for the operation, those were not required and were kept in a refrigerator in the ward. On day 9, when a CRC kept in the ward was transfused, he suddenly had a IHTR. In order to analyze a mechanism of IHTR, the anti-Jk(b) and anti-Di(b) antibodies, anti-HLA antibodies and the concentrations of inflammatory cytokines were measured in serum samples. The anti-Jk(b) and anti-Di(b) antibodies increased prior to IHTR experienced on day 9. The concentrations of IL-6 and IL-1beta increased from day 2, while the concentration of IL-8 increased from day 7. The anti-HLA class I antibody could be detected 2 days before IHTR. Thus, the anti-Jk(b) and anti-Di(b) antibodies induced the production of inflammatory cytokines and symptoms of DHTR and IHTR. The anti-HLA class I antibody could be produced in spite of using the filer for removing leukocytes, and may take part in the induction of IHTR. Further, blood products should be transfused soon after completing a crossmatch test in patients with anti-RBC alloantibodies.     

Heat-shock induced nuclear retention and recycling inhibition of importin alpha

Heat-shock induces a strong stress response and modifies all aspects of cellular physiology, which involves dynamic changes in the nucleocytoplasmic distributions of a variety of proteins. Many distinct nucleocytoplasmic transport pathways exist in eukaryotic cells, but how a particular transport pathway is regulated under different cellular conditions remains elusive. The finding of this study indicate that conventional nuclear import, which is mediated by importin alpha/beta, is down-regulated, while the nuclear import of 70 kD heat-shock cognate protein is up-regulated in heat-shock cells. Among the factors involved in the mediation of the conventional nuclear import, significant levels of importin alpha accumulate in the nucleus in response to heat-shock. An analysis of the behaviour of importin alpha with fluorescence recovery after photobleaching and fluorescence loss in photobleaching studies show that nuclear importin alpha becomes less mobile and its nucleocytoplasmic recycling is impaired in heat-shock cells. These data coincided well with biochemical and cytological studies. Our present data show that heat-shock induces the nuclear accumulation, nuclear retention, and recycling inhibition of importin alpha, resulting in the suppression of conventional nuclear import. This suggests a new regulatory mechanism for the adaptation of cells to environmental changes, such as heat-shock.     

An improved technique for repeated gavage administration to rat neonates

ABSTRACT  The technique for gavage administration to rat nurslings was improved to allow determination of the direct effects of chemical substances in the nurslings. Rat neonates were treated with distilled water from postnatal day 1 through 20 using this technique. The viability of neonates during the administration period was comparable to that of untreated neonates. No adverse effects of this technique on the development of neonates were found, and no histological alterations of the esophagus or pharynx. Therefore, we conclude that use of our improved gavage administration method will contribute to ensuring successful neonatal development and thus allowing accurate assessment of the toxicological effects of test compounds on rat nurslings.     

Expression of Functional Molecules in Non-Hodgkin's Lymphoma

It is well known that non Hodgkin's lymphoma (NHL) cells express various antigens which are normally involved in a variety of functions. In addition, NHL is diverse in its proliferative capacity. To investigate the relation between these factors and the clinical picture, 45 cases of NHL were studied by immunohistochemistry using snap-frozen materials obtained before therapy. Reactivities with 27 monoclonal antibodies were examined and the results were correlated with clinical findings. The expression of surface μ and CAM-1 in B-NHLs and CD25 in T-NHLs were significantly associated with bone marrow involvement. B-NHLs without expression of CD21(B2) and T-NHLs with CD25 were seen more frequently in cases with a LDH value of over 500 units/ml. The positivity rate of Ki-67 on B-NHLs was correlated with serum LDH value, NHL histologic classification, and overall survival. These data indicate that immunophenotyping and determination of the proliferative capacity of NHL are of value not only for confirmation of the histopathologic classification of the tumor but also for assessment of clinical behavior.     

Effect of N-methyl-2-pyrrolidone on skin permeation of estradiol.

N-Methyl-2-pyrrolidone (NMP) increased the skin permeation of estradiol (E2) in Yucatan micropig epidermis using a modified Franz-type diffusion cell. The addition of NMP significantly increased the fluxes of E2 from water and soybean oil. The flux and skin concentration of E2 were higher from soybean oil than from water and increased with increasing NMP concentrations in soybean oil. Correlation was observed with E2 flux and skin concentration (R(2) = 0.804) NMP enhanced E2 skin permeation because NMP made E2 skin concentration higher. Thus, NMP (10%) was added to the oily gel made by isocetyl isostearate and hydrogenated phospholipid. E2 permeation from the gel without NMP was the same as that from soybean oil suspension. The flux of E2 from the gel with NMP was 0.6 microg/h per cm(2) and might be sufficient for estrogen replacement therapy.     

Prolyl Isomerase Pin1 Expression in the Spinal Motor Neurons of Patients With Sporadic Amyotrophic Lateral Sclerosis

Background and PurposeAmyotrophic lateral sclerosis (ALS) is a fatal motor neuron disease. Selective deficiency of edited adenosine deaminase acting on RNA 2 (ADAR2), a key molecule in the acquisition of Ca²⁺ resistance in motor neurons, has been reported in sporadic ALS (sALS) spinal motor neurons. Since ADAR2 activity is positively regulated by prolyl isomerase Protein never in mitosis gene A interacting-1 (Pin1), a known phosphorylation-dependent peptidyl-prolyl cis/trans isomerase, we investigated Pin1 expression in spinal motor neurons in sALS.MethodsSpecimens of the spinal cord were obtained from the lumbar region in eight sALS patients and age-matched five controls after postmortem examinations. The specimens were double stained with anti-Pin1 and anti-TAR DNA-binding protein of 43 kDa (TDP-43) antibodies, and examined under a fluorescence microscope.ResultsThis study analyzed 254 and 422 spinal motor neurons from 8 sALS patients and 5 control subjects, respectively. The frequency of motor neurons with high cytoplasmic Pin1 expression from the spinal cord did not differ significantly between sALS specimens without cytoplasmic TDP-43 inclusions and control specimens. However, in sALS specimens, neurons for which the Pin1 immunoluminescence intensity in the cytoplasm was at least twice that in the background were more common in specimens with cytoplasmic TDP-43 inclusions (p<0.05 in χ² test).ConclusionsIn sALS, neurons with higher expression levels of Pin1 levels had more TDP-43 inclusions. Despite the feedback mechanism between Pin1 and ADAR2 being unclear, since Pin1 positively regulates ADAR2, our results suggest that higher Pin1 expression levels in motor neurons with TDP-43 pathology from sALS patients represent a compensatory mechanism.     

Human plasma concentrations of herbicidal carbamate molinate extrapolated from the pharmacokinetics established in in vivo experiments with chimeric mice with humanized liver and physiologically based pharmacokinetic modeling

To predict concentrations in humans of the herbicidal carbamate molinate, used exclusively in rice cultivation, a forward dosimetry approach was carried out using data from lowest-observed-adverse-effect-level doses orally administered to rats, wild type mice, and chimeric mice with humanized liver and from in vitro human and rodent experiments. Human liver microsomes preferentially mediated hydroxylation of molinate, but rat livers additionally produced molinate sulfoxide and an unidentified metabolite. Adjusted animal biomonitoring equivalents for molinate and its primary sulfoxide from animal studies were scaled to human biomonitoring equivalents using known species allometric scaling factors and human metabolic data with a simple physiologically based pharmacokinetic (PBPK) model. The slower disposition of molinate and accumulation of molinate sulfoxide in humans were estimated by modeling after single and multiple doses compared with elimination in rodents. The results from simplified PBPK modeling in combination with chimeric mice with humanized liver suggest that ratios of estimated parameters of molinate sulfoxide exposure in humans to those in rats were three times as many as general safety factor of 10 for species difference in toxicokinetics. Thus, careful regulatory decision is needed when evaluating the human risk resulting from exposure to low doses of molinate and related carbamates based on data obtained from rats.     

Inhibition of autophagy by chloroquine makes chemotherapy in nasopharyngeal carcinoma more efficient

Objectives

A combination of platinum-based chemotherapy and radiotherapy is the standard treatment for nasopharyngeal carcinoma (NPC). However, the efficacy of chemotherapy has reached a plateau. Many autophagy studies suggest that autophagy can either promote or suppress to cancer progression. Thus, a role of autophagy in the acquisition of chemoradioresistance has recently been a notable event. Therefore, we examined the relationship between autophagy and chemotherapy in NPC.