Recombinant adeno-associated viral (rAAV) vector-mediated overexpression of alpha-synuclein (alphaSyn) protein has been shown to cause neurodegeneration of the nigrostriatal dopaminergic pathway in rodents and primates. Using serotype-2 rAAV vectors, we recently reported the protective effect of Parkin on alphaSyn-induced nigral dopaminergic neurodegeneration in a rat model. Here we investigated the neuronal specificity of alphaSyn toxicity and the effect of Parkin co-expression in a primate model. We used another serotype (type-1) of AAV vector that was confirmed to deliver genes of interest anterogradely and retrogradely to neurons in rats. The serotype-1 rAAV (rAAV1) carrying alphaSyn cDNA (rAAV1-alphaSyn), and a cocktail of rAAV1-alphaSyn and rAAV1 carrying parkin cDNA (rAAV1-parkin) were unilaterally injected into the striatum of macaque monkeys, resulting in protein expression in striatonigral GABAergic and nigrostriatal dopaminergic neurons. Injection of rAAV1-alphaSyn alone decreased tyrosine hydroxylase immunoreactivity in the striatum compared with the contralateral side injected with a cocktail of rAAV1-alphaSyn and rAAV1-parkin. Immunostaining of striatonigral GABAergic neurons was similar on both sides. Overexpression of Parkin in GABAergic neurons was associated with less accumulation of alphaSyn protein and/or phosphorylation at Ser129 residue. Our results suggest that the toxicity of accumulated alphaSyn is not induced in non-dopaminergic neurons and that the alphaSyn-ablating effect of Parkin is exerted in virtually all neurons in primates.

Models of artificial intelligence (AI) that have billions of parameters can achieve high accuracy across a range of tasks1,2, but they exacerbate the poor energy efficiency of conventional general-purpose processors, such as graphics processing units or central processing units. Analog in-memory computing (analog-AI)3-7 can provide better energy efficiency by performing matrix-vector multiplications in parallel on 'memory tiles'. However, analog-AI has yet to demonstrate software-equivalent (SWeq) accuracy on models that require many such tiles and efficient communication of neural-network activations between the tiles. Here we present an analog-AI chip that combines 35 million phase-change memory devices across 34 tiles, massively parallel inter-tile communication and analog, low-power peripheral circuitry that can achieve up to 12.4 tera-operations per second per watt (TOPS/W) chip-sustained performance. We demonstrate fully end-to-end SWeq accuracy for a small keyword-spotting network and near-SWeq accuracy on the much larger MLPerf8 recurrent neural-network transducer (RNNT), with more than 45 million weights mapped onto more than 140 million phase-change memory devices across five chips.

Nitric oxide (NO) released from (Z)-1-[N-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1- ium-1,2-diolate (DETA/NO or NOC-18) induces apoptosis in human leukemia HL-60 cells. In this study, we isolated a HL-60 variant cell line, HL-NR6, that is resistant to DETA/NO toxicity as assessed by DNA fragmentation, morphology, and colony forming ability. The variant cells also showed resistance to reactive oxygen species (ROS) such as superoxide and hydrogen peroxide as well as NO donors, but not to anti-tumor drugs. We found that HL-NR6 cells when compared with HL-60 cells possessed twice the activities of Cu,Zn-superoxide dismutase (Cu,Zn-SOD) and catalase, but no change in Mn-SOD nor in glutathione peroxidase. Immunoblotting confirmed the high levels of both enzymes in the variant cell. We also observed that ROS generation following DETA/NO exposure was substantially higher in HL-60 cells than in HL-NR6 cells, using the 2',7'-dichlorofluorescein fluorometric method. Moreover, the SOD mimetic Mn(III) tetrakis(1-methyl-4-pyridyl) porphyrin and exogenous catalase effectively attenuated DETA/NO-elicited DNA fragmentation in HL-60 cells. Taken together, these data suggested that the NO resistance in HL-NR6 cells is associated with the increased Cu,Zn-SOD/catalase and that NO-mediated apoptosis in HL-60 cells is correlated with the generation of ROS and derived molecules like peroxynitrite.

We used a fluorescence differential display-PCR (FDD-PCR) technique to analyze the genes expressed in mouse brains collected at nine different developmental stages ranging from 3 days to 15 months after birth, and 5 age-dependently expressed genes were found. Age-dependent expression of each of these 5 genes was confirmed by quantitative real-time PCR analysis. Of the 5 genes, 4 (B1-B4) had high homology with the nucleotide sequences of cDNA clones of known mouse genes (myelin proteolipid protein, transferrin, embryo cDNA from the RIKEN full-length enriched library, and protein tyrosine phosphatase), and the rest (B5) with expressed sequence tags of an unknown gene. Sequencing analysis of the full-length cDNA constructed based on the B5 sequence demonstrated that the gene product of B5 was identical to G-substrate, a specific substrate for cGMP-dependent protein kinase. The expression patterns of known genes obtained in our study may provide a further opportunity to investigate the biological and physiological roles of the proteins they encode.

The mevalonate pathway is important for the biosynthesis of isoprenoids such as geranylgeranylpyrophosphate (GGPP) and farnesylpyrophosphate, as well as cholesterol. It has been reported that treatment with 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitor ameliorates glomerular injury in several experimental models of progressive glomerular disease. However, the effect of HMG-CoA reductase inhibitor on mesangial cell function has not been fully understood. This investigation was performed to elucidate the role of a mevalonate metabolite(s) in mesangial cell proliferation and extracellular matrix accumulation.

Deoxyribonuclease I (DNase I) was purified from the hen pancreas to electrophoretic homogeneity using six-step column chromatography. The purified enzyme showed a molecular mass of about 33 kDa and maximum activity at pH 7.0. It required divalent cations, Mg2+ and Ca2+, for its activity and was inhibited by EDTA, EGTA and an antibody specific to the purified enzyme but not by G-actin. A 1066-bp cDNA encoding hen DNase I was constructed from the total RNA of a hen pancreas using a combination of the reverse transcriptase-polymerase chain reaction and rapid amplification of cDNA ends methods, followed by sequencing. The cDNA was expressed in Escherichia coli, and the recombinant polypeptide exhibited significant enzyme activity. The mature hen DNase I protein was found to consist of 262 amino acids. In human and bovine DNase I four amino acid residues, Glu-13, Tyr-65, Val-67 and Ala-114 are involved in actin binding, whereas in the hen DNase I these positions were occupied by Asp, Phe, Ser and Phe, respectively. A survey of the DNase I distribution in 15 hen tissues showed that the pancreas had the highest levels of both DNase I enzyme activity and DNase I gene expression. The results of our phylogenetic and immunological analyses indicate that the hen DNase I is not closely related to the mammalian enzymes. This is the first report in which has been described the results of molecular, biochemical and immunological analyses on hen DNase I.

The relationship between the chemical structure and the mode of action of piperacillin-analogues (PIPC-analogues) against Escherichia coli and Klebsiella pneumoniae were investigated. The antibacterial activity of PIPC-analogues increased with an increase in the number of carbon atoms at the N-4 position of 2,3-dioxopiperazine. Their mode of action is discussed on the basis of the results of studies on outer membrane permeability, stability to beta-lactamase and binding affinity to penicillin-binding proteins (PBPs). The outer membrane permeability and stability to beta-lactamase were hardly affected by the chain length of the alkyl group at the N-4 position. On the other hand, the affinity to PBPs, especially to PBP 3, became stronger with increase of the number of carbon atoms at N-4 position. These results suggest that increased affinity to PBPs is the main reason for the increased antibacterial activity of the PIPC-analogues reported here.

In Escherichia coli, the SOS response is induced upon DNA damage and results in the enhanced expression of a set of genes involved in DNA repair and other functions. The initial step, self-cleavage of the LexA repressor, is promoted by the RecA protein which is activated upon binding to single-stranded DNA. In this work, induction of the SOS response by the addition of mitomycin C was found to be prevented by overexpression of the dinI gene. dinI is an SOS gene which maps at 24.6 min of the E.coli chromosome and encodes a small protein of 81 amino acids. Immunoblotting analysis with anti-LexA antibodies revealed that LexA did not undergo cleavage in dinI-overexpressed cells after UV irradiation. In addition, the RecA-dependent conversion of UmuD to UmuD' (the active form for mutagenesis) was also inhibited in dinI-overexpressed cells. Conversely, a dinI-deficient mutant showed a slightly faster and more extensive processing of UmuD and hence higher mutability than the wild-type. Finally, we demonstrated, by using an in vitro reaction with purified proteins, that DinI directly inhibits the ability of RecA to mediate self-cleavage of UmuD.

Accumulating evidence indicates that the Cbl protein plays a negative role in immune receptor signaling; however, the mode of Cbl action in B cell receptor (BCR) signaling still remains unclear. DT40 B cells deficient in Cbl showed enhanced BCR-mediated phospholipase C (PLC)-gamma2 activation, thereby leading to increased apoptosis. A possible explanation for the involvement of Cbl in PLC-gamma2 activation was provided by findings that Cbl interacts via its Src homology 2 (SH2) domain with B cell linker protein (BLNK) after BCR ligation. BLNK is a critical adaptor molecule for PLC-gamma2 tyrosine phosphorylation through its binding to the PLC-gamma2 SH2 domains. As a consequence of the interaction between Cbl and BLNK, the BCR-induced recruitment of PLC-gamma2 to BLNK and the subsequent PLC-gamma2 tyrosine phosphorylation were inhibited. Thus, our data suggest that Cbl negatively regulates the PLC-gamma2 pathway by inhibiting the association of PLC-gamma2 with BLNK.