Limited proteolysis patterns of the B chain of insulin.

The oxidized B chain of insulin was used as a simple model for further consideration of limited proteolysis with low substrate:enzyme ratios. With low B chain:trypsin ratios, the ordinarily slower cleavage rate of the -Lys₂₉-Ala₃₀ bond essentially equaled the cleavage saturation rate of the -Arg₂₂-Gly₂₃ bond. This led to the disappearance of octapeptide which ordinarily forms most rapidly. Heptapeptide and alanine, formed mainly by cleavage of the octapeptide, decreased somewhat at high enzyme relative levels. Trypsin added to B chain formed a single chromatographic peak.     

Identification of significant regions of transcription factor DP-1 (TFDP-1) involved in stability/instability of the protein

We previously reported the identification of DP-1 isoforms (α and β), which are structurally C-terminus-deleted ones, and revealed the low-level expression of these isoforms. It is known that wild-type DP-1 is degraded by the ubiquitin-proteasome system, but few details are known about the domains concerned with the protein stability/instability for the proteolysis of these DP-1 isoforms. Here we identified the domains responsible for the stability/instability of DP-1. Especially, the DP-1 “Stabilon” domain was a C-terminal acidic motif and was quite important for DP-1 stability. Moreover, we propose that this DP-1 Stabilon may be useful for the stability of other nuclear proteins when fused to them.     

Reversible hyperphosphorylation and reorganization of vimentin intermediate filaments by okadaic acid in 9L rat brain tumor cells.

Okadaic acid (OA), a protein phosphatase inhibitor, was found to induce hyperphosphorylation and reorganization of vimentin intermediate filaments in 9L rat brain tumor cells. The process was dose dependent. Vimentin phosphorylation was initially enhanced by 400 nM OA in 30 min and reached maximal level (about 26-fold) when cells were treated with 400 nM OA for 90 min. Upon removal of OA, dephosphorylation of the hyperphosphorylated vimentin was observed and the levels of phosphorylation returned to that of the controls after the cells recovered under normal growing conditions for 11 h. The phosphorylation and dephosphorylation of vimentin induced by OA concomitantly resulted in reversible reorganization of vimentin filaments and alteration of cell morphology. Cells rounded up as they were entering mitosis in the presence of OA and returned to normal appearance after 11 h of recovery. Immuno-staining with anti-vimentin antibody revealed that vimentin filaments were disassembled and clustered around the nucleus when the cells were treated with OA but subsequently returned to the filamentous states when OA was removed. Two-dimensional electrophoresis analysis further revealed that hyperphosphorylation of vimentin generated at least seven isoforms having different isoelectric points. Furthermore, the enhanced vimentin phosphorylation was accompanied by changes in the detergent-solubility of the protein. In untreated cells, the detergent-soluble and -insoluble vimentins were of equal amounts but the solubility could be increased when vimentins were hyperphosphorylated in the presence of OA. Taken together, the results indicated that OA could be involved in reversible hyperphosphorylation and reorganization of vimentin intermediate filaments, which may play an important role in the structure-function regulation of cytoskeleton in the cell.     

In vitro assessment of the toxicity of metal compounds

A review of in vitro mutagenesis assessment of metal compounds in mammalian and nonmammalian test systems has been compiled. Prokaryotic assays are ineffective or inconsistent in their detection of most metals as mutagens, with the notable exception of hexavalent chromium. Mammalian assay systems appear to be similarly inappropriate for the screening of metal compounds based upon the limited number of studies that have employed those compounds having known carcinogenic activity. Although of limited value as screening tests for the detection of potentially carcinogenic metal compounds, the well-characterized in vitro mutagenesis systems may prove to be of significant value as a means to elucidate mechanisms of metal genotoxicity.     

PHENOTYPIC PLASTICITY INDUCED IN TRANSPLANT EXPERIMENTS IN A MUTUALISTIC ASSOCIATION BETWEEN THE RED ALGA JANIA ADHAERENS (RHODOPHYTA,CORALLINALES) AND THE SPONGE HALICLONA CAERULEA (PORIFERA: HAPLOSCLERIDA): MORPHOLOGICAL RESPONSES OF THE ALGA1

The association between the red macroalga Jania adhaerens J. V. Lamour. and the sponge Haliclona caerulea is the most successful life‐form between 2 and 4 m depth in Mazatlán Bay (Mexican Pacific). J. adhaerens colonizes the rocky intertidal area and penetrates into deeper areas only when it lives in association with H. caerulea. The aposymbiotic form of the sponge has not been reported in the bay. To understand the ecological success of this association, we examined the capacity of J. adhaerens to acclimate in Mazatlán Bay using transplant experiments. The transplanted aposymbiotic J. adhaerens did not survive the first 2 weeks; however, J. adhaerens when living in association with H. caerulea, acclimated easily to depth, showing no sign of mortality during the 103 d of the experiment. We conclude that the ability of J. adhaerens to colonize in deeper areas in this hydrodynamic environment may in part rely on the protection provided by the sponge to the algal canopy. Both species contribute to the shape of the associated form. Nevertheless, the morphological variation in the association appears to be dominated by the variation in J. adhaerens canopy to regulate pigment self‐shading under light‐limited conditions and/or tissue resistance under high hydrodynamics. Consequently, our results are consistent with light as the abiotic controlling factor, which regulates the lower depth distribution of the association in Mazatlán Bay, through limiting the growth rate of J. adhaerens. Hydrodynamics may determine the upper limit of the association by imposing high mass losses.     

Hymenolepis diminuta: Worm loss and worm weight loss in long-term infections of the rat

Infections of one and two Hymenolepis diminuta established in newly weaned rats continued to grow for the duration of the experiment (238 days), whereas infections of 5 worms per rat became asymptotic around Day 55 postinfection and remained at or below this level thereafter as shown by biomass and mean weight per worm measurements. Infections of 50 worms established in newly weaned rats became asymptotic around Day 28 postinfection and thereafter worms were lost from the rats. Initially the biomass fell with the loss of worms, but by Day 56 a new lower biomass persisted for the remainder of the infection period. This level was maintained, despite diminishing numbers of worms, due to the growth of surviving individuals to a weight exceeding the original weight at maturity by a factor of more than 2. Experiments using rats that were mature at the time of infection demonstrated that the same response occurred, but approximately 3 weeks earlier.     

Biochemistry and genetics of taste- and odor-producing cyanobacteria

Cyanobacteria are one of the principal sources of volatile organic compounds (VOCs) which cause offensive taste and odor (T&O) in drinking and recreational water, fish, shellfish and other seafood. Although non-toxic to humans, these T&O compounds severely undermine public trust in these commodities, resulting in substantial costs in treatment, and lost revenue to drinking water, aquaculture, food and beverage and tourist/hospitality industries. Mitigation and control have been hindered by the complexity of the communities and processes which produce and modify T&O events, making it difficult to source-track the major producer(s) and the factors governing VOC production and fate. Over the past decade, however, advances in bioinformatics, enzymology, and applied detection technologies have greatly enhanced our understanding of the pathways, the enzymes and the genetic coding for some of the most problematic VOCs produced by cyanobacteria. This has led to the development of tools for rapid and sensitive detection and monitoring for the VOC production at source, and provided the basis for further diagnostics of endogenous and exogenous controls. This review provides an overview of current knowledge of the major cyanobacterial VOCs, the producers, the biochemistry and the genetics and highlight the current applications and further research needs in this area.     

ATM regulates Cdt1 stability during the unperturbed S phase to prevent re-replication

Ataxia-telangiectasia mutated (ATM) plays crucial roles in DNA damage responses, especially with regard to DNA double-strand breaks (DSBs). However, it appears that ATM can be activated not only by DSB, but also by some changes in chromatin architecture, suggesting potential ATM function in cell cycle control. Here, we found that ATM is involved in timely degradation of Cdt1, a critical replication licensing factor, during the unperturbed S phase. At least in certain cell types, degradation of p27^Kip1 was also impaired by ATM inhibition. The novel ATM function for Cdt1 regulation was dependent on its kinase activity and NBS1. Indeed, we found that ATM is moderately phosphorylated at Ser1981 during the S phase. ATM silencing induced partial reduction in levels of Skp2, a component of SCF^Skp2 ubiquitin ligase that controls Cdt1 degradation. Furthermore, Skp2 silencing resulted in Cdt1 stabilization like ATM inhibition. In addition, as reported previously, ATM silencing partially prevented Akt phosphorylation at Ser473, indicative of its activation, and Akt inhibition led to modest stabilization of Cdt1. Therefore, the ATM-Akt-SCF^Skp2 pathway may partly contribute to the novel ATM function. Finally, ATM inhibition rendered cells hypersensitive to induction of re-replication, indicating importance for maintenance of genome stability.     

Genome-wide CRISPR Screens Reveal Host Factors Critical for SARS-CoV-2 Infection

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Evolutionary assembly of cooperating cell types in an animal chemical defense system

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