Calpain inhibitor-1 protects the rat heart from ischemia-reperfusion injury: analysis by mechanical work and energetics

We hypothesized that calpain inhibitor-1 protected left ventricular (LV) function from ischemia-reperfusion injury by inhibiting the proteolysis of alpha-fodrin. To test this hypothesis, we investigated the effect of calpain inhibitor-1 on LV mechanical work and energetics in the cross-circulated rat hearts that underwent 15-min global ischemia and 60-min reperfusion (n = 9). After ischemia-reperfusion with calpain inhibitor-1, mean end-systolic pressure at midrange LV volume and systolic pressure-volume area (PVA) at midrange LV volume (total mechanical energy per beat) were hardly changed, although they were significantly (P < 0.01) decreased after ischemia-reperfusion without calpain inhibitor-1. Mean myocardial oxygen consumption per beat (Vo(2)) intercepts (PVA-independent Vo(2); Vo(2) for the total Ca(2+) handling in excitation-contraction coupling and basal metabolism) of Vo(2)-PVA linear relations were also unchanged after ischemia-reperfusion with calpain inhibitor-1, although they were significantly (P < 0.01) decreased after ischemia-reperfusion without calpain inhibitor-1. There were no significant differences in O(2) costs of LV PVA and contractility among the hearts in control (or normal) postischemia-reperfusion and postischemia-reperfusion with calpain inhibitor-1. Western blot analysis of alpha-fodrin and the immunostaining of 150-kDa products of alpha-fodrin confirmed that calpain inhibitor-1 almost completely protected the proteolysis of alpha-fodrin. Our results indicate that calpain inhibitor-1 prevents the heart from ischemia-reperfusion injury associated with the impairment of total Ca(2+) handling by directly inhibiting the proteolysis of alpha-fodrin.     

Overexpression of mutated Cu,Zn-SOD in neuroblastoma cells results in cytoskeletal change

Amyotrophic lateral sclerosis (ALS) involves the progressive degeneration of motor neurons in the spinal cord and the motor cortex. It has been shown that 15–20% of patients with familial ALS (FALS) have defects in the Sod1 gene, which encodes Cu,Zn-superoxide dismutase (SOD). To elucidate the pathological role of mutated Cu,Zn-SOD, we examined the issue of whether mutated Cu,Zn-SOD affects the cell cycle. Mouse neuroblastoma Neuro-2a cells were transfected with human wild-type or mutated (G37R, G93A) Cu,Zn-SOD. Mutated, Cu,Zn-SOD-transfected cells exhibited marked retardation in cell growth and G₂/M arrest. They also displayed lower reactivity to phalloidin, indicating that the cytoskeleton was disrupted. Immunoprecipitation, two-dimensional gel electrophoresis, and Western blot analysis indicated that mutated Cu,Zn-SOD associates with actin. Similar results were obtained by in vitro incubation experiments with purified actin and mutated Cu,Zn-SOD (G93A). These results suggest that mutated Cu,Zn-SOD in FALS causes cytoskeletal changes by associating with actin, which subsequently causes G₂/M arrest and growth retardation. amyotrophic lateral sclerosis; copper; zinc superoxide dismutase; G₂/M arrest; neurodegenerative disease     

Atypical virulence in a type III Toxoplasma gondii strain isolated in Japan

The virulence of a type III Toxoplasma gondii strain isolated in Japan and designated here as TgCatJpGi1/TaJ was examined in mice and micro minipigs in this study. Despite its type III genotype, oral or intraperitoneal inoculation of cysts from it resulted in severe virulence in C57BL/6J and BALB/c mice. In contrast, mice inoculated with a high dose of TgCatJpGi1/TaJ tachyzoites showed no obvious clinical signs of infection, and all of them survived for >21?days post-inoculation. Furthermore, no clinical signs of infection were seen when micro minipigs were inoculated with 900 cysts. Interestingly, our allelic type screening of the virulence-related rop5, rop16, rop17, and rop18 genes, as based on restriction fragment length polymorphism analysis (RFLP), revealed that the RFLP patterns for TgCatJpGi1/TaJ were identical to those from nonvirulent type III parasites. These results suggest that TgCatJpGi1/TaJ possesses an unknown virulence factor or factors.     

Optical study of spatiotemporal inhibition evoked by two-tone sequences in the guinea pig auditory cortex

Spatiotemporal response patterns in the anterior and dorsocaudal fields of the guinea pig auditory cortex after two-tone sequences were studied in anesthetized animals (Nembutal 30 mg kg⁻¹) using an optical recording method (voltage-sensitive dye RH795, 12 × 12 photodiode array). Each first (masker) and second (probe) tone was 30 ms long with a 10-ms rise-fall time. Masker-probe pair combinations of the same or different frequencies with probe delays of 30–150 ms were presented to the ear contralateral to the recording side. With same-frequency pairs, responses to the probe were inhibited completely after probe delays of less than 50 ms and the inhibition lasted for more than 150 ms, and the inhibition magnitudes in different isofrequency bands of the anterior field were essentially the same. With different-frequency (octave-separated) pairs, responses to the probe were not inhibited completely even after probe delays as short as 30 ms, and the inhibition lasted only for 110–130 ms. Inhibition magnitudes were different from location to location. Accepted: 4 August 1997     

Establishment of internal-image anti-idiotype monoclonal antibodies to a human antibody to lung cancer

 Internal-image anti-idiotype antibodies are expected to enhance anticancer effector mechanisms in vivo. The objective of this study was to establish hybridomas producing anti-idiotype monoclonal antibodies against a human monoclonal antibody (hmAb) 4G12 that reacts strongly with lung squamous cell carcinomas. BALB/c female mice 6 weeks old were immunized with 4G12. Splenocytes were hybridized with P3U1 cells and hybrid cells secreting anti-4G12 hmAb were cloned. Two clones reacted with 4G12 hmAb but not with 3H12 IgM hmAb, human IgM, human serum or fetal calf serum. These two Ab2 antibodies (IgG1κ) 2B12 and 2H1 demonstrated 91.5% and 90.3% inhibition in their reactivity with radiolabelled 4G12 on PC10 cells, indicating that 2B12 and 2H1 antibodies were of the Ab2β type. In criss-cross inhibition assays, the binding of 2B12 or 2H1 to 4G12 was not inhibited by 2H1 or 2B12. Thus 2B12 and 2H1 were thought to recognize the different epitopes on the antigen-binding sites. Antisera against 2B12 and 2H1 demonstrated specific reactivity to PC10 cells. The two Ab2β antibodies, 2B12 and 2H1, express internal images of lung squamous cell carcinoma recognized by the 4G12 antibody and may be useful for cancer immunotherapy. Received: 20 September 1996 / Accepted: 2 January 1997     

'Click peptide': a novel 'O-acyl isopeptide method' for peptide synthesis and chemical biology-oriented synthesis of amyloid beta peptide analogues.

After over a decade of studies on aspartic protease inhibitors and water-soluble prodrugs, we have been developing a novel method, since 2003, called 'O-acyl isopeptide method', for the synthesis of peptides containing difficult sequences. With our recent discoveries of 'O-acyl isodipeptide unit' and the 'racemization-free segment condensation method', this method has further evolved as a general synthetic method for peptides. Moreover, 'Click Peptide', which could be a powerful tool for identifying the pathological functions of amyloid beta peptides in Alzheimer's disease, represents a valuable use of the isopeptide method in Chemical Biology-oriented research.     

Three‐dimensional ultrastructure of chloroplast pockets formed under salinity stress

We investigated the invagination structure of a chloroplast that surrounds organelles such as mitochondria and peroxisomes within a thin layer of chloroplast stroma, which is called a chloroplast pocket. In this study, chloroplast pockets were observed in rice plants subjected to salinity stress but not under moderate growth condition. They included cytosol, transparent structure, lipid bodies, mitochondria, and peroxisomes. We constructed the three‐dimensional architecture of chloroplast pockets by using serial images obtained by transmission electron microscopy and focused ion beam‐scanning electron microscopy. Three types of chloroplast pockets were observed by transmission electron microscopy: Organelles were completely enclosed in a chloroplast pocket (enclosed type), a chloroplast pocket with a small gap in the middle part (gap type), and a chloroplast pocket with one side open (open type). Of the 70 pockets observed by serial imaging, 35 were enclosed type, and 21 and 14 were gap and open types, respectively. Mitochondria and peroxisomes were often in contact with the chloroplast pockets. Focused ion beam‐scanning electron microscopy revealed chloroplasts with a sheet structure partially surrounding peroxisomes. This fact suggests that chloroplasts might construct large sheet structures that would be related to the formation of chloroplast pockets.     

Fast liquid chromatographic assay of androgen aromatase activity

A rapid, sensitive nonradiometric assay method for the enzymatic aromatization of androgens has been developed using reversed-phase fast liquid chromatography. The use of a 3-microns-particle, 5-cm-long column allowed analysis of androstenedione, estrone, testosterone, and estradiol within 1 min. The reliability of the method was demonstrated by measuring the aromatase activity of human placental microsomes and that of the purified reconstituted aromatase system using both substrates. The rapid analysis enabled the processing of a large number of samples in a short time, which makes the present method especially suitable for the analysis of chromatographic fractions obtained during enzyme purifications and for enzyme kinetic studies.     

DNA-regulated arginine-specific mono(ADP-ribosyl)ation and de-ADP-ribosylation of endogenous acceptor proteins in human neutrophils

Arginine-specific mono(ADP-ribosyl)ation and de-ADP-ribosylation reactions of endogenous acceptor proteins were examined using human neutrophils. The cells contained arginine-specific ADP-ribosyltransferase, acceptor proteins and hydrolase catalyzing the release of ADP-ribose from the ADP-ribose/acceptor conjugate. One major acceptor protein with an apparent molecular mass of 27 kDa was detected in the neutrophils. The ADP-ribosylation of this protein was greatly enhanced when double-stranded DNA was added. The release of ADP-ribose from the ADP-ribosyl core-histones was suppressed. These findings provide clues as to the physiological function of neutrophil ADP-ribosyltransferase.