The Contribution of Non-catalytic Carbohydrate Binding Modules to the Activity of Lytic Polysaccharide Monooxygenases

Lignocellulosic biomass is a sustainable industrial substrate. Copper-dependent lytic polysaccharide monooxygenases (LPMOs) contribute to the degradation of lignocellulose and increase the efficiency of biofuel production. LPMOs can contain non-catalytic carbohydrate binding modules (CBMs), but their role in the activity of these enzymes is poorly understood. Here we explored the importance of CBMs in LPMO function. The family 2a CBMs of two monooxygenases, CfLPMO10 and TbLPMO10 from Cellulomonas fimi and Thermobispora bispora, respectively, were deleted and/or replaced with CBMs from other proteins. The data showed that the CBMs could potentiate and, surprisingly, inhibit LPMO activity, and that these effects were both enzyme-specific and substrate-specific. Removing the natural CBM or introducing CtCBM3a, from the Clostridium thermocellum cellulosome scaffoldin CipA, almost abolished the catalytic activity of the LPMOs against the cellulosic substrates. The deleterious effect of CBM removal likely reflects the importance of prolonged presentation of the enzyme on the surface of the substrate for efficient catalytic activity, as only LPMOs appended to CBMs bound tightly to cellulose. The negative impact of CtCBM3a is in sharp contrast with the capacity of this binding module to potentiate the activity of a range of glycoside hydrolases including cellulases. The deletion of the endogenous CBM from CfLPMO10 or the introduction of a family 10 CBM from Cellvibrio japonicus LPMO10B into TbLPMO10 influenced the quantity of non-oxidized products generated, demonstrating that CBMs can modulate the mode of action of LPMOs. This study demonstrates that engineered LPMO-CBM hybrids can display enhanced industrially relevant oxygenations.     

A Dual Interaction between the DNA Damage Response Protein MDC1 and the RAG1 Subunit of the V(D)J Recombinase

The first step in V(D)J recombination is the formation of specific DNA double-strand breaks (DSBs) by the RAG1 and RAG2 proteins, which form the RAG recombinase. DSBs activate a complex network of proteins termed the DNA damage response (DDR). A key early event in the DDR is the phosphorylation of histone H2AX around DSBs, which forms a binding site for the tandem BRCA1 C-terminal (tBRCT) domain of MDC1. This event is required for subsequent signal amplification and recruitment of additional DDR proteins to the break site. RAG1 bears a histone H2AX-like motif at its C terminus (R1Ct), making it a putative MDC1-binding protein. In this work we show that the tBRCT domain of MDC1 binds the R1Ct motif of RAG1. Surprisingly, we also observed a second binding interface between the two proteins that involves the Proline-Serine-Threonine rich (PST) repeats of MDC1 and the N-terminal non-core region of RAG1 (R1Nt). The repeats-R1Nt interaction is constitutive, whereas the tBRCT-R1Ct interaction likely requires phosphorylation of the R1Ct motif of RAG1. As the C terminus of RAG1 has been implicated in inhibition of RAG activity, we propose a model in which phosphorylation of the R1Ct motif of RAG1 functions as a self-initiated regulatory signal.     

Plasma Factor in Red Blood Cells Adhesion to Endothelial Cells: Humans and Rats

Erythrocyte adhesion to the vascular endothelium is one of the key determinants of microcirculatory blood flow. Adhesion is a complex process determined by the intricate interaction among red blood cells (RBC), plasma factors, and the vascular endothelium. Rats are commonly used as disease models to investigate the pathophysiology of various hematological disease processes occurring in humans and their response to prospective treatments. The aim of our study was to characterize the adhesion of RBC in adult blood from rat and human subjects, in order to test the validity of rat models for adhesion-related disease processes. We demonstrated that adhesion of RBC from rats (rRBC), to endothelial cells (EC) in plasma-free buffer, is stronger than from human subjects (hRBC). In addition, plasma proteins induced elevation of hRBC (eightfold) but depression of rRBC (threefold) adhesion to EC. It is thus suggested to be aware of the difference in RBC/EC interaction for human and rat subjects, when studying models of blood flow.     

Measuring the Quality of University Lectures: Development and Validation of the Instructional Skills Questionnaire (ISQ)

In higher education, student ratings are often used to evaluate and improve the quality of courses and professors’ instructional skills. Unfortunately, student-rating questionnaires rarely generate specific feedback for professors to improve their instructional skills. The impact of student ratings on professors’ instructional skills has proven to be low. This study concerns the psychometric properties of the Instructional Skills Questionnaire (ISQ), a new theory-based student-rating-of-teaching questionnaire with specific questions concerning lecturing skills. The ISQ is administered after a single lecture. This way, it serves as a formative feedback instrument for university professors during courses to assist them to improve and (re-) evaluate their skills if necessary. The ISQ contains seven dimensions of professors’ instructional skills and three student (self perceived) learning outcomes. In this study, Dutch students in 75 courses rated three 90-minute lectures (T1, T2 and T3) of their respective professors using the ISQ. In total, 14,298 ISQ-forms were used to rate 225 lectures. The teacher level reliabilities of the seven dimensions were found to be good at each measurement occasion. In addition, confirmatory multilevel factor analysis confirmed a seven dimensional factor structure at the teacher level at each measurement occasion. Furthermore, specific teacher level factors significantly predicted students’ (self-assessed) learning outcomes. These results partly supported the proposed theoretical framework on the relationship between the ISQ teaching dimensions and the student learning process, and provided evidence for the construct validity of the instrument. In sum, the ISQ is found to be a reliable and valid instrument, which can be used by professors and faculty development centers to assess and improve university teaching.     

Physiological evidence for central modulation of voice tremor

The present report presents an attempt to define the physiological parameter used to describe “voice tremor” in psychological stress evaluating machines, and to find its sources. This parameter was found to be a low frequency (5–20 Hz) random process which frequency modulates the vocal cord waveform and (independently) affects the frequency range of the third speech formant. The frequency variations in unstressed speakers were found to be the result of forced muscular undulations driven by central nervous signals and not of a passive resonant phenomenon. In this paper various physiological and clinical experiments which lead to the above conclusions are discussed. a) It is shown that induced muscular activity in the vocal tract and vocal cord regions can generate tremor in the voice. b) It is shown that relaxed subjects exhibit significant tremor correlation between spontaneously generated speech and EMG, with the EMG leading the speech tremor. c) Tremor in the electrical activity recorded from muscles overlapping vocal tract area was correlated with third formant demodulated signal and vocal cord demodulated pitch tremor was correlated with first formant demodulated tremor. d) Enhanced tremor was found in Parkinson patients and diminished tremor in patients with some traumatic brain injuries.     

Studies on thyroid cell surface glycoproteins: Isolation of plasma membranes and characterization of carbohydrate units

Plasma membranes were isolated from calf thyroid microsomes and further resolved into two subfractions by sucrose density gradient centrifugations. The lighter and major membrane fraction was obtained in a yield of 10 mg/100 g of thyroid and was enriched 38-fold with respect to 5′-nucleotidase activity compared to the homogenate. It differed from the denser plasma membrane fraction in containing greater amounts of phospholipid and cholesterol but had a similar total carbohydrate content (16 mg/100 mg protein) and monosaccharide composition. The membranes were found to retain most (80%) of their carbohydrate after delipidation. The major protein-bound sugars present in the lighter membrane fraction expressed as micromoles per 100 mg of peptide were: galactose 24, mannose 17, fucose 3, glucosamine 23, galactosamine 4, and sialic acid 9. Polyacrylamide gel electrophoresis in sodium dodecyl sulfate of the lipid-free membranes revealed at least 18 protein bands and 3 periodic acid-Schiffreactive glycoprotein components. Incubation of the delipidated membranes with Pronase resulted in the solubilization of 95% of the saccharide portion which upon filtration through Bio-Gel P-6 and P-10 columns yielded several glycopeptide fractions. While some of the carbohydrate was found in glycopeptides which appeared to contain the well-known complex and polymannose asparagine-bound oligosaccharides, as well as small O-glycosidically linked units, approximately half was recovered in high molecular weight components which contained galactose and glucosamine as their principal sugar constituents, and which were similar in composition to glycopeptides recently isolated (T. Krusius, J. Finne, and H. Rauvala, 1978, Eur. J. Biochem.92, 289–300) from human erythrocyte membranes.     

GROWTH FACTOR INTERACTIONS IN THE TISSUE CULTURE OF TUMOROUS AND NONTUMOROUS NICOTIANA GLAUCA-LANGSDORFFII

Schaeffer , Gideon W., Harold H. Smith and Marion P. Perkus . (Brookhaven Natl. Lab., Upton, N. Y.) Growth factor interactions in the tissue culture of tumorous and nontumorous Nicotiana glauca-langsdorffii. Amer. Jour. Bot. 50(8): 766–771. Illus. 1963.—Tissues representing tumorous and nontumorous Nicotiana glauca-langsdorffii were cultured on high (5 ×) and low (1 ×) concentrations of a modified White's basal medium containing 2.9 × 10^–6m indoleacetic acid. The growth responses of tissues of both the tumorous and nontumorous genotypes to supplements of kinetin, glutamine, inositol and nucleic acid constituents added singly and in all combinations were noted on high-salt media. The nucleic acid components inhibited growth and were omitted from low-salt media. The best growth response was observed with glutamine and inositol for tissues from the tumorous hybrid and with glutamine, inositol and kinetin in the nontumorous type. Kinetin was a distinct and consistent requirement for rapid growth of nontumorous tissues, but no appreciable kinetin effect could be observed with tissues from the tumorous genotype.