Occurrence of D-histidine residues in antimicrobial poly(arginyl-histidine), conferring resistance to enzymatic hydrolysis

The antimicrobial peptide poly(arginyl-histidine) is secreted by the ergot fungus Verticillium kibiense. We previously showed that poly(arginyl-histidine) from the fungus inhibits the growth of certain microorganisms more effectively than that chemically synthesized from the L-form of arginine and histidine, implying some substantial differences between the fungal and synthetic peptides. To elucidate what causes such differences, we here investigated the structural features of the fungal peptides. The acid hydrolysates of the fungal peptide contained d-histidine. When synthetic poly(L-arginyl-D-histidine) mimicking the fungal peptide was added to the culture of Salmonella typhimurium together with poly(L-arginyl-L-histidine), poly(L-arginyl-D-histidine) was not easily degraded during the incubation compared with poly(L-arginyl-L-histidine). We concluded that the d-form of histidine residues in the fungal peptide prolongs the life of the peptide leading to the enhancement of antimicrobial activity.     

Calcium dependence of native metabotropic glutamate receptor signaling in central neurons

Metabotropic glutamate receptors (mGluRs) are G protein-coupled receptors that are distributed throughout the brain and play important roles in regulation of synaptic efficacy. Some studies report that mGluRs heterologously expressed in nonneuronal cells are sensitive not only to glutamate but also to extracellular Ca²⁺ (Ca _o ²⁺ ). We studied the Ca _o ²⁺ -sensitivity of native mGluRs in mammalian central neurons. In cerebellar Purkinje cells that naturally express type-1 mGluR (mGluR1), physiological levels of Ca _o ²⁺ (around 2 mM) activate mGluR1-mediated intracellular Ca²⁺ mobilization. The activation of the native mGluR1 response to Ca _o ²⁺ appears to be slower than that to glutamate. Ca _o ²⁺ (2 mM) also augments glutamate analog-evoked, native mGluR1-mediated inward cation current and intracellular Ca _o ²⁺ mobilization. Detailed analysis of this effect suggests that Ca _o ²⁺ modulates the glutamate responsiveness of native and heterologously expressed mGluR1s in different manners. These findings suggest that Ca _o ²⁺ may enhance the basal level and glutamate responsiveness of neuronal mGluR signaling in vivo.     

The Wakayama epileptic rat (WER), a new mutant exhibiting tonic-clonic seizures and absence-like seizures.

A new mutant, the Wakayama epileptic rat (WER), exhibiting both spontaneous absence-like behavior and tonic-clonic convulsions, was identified in a colony of Wistar rats. To determine clear seizure characteristics of this mutant strain, we analyzed the mode of inheritance of the convulsion and observed patterns of electroencephalogram (EEG) during the seizures. F1 progeny were produced between the founder male and normal females of the same colony. Animals were monitored through the inbreeding course to analyze genetic control of epileptic behavior. EEGs were recorded using affected animals in the F3-4 and post F13 generations. After the F2 generation, affected rats spontaneously exhibited both absence-like immobile behavior and tonic-clonic convulsions. The absence-like seizures were characterized by motor arrest and head droop. The tonic-clonic convulsions began with neck and forelimb clonus, wild jumping/running, and opisthotonic posturing, and evolved to tonic, then clonic convulsions. Most convulsion onsets occurred between 25-70 days of age. Mating experiments revealed that 0%(0/18) of the animals in F1, 10%(3/26) in F2, 17%(1/6) in backcross progeny and 86% (100/116) in progeny of crosses between epileptic rats showed tonic-clonic convulsions. Ictal cortical EEGs were characterized by 4-6 (5.1 +/- 0.4, mean +/- SD) Hz spike-and-wave complexes in the absence-like seizures and by low-voltage fast waves in the tonic-clonic convulsions. This new mutant rat spontaneously exhibited both absence-like and tonic-clonic seizures. The tonic-clonic seizure was inherited as an autosomal recessive trait with 86% incidence. Thus, the new mutant rat may become a useful model for studying human inherited epilepsies.     

Identification of interleukin-8 converting enzyme as cathepsin L

IL-8 is produced by various cells, and the NH₂-terminal amino acid sequence of IL-8 displays heterogeneity among cell types. The mature form of IL-8 has 72 amino acids (72IL-8), while a precursor form (77IL-8) of IL-8 has five additional amino acids to the 72IL-8 NH₂-terminal. However, it has been unclear how IL-8 is processed to yield the mature form. In this study, converting enzyme was purified as a single 31-kDa band on silver-stained polyacrylamide gel from 160 l of cultured fibroblast supernatant by sequential chromatography. NH₂-terminal amino acid sequence analysis revealed a sequence, EAPRSVDWRE, which was identified as a partial sequence of cathepsin L. Polyclonal antibodies raised against cathepsin L recognized the purified converting enzyme on Western blot. Moreover, human hepatic cathepsin L cleaved 77IL-8 between Arg⁵ and Ser⁶, which is the same cleavage site as the putative converting enzyme, resulting in 72IL-8 formation. These data indicate that the converting enzyme of the partially purified fraction of the human fibroblast culture supernatant was cathepsin L. Furthermore, 72IL-8 was sevenfold more potent than 77IL-8 in a neutrophil chemotaxis assay. These results show that cathepsin L is secreted from human fibroblasts in response to external stimuli and plays an important role in IL-8 processing in inflammatory sites.     

Accumulation of Copper Induces DNA Strand Breaks in Brain Cells of Long-Evans Cinnamon (LEC) Rats, An Animal Model for Human Wilson Disease.

Copper accumulation and induction of DNA strand breaks were investigated in the brain of Long-Evans Cinnamon (LEC) rats, an animal model for human Wilson disease that is a heritable disease of copper accumulation and copper toxicity in the liver, kidney and brain. Copper contents in the brain of LEC rats increased from 20 weeks of age and were approximately 3.5 to 6 folds higher than those in the brain of WKAH rats at 24 weeks of age. Hepatic copper contents in LEC rats increased from 4 to 12 weeks of age in an age-dependent manner, and then decreased from 16 to 20 weeks of age. Thus, we consider that copper accumulated in the liver was released from severely damaged hepatocytes and deposited in the brain, although copper contents in the brain were 1/20-fold lower than those in the liver. We also evaluated the amounts of DNA single-strand breaks (SSBs) in the brain by comet analysis. The proportions of nuclei in the cerebrum and cerebellum without DNA damage decreased, and nuclei with severe DNA damage appeared in LEC rats at 24 weeks of age. The comet scores of cerebrum and cerebellum cells significantly increased in LEC rats and were significantly higher than those in WKAH rats at 24 weeks of age. The results show that SSBs in LEC rat brain cells are induced at a lower concentration of copper than are SSBs in hepatic cells.     

ATM-dependent DNA damage-response pathway as a determinant in chronic myelogenous leukemia

Chronic myelogenous leukemia (CML) begins with an indolent chronic phase, and subsequently progresses to an accelerated or blastic phase. Although several genes are known to be involved in the progression to blastic phase, molecular mechanisms for the evolution toward blast crisis have not been fully identified. Oncogenic stimuli enforce cell proliferation, which requires DNA replication. Unscheduled DNA replication enforced by oncogenic stimuli leads to double strand breaks on DNA. We found the DNA damage-response pathway is activated in bone marrow of chronic-phase CML patients possibly due to an enforced proliferation signal by BCR-ABL expression. Since ataxia telangiectasia mutated (ATM) is a central player of the DNA damage-response pathway, we studied whether loss of this pathway accelerates blast crisis. We crossed Atm-knockout mice with BCR-ABL transgenic mice to test this hypothesis. Interestingly, the loss of one of the Atm alleles was shown to be enough for the acceleration of the blast crisis, which is supported by the finding of increased genomic instability as assayed by breakage–fusion–bridge (BFB) cycle formation. In light of these findings, the DNA damage-response pathway plays a vital role for determination of susceptibility to blast crisis in CML.     

The establishment and validation of efficient assays for anti-IIa and anti-Xa activities of heparin sodium and heparin calcium

Heparin is used as an anticoagulant drug. The anticoagulation process is mainly caused by the interaction of heparin with antithrombin followed by inhibition of anticoagulant factor IIa and factor Xa. The anti-factor IIa and anti-factor Xa activities of heparin are critical for its anticoagulant effect; however, physicochemical methods that can reflect these activities have not been established. Thus, the measurements of anti-IIa and anti-Xa activities by biological assay are critical for the quality control of heparin products. Currently in the Japanese Pharmacopoeia (JP), the activities of heparin sodium and heparin calcium are measured by an anti-Xa activity assay (anti-Xa assay), but anti-IIa activity is not measured. Here, we established an anti-IIa activity assay (anti-IIa assay) and an anti-Xa assay having good accuracy and precision. When samples having a relative activity of 0.8, 1.0 and 1.2 were measured by the established anti-IIa and anti-Xa assays in nine laboratories, good accuracy (100.0–102.8% and 101.6–102.8%, respectively), good intermediate precision (1.9–2.1% and 2.4–4.2%, respectively) and good reproducibility (4.0–4.8% and 3.6–6.4%, respectively) were obtained. The established anti-IIa and anti-Xa assays have similar protocols, and could be performed by a single person without a special machine. The established assays would be useful for quality control of heparin.     

All APOBEC3 family proteins differentially inhibit LINE-1 retrotransposition

Approximately 17% of the human genome is comprised of long interspersed nuclear element 1 (LINE-1, L1) non-LTR retrotransposons. L1 retrotransposition is known to be the cause of several genetic diseases, such as hemophilia A, Duchene muscular dystrophy, and so on. The L1 retroelements are also able to cause colon cancer, suggesting that L1 transposition could occur not only in germ cells, but also in somatic cells if innate immunity would not function appropriately. The mechanisms of L1 transposition restriction in the normal cells, however, are not fully defined. We here show that antiretroviral innate proteins, human APOBEC3 (hA3) family members, from hA3A to hA3H, differentially reduce the level of L1 retrotransposition that does not correlate either with antiviral activity against Vif-deficient HIV-1 and murine leukemia virus, or with patterns of subcellular localization. Importantly, hA3G protein inhibits L1 retrotransposition, in striking contrast to the recent reports. Inhibitory effect of hA3 family members on L1 transposition might not be due to deaminase activity, but due to novel mechanism(s). Thus, we conclude that all hA3 proteins act to differentially suppress uncontrolled transposition of L1 elements.     

Iron dependent degradation of an isothiazolone biocide (5-chloro-2-methyl-4-isothiazolin-3-one)

An isothiazolone biocide, 5-chloro-2-methyl-4-isothiazolin-3-one (CMI), was degraded in the presence of iron. According to the Fe-dependent degradation of CMI, stoichiometric production of chloride was observed. Copper and stainless steel did not enhance the physico-chemical degradation of CMI, whilst phosphate inhibited the Fe-dependent degradation. Neither aerobic nor anaerobic conditions influenced the Fe-dependent CMI degradation. Furthermore, FeO(OH)-powder and Fe(3)O(4)-powder did not lead to the physico-chemical degradation of CMI. Rapid disappearance of CMI was observed in an operating cooling water plant. CMI added to the cooling tower declined from 1.4 mg l(-1) to < 0.1 mg l(-1) in 2 d. This finding is important in optimising the use of CMI and combating resistance if encountered.