Characterization of recombinant human renin: Kinetics,pH-stability, and peptidomimetic inhibitor binding

The kinetic behavior andpH-stability of recombinant human renin was analyzed using a new fluorogenic substrate based on the normal P₆-P₃ renin cleavage sequence in human angiotensinogen. The design of this fluorogenic substrate makes possible, for the first time, direct monitoring of the kinetics of proteolytic conversion of prorenin to renin. ThepH-stability profile for renin, measured with the substrate at 25°C, indicated a broad plateau of stability betweenpH 6.0 and 10.0. Analysis of thepH-activity profile of renin for the substrate indicated a minimumK _m (1.8 µM) atpH 7.4 and a maximumV _m betweenpH 7.4 and 8.0. The thermodynamics of the binding of a novel, soluble, peptidomimetic inhibitor to renin indicated it is possible to retain the tight-binding characteristics and enthalpy contributions to binding of larger peptide-derived inhibitors, while reducing inhibitor size and entropic contributions to binding. A novel derivative of the fluorogenic substrate, containing a 3-methyl histidine substitution at the P₂ site, was used to test the recent hypothesis that renin functions by virtue of substrate-directed catalysis.     

Acetylation changes tau interactome to degrade tau in Alzheimer’s disease animal and organoid models

Alzheimer's disease (AD) is an age‐related neurodegenerative disease. The most common pathological hallmarks are amyloid plaques and neurofibrillary tangles in the brain. In the brains of patients with AD, pathological tau is abnormally accumulated causing neuronal loss, synaptic dysfunction, and cognitive decline. We found a histone deacetylase 6 (HDAC6) inhibitor, CKD‐504, changed the tau interactome dramatically to degrade pathological tau not only in AD animal model (ADLP^APT) brains containing both amyloid plaques and neurofibrillary tangles but also in AD patient‐derived brain organoids. Acetylated tau recruited chaperone proteins such as Hsp40, Hsp70, and Hsp110, and this complex bound to novel tau E3 ligases including UBE2O and RNF14. This complex degraded pathological tau through proteasomal pathway. We also identified the responsible acetylation sites on tau. These dramatic tau‐interactome changes may result in tau degradation, leading to the recovery of synaptic pathology and cognitive decline in the ADLP^APT mice.     

Targeting of cancer cells with monoclonal antibodies specific for C3b(i)

Purpose: The goal of this research is to determine the feasibility of an immunotherapeutic approach based on the use of monoclonal antibodies (mAb) to target complement activation fragments on opsonized cancer cells. Methods: We investigated whether treatment of LNCaP and C4-2 human prostate cancer cell lines with normal human serum would allow for deposition of sufficient amounts of the complement-activation protein C3b and its fragments [collectively referred to as C3b(i)] such that these proteins could serve as cancer-cell-associated antigens for targeting by mAb. Radioimmunoassays, flow cytometry, and magnetic purging with specific immunomagnetic beads were used for the analyses. Results: In vitro opsonization of human prostate cancer cells with normal human serum resulted in deposition of C3b(i) in sufficient quantity (approx. 100,000 molecules/cell) for the cells to be targeted in a variety of protocols. We found that ⁵¹Cr-labeled and C3b(i)-opsonized cancer cells could be specifically purged at high efficiency (95%–99%) using anti-C3b(i) mAb covalently coupled to magnetic beads. Flow-cytometry experiments indicated that most normal white cells were not removed under similar conditions. Opsonization of cancer cells with sera from men with prostate cancer led to lower levels of cell-associated IgM and, subsequently, lower amounts of C3b(i) deposited than in normal subjects. Prototype experiments suggested that this deficiency could be corrected by addition of IgM from normal donor plasma. Conclusion: mAb directed against complement-activation products may provide new opportunities to deliver diagnostic and therapeutic agents selectively to cancer cells and tumor deposits. These opportunities may include ex vivo purging of C3b(i)-opsonized cancer cells prior to autologous bone marrow or stem cell transplantation. Received: 17 February 2000 / Accepted: 1 August 2000     

Concurrent expression of heme oxygenase-1 and p53 in human retinal pigment epithelial cell line

Heme oxygenase-1 (HO-1) is a stress-responsive protein that is known to regulate cellular functions such as cell proliferation, inflammation, and apoptosis. Here, we investigated the effects of HO activity on the expression of p53 in the human retinal pigment epithelium (RPE) cell line ARPE-19. Cobalt protoporphyrin (CoPP) induced the expression of both HO-1 and p53 without significant toxicity to the cells. In addition, the blockage of HO activity with the iron chelator DFO or with HO-1 siRNA inhibited the CoPP-induced expression of p53. Similarly, zinc protoporphyrin (ZnPP), an inhibitor of HO, suppressed p53 expression in ARPE-19 cells, although ZnPP increased the level of HO-1 protein while inhibiting HO activity. Also, CoPP-induced p53 expression was not affected by the formation of reactive oxygen species (ROS). Based on these results, we conclude that HO activity is involved in the regulation of p53 expression in a ROS-independent mechanism, and also suggest that the expression of p53 in ARPE-19 cells is associated with heme metabolites such as biliverdin/bilirubin, carbon monoxide, and iron produced by the activity of HO.     

6-O-Angeloylenolin induced cell-cycle arrest and apoptosis in human nasopharyngeal cancer cells

High incidence of lymph node spread and distant metastasis make poor prognosis in human nasopharyngeal carcinoma (NPC). Therefore, better treatments for NPC are needed. This study investigated the anticancer activity of 6-O-angeloylenolin, a plant-derived sesquiterpene lactone, on human nasopharyngeal cancer (CNE) cells. 6-O-Angeloylenolin was found to significantly inhibit the proliferation of CNE cells. The rate of inhibition was comparable to that of cisplatin, a well known chemotherapeutic agent used to treat NPC. Further mechanistic studies revealed that 6-O-angeloylenolin caused cell-cycle arrest in the S and G2/M phases and, subsequently, the induction of apoptosis. Rapid repressions of cyclin D1, cyclin D3, p27, cdc25c and p-cdc25c (Ser216) were observed after 1-h treatment, followed by decreases in the expression of CDK4, cdc2 and p-cdc2 (Tyr15) after 12 h. Down-regulations of p-Rb (Ser780), p21^Waf1/Cip1, cyclin A, and cyclin E were also detected as later events. Two early events that marked the occurrence of apoptosis were phosphatidylserine exposure and mitochondria membrane potential depletion, which occurred after 12 h of treatment, while a sub-G1 peak was also detected after 36-h treatment. Apoptosis induction was further confirmed by other apoptotic features, including nuclear fragmentation, and PARP cleavage. Moreover, 6-O-angeloylenolin caused the release of cytochrome c and AIF to the cytosol by regulating the expression of the Bcl-2 family proteins. However, pretreatment of the general caspase inhibitor failed to attenuate the apoptosis induction effect, suggesting that apoptosis induction of 6-O-angeloylenolin was independent of caspase activation. While 6-O-angeloylenolin also triggered the activation of Akt, ERK and JNK, only the JNK inhibitor significantly decreased the extent of cell death and apoptosis in CNE cells. Taken together, these results suggest the potential applicability of 6-O-angeloylenolin as a candidate for NPC treatment.     

Floral Spray Transformation Can Efficiently Generate Arabidopsis

In this study, floral spray and floral dip were used to replace the vacuum step in the Agrobacterium-mediated transformation of a superoxide dismutase (SOD) gene into Arabidopsis. The transgene was constructed by using a CaMV 35S promoter to drive a rice cytosolic CuZnSOD coding sequence in Arabidopsis. The transgene construct was developed in binary vectors and mobilized into Agrobacterium. When Arabidopsis plants started to initiate flower buds, the primary inflorescence shoots were removed and then transformed by floral spray or floral dip. More than 300 transgenic plants were generated to assess the feasibility of floral spray used in the in planta transformation. The result indicates that the floral spray method of Agrobacterium can achieve rates of in planta transformation comparable to the vacuum-infiltration and floral dip methods. The floral spray method opens up the possibility of in planta transformation of plant species which are too large for dipping or vacuum infiltration.     

The Postnatal Development of the Cerebellum— A fMRI and Silver Study

Summary The aim of this study was to evaluate the postnatal development of the cerebella of the pig and to compare this with the activation of the fMRI. The cells in the cerebella were studied by silver technique and the activation of the fMRI in the cerebella was initiated by flexion and extension of the hind paw. Our results showed an increase of the branching of the cells of the cerebellar cortex postnatally, coordinated with registration of fMRI active sites in the cerebella at 6-month postnatal. We concluded that the full maturation of the cerebella was around 6-month postnatal in the pig.