SV40 T-antigen is required for maintenance of immortal growth in SV40-transformed human fibroblasts.

Two lines of immortal human fibroblasts were isolated following transfection of TIG-3 cells with plasmid DNA, pMT-1ODtsA, that contained SV40 early gene with a deletion in replication origin and ts mutation in coding sequence for T-antigen. These cells continued proliferation at 34 degrees C, over 565 population doubling level (PDL) which is far over the limited division potential of untransformed normal TIG-3 of 70-80 PDL. When the culture temperature was shifted to 40 degrees C after 70 PDL, they ceased proliferation immediately. One of these immortal clones, SVts8, lost its ts phenotype after retransformation with wtT-antigen gene. These results indicated that the function of intact T-antigen is required for maintenance of immortal proliferation, at least in one of the SV40 transformed immortal clones.     

Calcium/phospholipid-dependent kinase recognizes sites in microtubule-associated protein 2 which are phosphorylated in living brain and are not accessible to other kinases

Microtubule-associated protein 2 (MAP-2) purified after microtubule assembly cycles from bovine brain had been shown to contain about 10 esterified phosphates (mol/mol), which were relatively phosphatase resistant and essentially confined to the projection domain which contributes to the visible arms on microtubules. The kinase responsible for phosphorylating these sites had not been identified. We have approached this question by using a phosphatase that releases the bulk of these residues and then determining which kinase can now add additional residues corresponding to those released. Three kinases were chosen because of their abundance in brain and/or proximity to microtubules. Of these only Ca/phospholipid-dependent kinase was able to recognize the previously occupied sites. We also found that MAP-2 isolated from rat brain without assembly cycles contained more phosphate than previously recognized, greater than 30 mol/mol, suggesting that 20 of these had been inadvertently released by phosphatase during assembly cycles. All 3 kinases (Ca/phospholipid-dependent, cAMP-dependent, and Ca/calmodulin-dependent kinase II) recognized more sites in the bovine than in the rat MAP-2.     

Induction of histidine decarboxylase activity in mouse skin after application of indole alkaloids,a new class of tumor promoter

The effects of various promoters in two-step carcinogenesis on the induction of histidine decarboxylase in the skin of mice was investigated. The potencies of various phorbol esters in inducing histidine decarboxylase activity were parallel with their tumor-promoting activities. Indole alkaloids such as dihydroteleocidin B and lyngbyatoxin A, which induced ornithine decarboxylase and promoted tumor development in the skin of mice with the same potency as 12-O-tetradecanoylphorbol-13-acetate (TPA), also induced histidine decarboxylase activity. These results suggest that histamine produced by this inducible histidine decarboxylase may play some role in tumor promotion.     

DAJIN enables multiplex genotyping to simultaneously validate intended and unintended target genome editing outcomes

Genome editing can introduce designed mutations into a target genomic site. Recent research has revealed that it can also induce various unintended events such as structural variations, small indels, and substitutions at, and in some cases, away from the target site. These rearrangements may result in confounding phenotypes in biomedical research samples and cause a concern in clinical or agricultural applications. However, current genotyping methods do not allow a comprehensive analysis of diverse mutations for phasing and mosaic variant detection. Here, we developed a genotyping method with an on-target site analysis software named Determine Allele mutations and Judge Intended genotype by Nanopore sequencer (DAJIN) that can automatically identify and classify both intended and unintended diverse mutations, including point mutations, deletions, inversions, and cis double knock-in at single-nucleotide resolution. Our approach with DAJIN can handle approximately 100 samples under different editing conditions in a single run. With its high versatility, scalability, and convenience, DAJIN-assisted multiplex genotyping may become a new standard for validating genome editing outcomes.

Genome editing can introduce designed mutations into a target genomic site, but also into unintended off-target sites. DAJIN, a novel nanopore sequencing data analysis tool, identifies and quantifies allele numbers and their mutation patterns, reporting consensus sequences and visualizing mutations in alleles at single-nucleotide resolution.     

Habitat occupancy of sloth bear Melursus ursinus in Chitwan National Park,Nepal

Mammals have experienced a massive decline in their populations and geographic ranges worldwide. The sloth bear, Melursus ursinus (Shaw, 1791), is one of many species facing conservation threats. Despite being endangered in Nepal, decades of inattention to the situation have hindered their conservation and management. We assessed the distribution and patterns of habitat use by sloth bears in Chitwan National Park (CNP), Nepal. We conducted sign surveys from March to June, 2020, in 4 × 4 km grids (n = 45). We collected detection/non‐detection data along a 4‐km trail that was divided into 20 continuous segments of 200 m each. We obtained environmental, ecological, and anthropogenic covariates to understand determinants of sloth bear habitat occupancy. The data were analyzed using the single‐species single‐season occupancy method, with a spatially correlated detection. Using repeated observations, these models accounted for the imperfect detectability of the species to provide robust estimates of habitat occupancy. The model‐averaged occupancy estimate for the sloth bear was 69% and the detection probability was 0.25. The probability of habitat occupancy by sloth bears increased with the presence of termites and fruits and in rugged, dry, open, undisturbed habitats. Our results indicate that the sloth bear is elusive, functionally unique, and widespread in CNP. Future conservation interventions and action plans aimed at sloth bear management must adequately consider their habitat requirements.     

Resonance Raman study on synergistic activation of soluble guanylate cyclase by imidazole, YC-1 and GTP

Soluble guanylate cyclase (sGC), a physiological nitric oxide (NO) receptor, is a heme-containing protein and catalyzes the conversion of GTP to cyclic GMP. We found that 200 mM imidazole moderately activated sGC in the coexistence with 3-(5'-hydroxymethyl-2'-furyl)-1-benzylindazole (YC-1), although imidazole or YC-1 alone had little effect for activation. GTP facilitated this process. Resonance Raman spectra of imidazole complex of native sGC and CO-bound sGC (CO-sGC) have demonstrated that a simple heme adduct with imidazole at the sixth coordination position is not present for both sGC and CO-sGC below 200 mM of the imidazole concentration and that the Fe-CO stretching band (nuFe-CO)) appears at 492 cm(-1) in the presence of imidazole compared with 473 cm(-1) in its absence. Both frequencies fall on the line of His-coordinated heme proteins in the nuFe-CO vs nuC-O plot. However, it is stressed that the CO-heme of sGC becomes apparently photo-inert in a spinning cell in the presence of imidazole, suggesting the formation of five-coordinate CO-heme or of six-coordinate heme with a very weak trans ligand. These observations suggest that imidazole alters not only the polarity of heme pocket but also the coordination structure at the fifth coordination side presumably by perturbing the heme-protein interactions at propionic side chains. Despite the fact that the isolated sGC stays in the reduced state and is not oxidized by O(2), sGC under the high concentration of imidazole (1.2 M) yielded nu4 at 1373 cm(-1) even after its removal by gel-filtration, but addition of dithionite gave the strong nu4 band at 1360 cm(-1). This indicated that imidazole caused autoxidation of sGC.