Neurologic Abnormalities in Two Dogs Suspected Lyme Disease

A 2-year-old mongrel dog developed neurological signs following tick bite. These included astasia, persistent tonic convulsions and hyper-reflexia. Both serum IgG and IgM antibody titers against Borrelia burgdorferi were positive in enzyme-linked immunosorbent assay (ELISA). The neurological signs subsided after high-dose penicillin and streptomycin treatment. A strain of spirochetes (P427a) was isolated from the midgut of Ixodes persulcatus feeding on the dog. Morphological characteristic, immunological property and protein profile revealed that the isolate was B. burgdorferi. Similarly, a 2-year-old Labrador retriever dog developed neurological signs after tick bite and showed a positive IgG antibody titer against B. burgdorferi. Antibiotic treatment was effective also in this case. These findings suggest that neurological symptoms shown in both dogs were caused by infection with B. burgdorferi.     

Targeting age‐specific changes in CD4+ T cell metabolism ameliorates alloimmune responses and prolongs graft survival

Age impacts alloimmunity. Effects of aging on T‐cell metabolism and the potential to interfere with immunosuppressants have not been explored yet. Here, we dissected metabolic pathways of CD4⁺ and CD8⁺ T cells in aging and offer novel immunosuppressive targets. Upon activation, CD4⁺ T cells from old mice failed to exhibit adequate metabolic reprogramming resulting into compromised metabolic pathways, including oxidative phosphorylation (OXPHOS) and glycolysis. Comparable results were also observed in elderly human patients. Although glutaminolysis remained the dominant and age‐independent source of mitochondria for activated CD4⁺ T cells, old but not young CD4⁺ T cells relied heavily on glutaminolysis. Treating young and old murine and human CD4⁺ T cells with 6‐diazo‐5‐oxo‐l‐norleucine (DON), a glutaminolysis inhibitor resulted in significantly reduced IFN‐γ production and compromised proliferative capacities specifically of old CD4⁺ T cells. Of translational relevance, old and young mice that had been transplanted with fully mismatched skin grafts and treated with DON demonstrated dampened Th1‐ and Th17‐driven alloimmune responses. Moreover, DON diminished cytokine production and proliferation of old CD4⁺ T cells in vivo leading to a significantly prolonged allograft survival specifically in old recipients. Graft prolongation in young animals, in contrast, was only achieved when DON was applied in combination with an inhibition of glycolysis (2‐deoxy‐d‐glucose, 2‐DG) and OXPHOS (metformin), two alternative metabolic pathways. Notably, metabolic treatment had not been linked to toxicities. Remarkably, immunosuppressive capacities of DON were specific to CD4⁺ T cells as adoptively transferred young CD4⁺ T cells prevented immunosuppressive capacities of DON on allograft survival in old recipients. Depletion of CD8⁺ T cells did not alter transplant outcomes in either young or old recipients. Taken together, our data introduce an age‐specific metabolic reprogramming of CD4⁺ T cells. Targeting those pathways offers novel and age‐specific approaches for immunosuppression.     

In Vitro Reconstruction of Neuronal Networks Derived from Human iPS Cells Using Microfabricated Devices

Morphology and function of the nervous system is maintained via well-coordinated processes both in central and peripheral nervous tissues, which govern the homeostasis of organs/tissues. Impairments of the nervous system induce neuronal disorders such as peripheral neuropathy or cardiac arrhythmia. Although further investigation is warranted to reveal the molecular mechanisms of progression in such diseases, appropriate model systems mimicking the patient-specific communication between neurons and organs are not established yet. In this study, we reconstructed the neuronal network in vitro either between neurons of the human induced pluripotent stem (iPS) cell derived peripheral nervous system (PNS) and central nervous system (CNS), or between PNS neurons and cardiac cells in a morphologically and functionally compartmentalized manner. Networks were constructed in photolithographically microfabricated devices with two culture compartments connected by 20 microtunnels. We confirmed that PNS and CNS neurons connected via synapses and formed a network. Additionally, calcium-imaging experiments showed that the bundles originating from the PNS neurons were functionally active and responded reproducibly to external stimuli. Next, we confirmed that CNS neurons showed an increase in calcium activity during electrical stimulation of networked bundles from PNS neurons in order to demonstrate the formation of functional cell-cell interactions. We also confirmed the formation of synapses between PNS neurons and mature cardiac cells. These results indicate that compartmentalized culture devices are promising tools for reconstructing network-wide connections between PNS neurons and various organs, and might help to understand patient-specific molecular and functional mechanisms under normal and pathological conditions.     

Inhibitory effect of a synthetic polypeptide, poly(Tyr-Ile-Gly-Ser-Arg), on the metastatic formation of malignant tumour cells

A new type of polypeptide containing the repeating sequence of cell attachment site in laminin, poly(Tyr-Ile-Gly-Ser-Arg), was synthesized by the polymerization procedure with diphenylphosphoryl azide (DPPA). We found that this polypeptide inhibited the metastatic formation in the lung of malignant tumour cells far more effectively than pentapeptide.     

Partial Asymmetric Synthesis in α-Alkylation of Enamine with Electrophilic Olefin

About 30 dipeptides and some tripeptides were led to new benzimidazole derivatives by incorporating their carboxyl groups into benzimidazole ring by the reaction with o-phenylenediamine. The ring closure to benzimidazole was well achieved by heating mildly at a moderate temperature in acetic acid.

Some benzimidazole derivatives of peptides had remarkable phytotoxicities.     

2′-C-Cyano-2′-deoxy-1-β-D-arabinofuranosylcytosine (CNDAC): A Mechanism-Based DNA-Strand-Breaking Antitumor Nucleoside1

Abstract

The antitumor mechanism of action of 2′-C-cyano-2′-deoxy-1-β-d-arabinofuranosylcytosine (CNDAC) has been examined. CNDAC was designed as a potentially DNA-self-strand-breaking nucleoside. It had potent antitumor effects against various solid tumors in vitro as well as in vivo. Using a chain-extension method with Vent (exo^?) DNA polymerase and a short primer/template system, we found that 5′-triphosphate of CNDAC (CNDACTP) was incorporated into the primer at a site opposite a guanine residue in the template. After further chain-extension reaction of the primer containing CNDAC at the 3′-terminus, chain elongation was not observed. Therefore, CNDACTP appeared to act as a chain-terminator. Analyses of the structure of the 3′-terminus in the primer revealed 2′-C-cyano-2′,3′-didehydro-2′,3′-dideoxycytidine (ddCNC) together with CNDAC and 2′-C-cyano-2′-deoxy-1-β-d-ribofuranosylcytosine (CNDC). The existence of ddCNC in the 3′-end of the primer would be due to the self-strand-break by the nucleotide incorporated next to CNDAC. We also found that CNDAC was epimerized to CNDC in near-neutral to alkaline media. Therefore, CNDC found in the primer was epimerized after incorporation of CNDACTP into the primer. We also described the metabolism of CNDAC.     

Alkaline phosphatase isozymes in the midgut of silkworm: purification of high pH-stable microvillus and labile cytosolic enzymes

Summary Genetically defined alkaline phosphatase (ALP) isozymes from the larval midgut tissues ofBombyx mori were purified and characterized. The membrane-bound form (m-ALP) was solubilized with 1% Triton X-100, then purified by DEAE-Sephacel, Con A-Sepharose 4B and Ultrogel AcA 34 column chromatography. The soluble form (s-ALP) was purified by DEAE-Sephacel, epoxy Toyopearl coupled with phosphonic acid and Ultrogel AcA 34 column chromatography. About 840- and 650-fold purification were achieved for m-ALP and s-ALP, respectively, and both ALPs were homogeneous as judged by poly-acrylamide gel electrophoresis. Both forms were found to be similar (MW=68 000 in gel permeation chromatography, and a single subunit as a monomer in denaturing SDS-polyacrylamide gels with MW=58 000 for m-ALP and MW=61 000 for s-ALP). The pH optima of ALPs were shown to lie at 10.9 (m-ALP) and 9.8 (s-ALP), the former being extremely stable even in pH 10–12 which accords with the physiological milieu inBombyx midgut lumen. The K_m values of the m-ALP and s-ALP forp-nitrophenyl phosphate were 1.99 and 1.49 mM, respectively. Both ALPs had a similar substrate specificity.l-Cysteine strongly inhibited both ALPs, but inhibitory effects ofl-phenylalanine,l-homoarginine andl-leucine were undetectable for s-ALP and very weak for m-ALP. A comparison of enzymatic properties on two ALPs suggested that each isozyme plays different roles.Abbreviations m-ALP membrane-bound alkaline phosphatase - s-ALP soluble alkaline phosphatase