The PRiMA-linked Cholinesterase Tetramers Are Assembled from Homodimers: HYBRID MOLECULES COMPOSED OF ACETYLCHOLINESTERASE AND BUTYRYLCHOLINESTERASE DIMERS ARE UP-REGULATED DURING DEVELOPMENT OF CHICKEN BRAIN*

Acetylcholinesterase (AChE) is anchored onto cell membranes by the transmembrane protein PRiMA (proline-rich membrane anchor) as a tetrameric globular form that is prominently expressed in vertebrate brain. In parallel, the PRiMA-linked tetrameric butyrylcholinesterase (BChE) is also found in the brain. A single type of AChE-BChE hybrid tetramer was formed in cell cultures by co-transfection of cDNAs encoding AChE_T and BChE_T with proline-rich attachment domain-containing proteins, PRiMA I, PRiMA II, or a fragment of ColQ having a C-terminal GPI addition signal (Q_N-GPI). Using AChE and BChE mutants, we showed that AChE-BChE hybrids linked with PRiMA or Q_N-GPI always consist of AChE_T and BChE_T homodimers. The dimer formation of AChE_T and BChE_T depends on the catalytic domains, and the assembly of tetramers with a proline-rich attachment domain-containing protein requires the presence of C-terminal “t-peptides” in cholinesterase subunits. Our results indicate that PRiMA- or ColQ-linked cholinesterase tetramers are assembled from AChE_T or BChE_T homodimers. Moreover, the PRiMA-linked AChE-BChE hybrids occur naturally in chicken brain, and their expression increases during development, suggesting that they might play a role in cholinergic neurotransmission.     

Evaluation of Bottlenecks in the Late Stages of Protein Secretion in Bacillus subtilis

Despite a high capacity for secretion of homologous proteins, the secretion of heterologous proteins by Bacillus subtilis is frequently inefficient. In the present studies, we have investigated and compared bottlenecks in the secretion of four heterologous proteins: Bacillus lichenifomis α-amylase (AmyL), Escherichia coli TEM β-lactamase (Bla), human pancreatic α-amylase (HPA), and a lysozyme-specific single-chain antibody. The same expression and secretion signals were used for all four of these proteins. Notably, all identified bottlenecks relate to late stages in secretion, following translocation of the preproteins across the cytoplasmic membrane. These bottlenecks include processing by signal peptidase, passage through the cell wall, and degradation in the wall and growth medium. Strikingly, all translocated HPA was misfolded, its stability depending on the formation of disulfide bonds. This suggests that the disulfide bond oxidoreductases of B. subtilis cannot form the disulfide bonds in HPA correctly. As the secretion bottlenecks differed for each heterologous protein tested, it is anticipated that the efficient secretion of particular groups of heterologous proteins with the same secretion bottlenecks will require the engineering of specifically optimized host strains.     

Analysis of ribosomal DNA restriction patterns in the genusKluyveromyces

Riboprinting was used to determine the relationships among strains belonging to 15 species of the genusKluyveromyces. The small subunit ribosomal RNA gene (SSU rDNA) was amplified using the Polymerase Chain Reaction (PCR) and subjected to a battery of nine restriction enzymes. Similarity coefficients between strains were calculated based on shared and unique restriction fragments. Cluster analysis revealed three major groups that generally correlated with previously reported relationships based on other molecular data. Variations in SSU rDNA restriction fragments may be used for differentiation of theKluyveromyces strains included in this study.The U.S. Government right to retain a non-exclusive, royalty free licence in and to any copyright is acknowledged.     

Solid cultures of thrips-pathogenic fungi Isaria javanica strains for enhanced conidial productivity and thermotolerance

Entomopathogenic fungi have great potential to control agricultural and horticultural insect pests, however optimizing conidial production systems to demonstrate high productivity and stability still needs additional efforts for successful field application and industrialization. Although many virulent entomopathogenic fungal isolates have been viewed as potential candidates in a laboratory environment, very few of the isolates are being used in practice for application in agricultural fields as commercial products. I. javanicus is an entomopathogenic fungus that is parasitic to various diverse coleopteran and lepidopteran insects and thought good candidate as biopesticdes. In this work, the basic characteristics of two entomopathogenic fungi, I. javanica FG340 and Pf04, were investigated in morphological examinations, genetic identification, and virulence against Thrips palmi, and then the feasibility of various grains substrates for conidial production was assessed, particularly focusing on conidial productivity and thermotolerance. Isaria javanica FG340 and Pf04 conidia were solid-cultured on 12 grains for 14?days in a Petri dish. Of the tested Italian millet, perilla seed, millet and barley-based cultures showed high conidial production. The four-grain media yielded >1?×?10⁹ conidia/g of I. javanica FG340 and Pf04. Pf04 strain had enhanced thermotolerance up to 45?°C when cultured on Italian millet. In application, it was easy to make a conidial suspension using the cultured grains, and several surfactants were tested to release the conidia. This work suggests several possible inexpensive grain substrates by which to promote conidial production combined with enhanced stability against exposure to high temperature.     

Characterization of xylanase from Lentinus edodes M290 cultured on waste mushroom logs

Extracellular enzymes from Lentinus edodes M290 on normal woods (Quercus mongolica) and waste logs from oak mushroom production were comparatively investigated. Endoglucanase, cellobiohydrolase, beta-glucosidase, and xylanase activities were higher on waste mushroom logs than on normal woods after L. edodes M290 inoculation. Xylanase activity was especially different, with a three times higher activity on waste mushroom logs. When the waste mushroom logs were used as a carbon source, a new 35 kDa protein appeared. After the purification, the optimal pH and temperature for xylanase activity were determined to be 4.0 and 50 degrees C, respectively. More than 50% of the optimal xylanase activity was retained when the temperature was increased from 20 to 60 degrees C, after a 240 min reaction. At 40 degrees C, the xylanase maintained 93% of the optimal activity, after a 240 min reaction. The purified xylanase showed a very high homology to the xylanase family 10 from Aspergillus terreus by LC/MS-MS analysis. The highest Xcorr (1.737) was obtained from the peptide KWI SQGIPIDGIG SQTHLGSGGS WTVK originated from Aspergillus terreus, indicating that the 35 kDa protein was xylanase. This protein showed low homology to a previously reported L. edodes xylanase sequence.     

An Imported Case of Cystic Echinococcosis in the Liver

A 25-year-old Uzbek male presented with right upper abdominal pain for 20 days. On radiologic studies, a huge cystic mass was noticed in the right liver which was suspected as parasitic. The patient received right hepatic segmentectomy (segment 7), and the surgically resected mass was confirmed as cystic echinococcosis (CE), measuring 10.5 cm in its diameter. The inner surface of the cyst was bile-stained. The patient was discharged on the 8th hospital day, and was rechecked 6 months after the surgical intervention without any evidence of recurrence. The present report describes findings of an imported case of CE which represented ultrasound images of the ''ball of wool''.     

Detection of an intermediate during the unfolding process of the dimeric ketosteroid isomerase

Failure to detect the intermediate in spite of its existence often leads to the conclusion that two-state transition in the unfolding process of the protein can be justified. In contrast to the previous equilibrium unfolding experiment fitted to a two-state model by circular dichroism and fluorescence spectroscopies, an equilibrium unfolding intermediate of a dimeric ketosteroid isomerase (KSI) could be detected by small angle X-ray scattering (SAXS) and analytical ultracentrifugation. The sizes of KSI were determined to be 18.7A in 0M urea, 17.3A in 5.2M urea, and 25.1A in 7M urea by SAXS. The size of KSI in 5.2M urea was significantly decreased compared with those in 0M and 7M urea, suggesting the existence of a compact intermediate. Sedimentation velocity as obtained by ultracentrifugation confirmed that KSI in 5.2M urea is distinctly different from native and fully-unfolded forms. The sizes measured by pulse field gradient nuclear magnetic resonance (NMR) spectroscopy were consistent with those obtained by SAXS. Discrepancy of equilibrium unfolding studies between size measurement methods and optical spectroscopies might be due to the failure in detecting the intermediate by optical spectroscopic methods. Further characterization of the intermediate using (1)H NMR spectroscopy and Kratky plot supported the existence of a partially-folded form of KSI which is distinct from those of native and fully-unfolded KSIs. Taken together, our results suggest that the formation of a compact intermediate should precede the association of monomers prior to the dimerization process during the folding of KSI.     

Topological constraints in nucleic acid hybridization kinetics

A theoretical examination of kinetic mechanisms for forming knots and links in nucleic acid structures suggests that molecules involving base pairs between loops are likely to become topologically trapped in persistent frustrated states through the mechanism of ‘helix-driven wrapping’. Augmentation of the state space to include both secondary structure and topology in describing the free energy landscape illustrates the potential for topological effects to influence the kinetics and function of nucleic acid strands. An experimental study of metastable complementary ‘kissing hairpins’ demonstrates that the topological constraint of zero linking number between the loops effectively prevents conversion to the minimum free energy helical state. Introduction of short catalyst strands that break the topological constraint causes rapid conversion to full duplex.