A novel thermophilic endo-β-1,4-mannanase from Aspergillus nidulans XZ3: functional roles of carbohydrate-binding module and Thr/Ser-rich linker region

The gene man5XZ3 from Aspergillus nidulans XZ3 encodes a multimodular β-mannanase of glycoside hydrolase family 5 that consists of a family 1 carbohydrate-binding module (CBM1), a Thr/Ser-rich linker region, and a catalytic domain. Recombinant Man5XZ3 and its two truncated derivatives, Man5ΔCBM (removing the CBM1) and Man5ΔCL (removing both the CBM1 and linker region), were produced in Pichia pastoris and showed significant variance in the secondary structure. The three enzymes had similar biochemical properties, such as optimal pH and temperature (pH 5.0 and 80 °C) and excellent pH stability at pH 4.0–10.0. Removal of the CBM1 alone could improve the thermostability of Man5XZ3, but further removal of the linker region resulted in worse thermostability. Man5XZ3 retained greater enzyme activity in the presence of an organic solvent (acetone), two detergents (SDS and Triton X-100), and a chaotropic agent (urea) compared with Man5ΔCBM and Man5ΔCL. This study provides an excellent β-mannanase candidate favorable for various industries and primarily demonstrates the relationship between enzyme structure and function.     

Protein kinase C-associated kinase (PKK) mediates Bcl10-independent NF-kappa B activation induced by phorbol ester

Protein kinase C-associated kinase (PKK) is a recently described kinase of unknown function that was identified on the basis of its specific interaction with PKC beta. PKK contains N-terminal kinase and C-terminal ankyrin repeats domains linked to an intermediate region. Here we report that the kinase domain of PKK is highly homologous to that of two mediators of nuclear factor-kappa B (NF-kappa B) activation, RICK and RIP, but these related kinases have different C-terminal domains for binding to upstream factors. We find that expression of PKK, like RICK and RIP, induces NF-kappa B activation. Mutational analysis revealed that the kinase domain of PKK is essential for NF-kappa B activation, whereas replacement of serine residues in the putative activation loop did not affect the ability of PKK to activate NF-kappa B. A catalytic inactive PKK mutant inhibited NF-kappa B activation induced by phorbol ester and Ca(2+)-ionophore, but it did not block that mediated by tumor necrosis factor alpha, interleukin-1 beta, or Nod1. Inhibition of NF-kappa B activation by dominant negative PKK was reverted by co-expression of PKC beta I, suggesting a functional association between PKK and PKC beta I. PKK-mediated NF-kappa B activation required IKK alpha and IKK beta but not IKK gamma, the regulatory subunit of the IKK complex. Moreover, NF-kappa B activation induced by PKK was not inhibited by dominant negative Bimp1 and proceeded in the absence of Bcl10, two components of a recently described PKC signaling pathway. These results suggest that PKK is a member of the RICK/RIP family of kinases, which is involved in a PKC-activated NF-kappa B signaling pathway that is independent of Bcl10 and IKK gamma.     

The cholesterol turnover, synthesis, and absorption in two sisters with familial hypercholesterolemia (type IIa).

To explore the mechanisms of the profound plasma cholesterol elevations in familial homozygous hypercholesterolemia (type IIa), cholesterol turnover, sterol balance, cholesterol absorption, and low density lipoprotein studies were carried out under controlled dietary conditions in two sisters (aged 13 and 16). Cholesterol turnover was prolonged. The half-life of the first exponential of the plasma cholesterol specific activity decay curve was double that of normal adults. The rate constants for the removal of cholesterol from pool A (KAA = 0.0652) and for the excretion of cholesterol from the system (Kaa = 0.0197) were less than half of normal. The production rates of cholesterol were low, only 6.30 and 6.86 mg/kg per day as measured by cholesterol turnover and sterol balance techniques, respectively. Fecal neutral steroid and bile acid excretion were 5.22 and 1.64 mg/kg per day, which is remarkably low in comparison to those of normal and heterozygous children. Cholesterol absorption was within the upper limit of the values reported for normal adults. THE HDL cholesterol values were extremely low (27 mg/dl) in contrast to profoundly elevated LDL levels. The fractional catabolic rate of LDL (0.127 per day) and the rate of synthesis and catabolism of apo-LDL (15 mg/kg per day) were low in comparison to previously reported values in homozygotes. These composite data indicated that the definable metabolic defects of these two sisters with homozygous familial hypercholesterolemia were the sluggish clearance of cholesterol from the body coupled with low total body synthesis of cholesterol.     

The kic1 kinase of schizosaccharomyces pombe is a CLK/STY orthologue that regulates cell-cell separation

The CLK/STY kinases are a family of dual-specificity protein kinases implicated in the regulation of cellular growth and differentiation. Some of the kinases in the family are shown to phosphorylate serine-arginine-rich splicing factors and to regulate pre-mRNA splicing. However, the actual cellular mechanism that regulates cell growth, differentiation, and development by CLK/STY remains unclear. Here we show that a functionally conserved CLK/STY kinase exists in Schizosaccharomyces pombe, and this orthologue, called Kic1, regulates the cell surface and septum formation as well as a late step in cytokinesis. The Kic1 protein is modified in vivo, likely by phosphorylation, suggesting that it can be involved in a control cascade. In addition, kic1(+) together with dsk1(+), which encodes a related SR-specific protein kinase, constitutes a critical in vivo function for cell growth. The results provide the first in vivo evidence for the functional conservation of the CLK/STY family through evolution from fission yeast to mammals. Furthermore, since cell division and cell-cell interaction are fundamental for the differentiation and development of an organism, the novel cellular role of kic1(+) revealed from this study offers a clue to the understanding of its counterparts in higher eukaryotes.     

Does Optic Nerve Head Surface Topography Change Prior to Loss of Retinal Nerve Fiber Layer Thickness: A Test of the Site of Injury Hypothesis in Experimental Glaucoma

Purpose

To test the hypothesis that optic nerve head (ONH) deformation manifesting as changes in its mean surface height precedes thinning of the peripapillary retinal nerve fiber layer (RNFL) in experimental glaucoma (EG).

Methods

68 rhesus macaque monkeys each had three or more baseline imaging sessions under manometric intraocular pressure (IOP) control to obtain average RNFL thickness (RNFLT) and the ONH surface topography parameter mean position of the disc (MPD). Laser photocoagulation was then applied to the trabecular meshwork of one eye to induce chronic, mild-to-moderate IOP elevation and bi-weekly imaging continued. Event analysis was applied to determine for each parameter when an ‘endpoint’ occurred (signficant change from baseline) for eight different endpoint criteria. Specificity was assessed in the group of 68 fellow control eyes. Classical signal detection theory and survival analysis were used to compare MPD with RNFLT.

Results

Regardless of the endpoint criterion, endpoints were always more frequent for MPD than for RNFLT. The discriminability index (d’) was 2.7 ± 0.2 for MPD and 1.9 ± 0.2 for RNFLT (p<0.0001). Endpoints were reached by MPD an average of 1-2 months earlier than by RNFLT (p<0.01). At the onset of the first specific, detectable MPD change in EG eyes, there was still no significant change in RNFLT on average (p=0.29) and only 25% of individual eyes exhibited signficant reduction. In contrast, at onset of signficant RNFLT change, MPD had already changed an average of 101 µm from baseline (p<0.0001) and 71% of the individual eyes had exhibited significant change. The magnitude of MPD change was more than could be explained on the basis of axon loss alone.

Conclusions

This study demonstrates that the average surface height of the ONH changes prior to any detectable loss of average peripapillary RNFL thickness in non-human primate eyes with experimental glaucoma.     

Construction of novel chimeric proteins through the truncation of SEC2 and Sak from Staphylococcus aureus

It is an usual clinical phenomenon that cancer patients are prone to thrombosis. Until now, there have been no efficient methods or appropriate drugs to prevent and cure tumor thrombus. Therefore, the construction of a bifunctional chimeric protein for the treatment of cancer, complicated with thrombosis, is of great significance. Utilizing the superantigenic activity of staphylococcal enterotoxin C2 (SEC2) and the thrombolytic activity of staphylokinase (Sak), Sak-linker-SEC2 and SEC2-linker-Sak were constructed which had good anti-tumor and thrombolytic activities at the same time. Due to the intrinsic emetic activity of SEC2 and high molecular weight (MW) of chimeric proteins (44?kDa), their clinical applications will be restricted. In this study, novel chimeric proteins including ΔSEC2–ΔSak and ΔSak–ΔSEC2 were constructed through the truncation of SEC2 and Sak without 9-Ala linker and His-tag. Compared with the former, both the truncated proteins preserved nearly the same anti-tumor and thrombolytic activities. In addition, their MWs were only 29?kDa and their immunoreactivities were slightly lower than that of Sak-linker-SEC2 and SEC2-linker-Sak, respectively. Therefore, the novel chimeric proteins possessed merits and characteristics, such as low MS, low immunogenicity, and difunctionality which the former had not. It will be of great interest if the above-mentioned proteins can be used to cure Trousseau syndrome in clinic.