Purification and Characterization of Three Distinct Protein Kinases from Marchantia polymorpha

Three protein kinases (HK-I, HK-II and HK-III) have been partiallypurified from the 1.0 M KC1 extract of Marchantia polymorphaand biochemically characterized. It was found that (i) the molecularweights of HK-I, HK-II and HK-III were approximately 23 kDa,47 kDa and 28 kDa, respectively; (ii) these three kinases requireddivalent cations, such as Mn²⁺ and Mg²⁺, but not Ca²⁺, for activity;and (iii) histone H1 was an effective phosphate acceptor forboth HK-I and HK-II, whereas the other kinase (HK-III) effectivelyphosphorylated whole histone (Type II-A from calf thymus) ratherthan histone H1. Heparin (20µg/ml), an inhibitor of caseinkinase II, significantly stimulated the phosphorylation of cellularpolypeptides by HK-II, which was thermo sensitive even at 30?C,rather than that by the other kinases (HK-I and HK-III). Moreover,experiments in vitro and in vivo to determine the native phosphateacceptors for HK-II indicated that a 60-kDa cellular polypeptidemay be one of the native phosphate acceptors for the proteinkinase. In addition, the similarity in properties of cdc2-kinase,which plays an important role in the cell cycle (in the transitionfrom the G₂ phase to mitosis) of yeast and many eukaryotic cells,to HK-II is discussed. (Received May 2, 1990; Accepted December 6, 1990)     

Molecular evidence of digestion and absorption of epibiotic bacterial community by deep-sea crab Shinkaia crosnieri

The hydrothermal vent crab Shinkaia crosnieri is considered to obtain nutrition from the epibiotic bacteria found on the setae, but previous studies have not shown how nutrients can be transferred from the epibionts to the host. In this study, microscopic observations of S. crosnieri intestinal components detected autofluorescent setae fragments and pigmentation derived from the digestion of epibionts in a dye-stained epibiont tracer experiment. An in vitro digestion experiment with epibiotic populations using an intestinal extract demonstrated the degradation of epibiotic cells by digestive enzymes. A phylogenetic analysis showed that many of the bacterial 16S ribosomal RNA gene sequences obtained from the intestine were closely related to the sequences of the epibionts, thus they were probably derived from the epibionts. A stable isotope tracer experiment also indicated that ¹³C assimilated by the epibionts provided a carbon (nutrition) source for the host. Both activity measurements and isotope studies showed that chemosynthetic metabolism by the gut microbial components were inactive. Together with the feeding behaviour of living S. crosnieri, these results indicate that S. crosnieri ingests the epibionts using maxillipeds and assimilates them via its digestive organs as a nutrient source. The results of this study elucidate the mechanism of nutritional transfer in ectosymbiosis between chemosynthetic bacteria and deep-sea invertebrates.     

MicroRNA Stability in Postmortem FFPE Tissues: Quantitative Analysis Using Autoptic Samples from Acute Myocardial Infarction Patients

MicroRNAs (miRNAs) are very short (18–24 nucleotides) nucleic acids that are expressed in a number of biological tissues and have been shown to be more resistant to extreme temperatures and pH compared to longer RNA molecules, like mRNAs. As miRNAs contribute to diverse biological process and respond to various kinds of cellular stress, their utility as diagnostic biomarkers and/or therapeutic targets has recently been explored. Here, we have evaluated the usefulness of miRNA quantification during postmortem examination of cardiac tissue from acute myocardial infarction (AMI) patients. Cardiac tissue was collected within one week of the patient’s death and either frozen (19 samples) or fixed in formalin for up to three years (36 samples). RNA integrity was evaluated with an electropherogram, and it appears that longer RNAs are fragmented after death in the long-term fixed samples. Quantitative PCR was also performed for seven miRNAs and three other small RNAs in order to determine the appropriate controls for our postmortem analysis. Our data indicate that miR-191 and miR-26b are more suitable than the other types of small RNA molecules as they are stably detected after death and long-term fixation. Further, we also applied our quantitation method, using these endogenous controls, to evaluate the expression of three previously identified miRNA biomarkers, miR-1, miR-208b, and miR-499a, in formalin-fixed tissues from AMI patients. Although miR-1 and miR-208b decreased (1.4-fold) and increased (1.2-fold), respectively, in the AMI samples compared to the controls, the significance of these changes was limited by our sample size. In contrast, the relative level of miR-499a was significantly decreased in the AMI samples (2.1-fold). This study highlights the stability of miRNAs after death and long-term fixation, validating their use as reliable biomarkers for AMI during postmortem examination.     

Relationship between Trusting Behaviors and Psychometrics Associated with Social Network and Depression among Young Generation: A Pilot Study

Maladaptive social interaction and its related psychopathology have been highlighted in psychiatry especially among younger generations. In Japan, novel expressive forms of psychiatric phenomena such as “modern-type depression” and “hikikomori” (a syndrome of severe social withdrawal lasting for at least six months) have been reported especially among young people. Economic games such as the trust game have been utilized to evaluate real-world interpersonal relationships as a novel candidate for psychiatric evaluations. To investigate the relationship between trusting behaviors and various psychometric scales, we conducted a trust game experiment with eighty-one Japanese university students as a pilot study. Participants made a risky financial decision about whether to trust each of 40 photographed partners. Participants then answered a set of questionnaires with seven scales including the Lubben Social Network Scale (LSNS)-6 and the Patient Health Questionnaire (PHQ)-9. Consistent with previous research, male participants trusted partners more than female participants. Regression analysis revealed that LSNS-family (perceived support from family) for male participants, and item 8 of PHQ-9 (subjective agitation and/or retardation) for female participants were associated with participants’ trusting behaviors. Consistent with claims by social scientists, our data suggest that, for males, support from family was negatively associated with cooperative behavior toward non-family members. Females with higher subjective agitation (and/or retardation) gave less money toward males and high attractive females, but not toward low attractive females in interpersonal relationships. We believe that our data indicate the possible impact of economic games in psychiatric research and clinical practice, and validation in clinical samples including modern-type depression and hikikomori should be investigated.     

Antibody expression in protease-deficient strains of the methylotrophic yeast Ogataea minuta

When human antibody genes were expressed in the methylotrophic yeast Ogataea minuta, the secreted antibody became partially degraded. To suppress the degradation, a vacuolar protease-deficient strain was constructed and its antibody production was evaluated. Although antibody productivity was improved in the vacuolar protease-deficient strain, the secreted antibody still became partially degraded. Peptide sequencing revealed that the cleavage occurred in the CH1 region of the heavy chain, implying that the cleavage was caused by an aspartic protease, Yps1p. To inhibit this cleavage, Yps1p-deficient strains were constructed and their antibody production was evaluated. As a result, the partial degradation of the antibody was suppressed in the O. minuta multiple-protease-deficient strains.     

Annotating gene function by combining expression data with a modular gene network

MOTIVATION: A promising and reliable approach to annotate gene function is clustering genes not only by using gene expression data but also literature information, especially gene networks. RESULTS: We present a systematic method for gene clustering by combining these totally different two types of data, particularly focusing on network modularity, a global feature of gene networks. Our method is based on learning a probabilistic model, which we call a hidden modular random field in which the relation between hidden variables directly represents a given gene network. Our learning algorithm which minimizes an energy function considering the network modularity is practically time-efficient, regardless of using the global network property. We evaluated our method by using a metabolic network and microarray expression data, changing with microarray datasets, parameters of our model and gold standard clusters. Experimental results showed that our method outperformed other four competing methods, including k-means and existing graph partitioning methods, being statistically significant in all cases. Further detailed analysis showed that our method could group a set of genes into a cluster which corresponds to the folate metabolic pathway while other methods could not. From these results, we can say that our method is highly effective for gene clustering and annotating gene function.     

Region specificity of rectus femoris muscle for force vectors in vivo

To examine the region specificity within the rectus femoris (RF) for knee extension and hip flexion force directions, three force components around the ankle were measured during intramuscular electrical stimulation applied to six parts of the RF: a proximal and medial part, a proximal and lateral part, a middle and medial part, a middle and lateral part, a distal and medial part, and a distal and lateral part. As a result, the exerted force directions in all of the subjects were variable in all regions, and the proximal region of the RF was the dominant contributor to the hip flexion moment. In addition, the force in the lateral region of the RF, rather than that in the medial region, denoted the lateral direction. These results suggest that divergent regions of muscle fibers within the RF are responsible for different functions in determining the force direction.     

Calcium-induced Mechanical Change in the Neck Domain Alters the Activity of Plant Myosin XI

Plant myosin XI functions as a motor that generates cytoplasmic streaming in plant cells. Although cytoplasmic streaming is known to be regulated by intracellular Ca(2+) concentration, the molecular mechanism underlying this control is not fully understood. Here, we investigated the mechanism of regulation of myosin XI by Ca(2+) at the molecular level. Actin filaments were easily detached from myosin XI in an in vitro motility assay at high Ca(2+) concentration (pCa 4) concomitant with the detachment of calmodulin light chains from the neck domains. Electron microscopic observations showed that myosin XI at pCa 4 shortened the neck domain by 30%. Single-molecule analysis revealed that the step size of myosin XI at pCa 4 was shortened to 27 nm under low load and to 22 nm under high load compared with 35 nm independent of the load for intact myosin XI. These results indicate that modulation of the mechanical properties of the neck domain is a key factor for achieving the Ca(2+)-induced regulation of cytoplasmic streaming.     

RNA processing bodies, peroxisomes, Golgi bodies, mitochondria, and endoplasmic reticulum tubule junctions frequently pause at cortical microtubules

Organelle motility, essential for cellular function, is driven by the cytoskeleton. In plants, actin filaments sustain the long-distance transport of many types of organelles, and microtubules typically fine-tune the motile behavior. In shoot epidermal cells of Arabidopsis thaliana seedlings, we show here that a type of RNA granule, the RNA processing body (P-body), is transported by actin filaments and pauses at cortical microtubules. Interestingly, removal of microtubules does not change the frequency of P-body pausing. Similarly, we show that Golgi bodies, peroxisomes, and mitochondria all pause at microtubules, and again the frequency of pauses is not appreciably changed after microtubules are depolymerized. To understand the basis for pausing, we examined the endoplasmic reticulum (ER), whose overall architecture depends on actin filaments. By the dual observation of ER and microtubules, we find that stable junctions of tubular ER occur mainly at microtubules. Removal of microtubules reduces the number of stable ER tubule junctions, but those remaining are maintained without microtubules. The results indicate that pausing on microtubules is a common attribute of motile organelles but that microtubules are not required for pausing. We suggest that pausing on microtubules facilitates interactions between the ER and otherwise translocating organelles in the cell cortex.