Production of monoclonal antibodies and immunohistochemical studies of brain myo-inositol monophosphate phosphatase

Five monoclonal antibodies that recognize porcine brain myo-inositol monophosphate phosphatase (IMPase) have been selected and designated as mAb IMPP 9, IMPP 10, IMPP 11, IMPP 15, and IMPP 17. These antibodies recognize different epitopes of the enzyme and one of these inhibited the enzyme activity. When the total proteins of the porcine brain homogenate separated by SDS-PAGE were probed with monoclonal antibodies, a single reactive protein band of 29 kDa, co-migrating with the purified porcine brain IMPase, was detected. Using the anti-IMPase antibodies as probes, the cross reactivities of the brain IMPase from human and other mammalian tissues, as well as from avian sources, were investigated. Among the human and animal tissues tested, the immunoreactive bands on Western blots appeared to have the same molecular mass of 29 kDa. In addition, there was IMPase immunoreactivity in the various neuronal populations in the rat brain. These results indicate that mammalian brains contain only one major type of immunologically similar IMPase, although some properties of the enzymes that were previously reported differ from each another. The first demonstration of the IMPase localization in the brain may also provide useful data for future investigations on the function of this enzyme in relation to various neurological diseases.     

Mitochondrial DNA polymorphism among five Asian populations

Mitochondrial DNA (mtDNA) polymorphisms were detected using 13 restriction enzymes on the total DNA obtained from blood samples of five Asian populations: Japanese and Ainu of northern Japan, Korean, Negrito (Aeta) of the Philippines, and Vedda of Sri Lanka. Of a total of 28 restriction-enzyme morphs detected, eight had not been reported previously. By combining the morphs, we were able to classify mtDNAs of 243 individuals into 20 mtDNA types. Phylogenetic analyses using maximum parsimony and genetic distance methods both showed that the Japanese, Ainu, and Korean populations were closely related to each other. Aeta was found to show a relatively close relationship to these three populations, confirming the conclusion from previous studies of blood markers. In contrast, Vedda was quite different from the other four populations.     

The identification of an eukaryotic initiation factor 4D precursor in spermidine-depleted Chinese hamster ovary cells

When Chinese hamster ovary cells are incubated with [terminal methylenes-3H]spermidine, radioactivity is incorporated into a single cellular protein, eukaryotic initiation factor 4D (eIF-4D), through posttranslational synthesis of the amino acid hypusine (N epsilon-(4-amino-2-hydroxybuyly)lysine). The effect of spermidine depletion on this protein modification reaction was studied by high resolution two-dimensional gel electrophoresis. Factor eIF-4D containing both [3H]lysine and [3H]hypusine was detected as one of the major labeled cellular proteins on the fluorographic map of the proteins from Chinese hamster ovary cells that had been incubated with [3H]lysine. When these cells were depleted of spermidine by the use of DL-alpha-difluoromethylornithine before addition of [3H]lysine, no radiolabeling of this mature eIF-4D (hypusine form, Mr approximately 18,000; pI approximately 5.3) occurred. Instead, a new radiolabeled protein (Mr 18,000; pI 5.1) that contained [3H]lysine but no [3H]hypusine or [3H]deoxyhypusine was seen. This protein was identified as an eIF-4D precursor by comparison of the two-dimensional map of its tryptic peptides with that of the tryptic peptides from [3H]lysine-labeled eIF-4D. Further comparisons also suggest that additional post-translational modification processes are involved in the biogenesis of eIF-4D.     

Isolation and characterization of a novel glycosyl hydrolase family 74 (GH74) cellulase from the black goat rumen metagenomic library

This study aimed to isolate and characterize a novel cellulolytic enzyme from black goat rumen by using a culture-independent approach. A metagenomic fosmid library was constructed from black goat rumen contents and screened for a novel cellulase. The KG37 gene encoding a protein of 858 amino acid residues (92.7 kDa) was isolated. The deduced protein contained a glycosyl hydrolase family 74 (GH74) domain and showed 77% sequence identity to two endo-1,4-β-glucanases from Fibrobacter succinogenes. The novel GH74 cellulase gene was overexpressed in Escherichia coli, and its protein product was functionally characterized. The recombinant GH74 cellulase showed a broad substrate spectrum. The enzyme exhibited its optimum activity at pH 5.0 and temperature range of 20–50 °C. The enzyme was thermally stable at pH 5.0 and at a temperature of 20–40 °C. The novel GH74 cellulase can be practically exploited to convert lignocellulosic biomass to value-added products in various industrial applications in future.     

Structure of monomeric Interleukin-8 and its interactions with the N-terminal Binding Site-I of CXCR1 by solution NMR spectroscopy

The structure of monomeric human chemokine IL-8 (residues 1–66) was determined in aqueous solution by NMR spectroscopy. The structure of the monomer is similar to that of each subunit in the dimeric full-length protein (residues 1–72), with the main differences being the location of the N-loop (residues 10–22) relative to the C-terminal α-helix and the position of the side chain of phenylalanine 65 near the truncated dimerization interface (residues 67–72). NMR was used to analyze the interactions of monomeric IL-8 (1–66) with ND-CXCR1 (residues 1–38), a soluble polypeptide corresponding to the N-terminal portion of the ligand binding site (Binding Site-I) of the chemokine receptor CXCR1 in aqueous solution, and with 1TM-CXCR1 (residues 1–72), a membrane-associated polypeptide that includes the same N-terminal portion of the binding site, the first trans-membrane helix, and the first intracellular loop of the receptor in nanodiscs. The presence of neither the first transmembrane helix of the receptor nor the lipid bilayer significantly affected the interactions of IL-8 with Binding Site-I of CXCR1.     

Overexpression of the Bx7 high molecular weight glutenin subunit on the <Emphasis Type="Italic">Glu</Emphasis>-<Emphasis Type="Italic">B1</Emphasis> locus in a Korean wheat landrace

The Glu-B1al (Bx7^OE + By8) allele is important for bread-making quality. The allele was found in a Korean wheat landrace using specific DNA markers. Molecular analyses were conducted to identify the overexpressed Bx7 (Bx7^OE) subunit of the allele. The Korean wheat landrace (accession ID: IT166460) showed a similar protein expression level of Bx7 subunit, i.e., overexpression of Bx7 subunit towards cv. Glenlea, Canadian Western Red Spring wheat, which harbors Bx7^OE subunit of Glu-B1al as detected on SDS–PAGE (sodium dodecyl sulfate poly-acrylamide gel electrophoresis). In addition, 2-DE (two-dimensional electrophoresis) analysis revealed similar protein expression patterns of the Bx7 subunit regions of IT166460 and Glenlea. The proportion of Bx7 to total HMW-GSs (high molecular weight glutenin subunits) in IT166460 (56.17 ± 0.22%) was higher than that of Chinese Spring (34.75 ± 1.03%) and even that of Glenlea (46.25 ± 1.76%) as assessed by RP-HPLC (reverse-phase high-performance liquid chromatography). Overexpression of Bx7 subunit was caused by gene duplication and indels of the promoter region of the Bx7 gene. IT166460 attained the 43 bp indel of the promoter region, as did Glenlea, i.e., the amplicon size of IT166460 was the same as that of Glenlea. In addition, the nucleotides present in the duplicated gene in IT166460 were the same as those in Glenlea. Bx7^OE subunit is critical for dough strength. However, most wheat accessions harboring the subunit are distributed in America. Furthermore, most Korean wheats have little genetic variation in glutenin composition and are associated with inferior bread quality. Hence, IT166460 could be used to improve bread-making quality in the Korean wheat breeding program.     

Dynamic localization of HmpF regulates type IV pilus activity and directional motility in the filamentous cyanobacterium Nostoc punctiforme

Many cyanobacteria exhibit surface motility powered by type 4 pili (T4P). In the model filamentous cyanobacterium Nostoc punctiforme, the T4P systems are arrayed in static, bipolar rings in each cell. The chemotaxis‐like Hmp system is essential for motility and the coordinated polar accumulation of PilA on cells in motile filaments, while the Ptx system controls positive phototaxis. Using transposon mutagenesis, a gene, designated hmpF, was identified as involved in motility. Synteny among filamentous cyanobacteria and the similar expression patterns for hmpF and hmpD imply that HmpF is part of the Hmp system. Deletion of hmpF produced a phenotype distinct from other hmp genes, but indistinguishable from pilB or pilQ. Both an HmpF‐GFPuv fusion protein, and PilA, as assessed by in situ immunofluorescence, displayed coordinated, unipolar localization at the leading pole of each cell. Reversals were modulated by changes in light intensity and preceded by the migration of HmpF‐GFPuv to the lagging cell poles. These results are consistent with a model where direct interaction between HmpF and the T4P system activates pilus extension, the Hmp system facilitates coordinated polarity of HmpF to establish motility, and the Ptx system modulates HmpF localization to initiate reversals in response to changes in light intensity.     

Cholesterol efflux from larval Manduca sexta fat body in vitro: high-density lipophorin as the acceptor

The objective of this study was to characterize the transfer of cholesterol from Manduca sexta larvae fat body to high-density lipophorin. [³H]-Cholesterol-labeled fat body was incubated with lipophorin under different conditions and cholesterol transfer was determined. Transfer rate exhibited a hyperbolic dependency on lipophorin concentration with an apparent K_m of 3.6 mg/ml, which is consistent with either an aqueous diffusion mechanism of cholesterol transfer or a receptor-mediated process. Several results, including the high K_m, the high activation energy, and the lack of Ca²⁺ dependence favor aqueous diffusion model. In addition, anti-lipid transfer particle antibodies had only a small inhibitory effect, suggesting it is not involved in cholesterol transfer. However, the transfer was inhibited in the presence of suramin, which would be consistent with a receptor-mediated process. The effects of suramin may be complex because it can change membrane properties when bound to the lipophorin receptor and affect the rate of cholesterol desorption. The preponderance of data suggests that the export of cholesterol from fat body to lipophorin follows a simple aqueous diffusion pathway. Although we cannot completely exclude some contribution from a receptor-mediated pathway, it seems that if such a pathway were present, it represents a minor route.     

A role for cell cycle proteins in the serum-starvation resistance of Epstein-Barr virus immortalized B lymphocytes.

Epstein-Barr virus (EBV) is a B-lymphotropic human herpes virus that infects B lymphocytes and is associated with a broad spectrum of benign and malignant diseases. B cell infection by EBV causes indefinite cell proliferation that results in the development of immortalized lymphoblastoid cell lines (LCLs). We found that SNU-1103, a latency type III EBV-transformed LCL developed from a Korean cancer patient, resisted the G1 arrest that was normally caused by serum starvation. Western blot analyses revealed several alterations in the expression of key regulatory cell cycle proteins involved in the G1 phase. High expression of cyclin D2 and time-dependent increases in cyclin-dependent kinase 6 (CDK6) and cyclin D3 were observed in SNU-1103 during serum starvation. Very unexpectedly, in SNU-1103, the key G1 phase CDK inhibitor p21CiP1 was expressed at a consistently high level, while p27KiP1 expression was increased. Of three pRb family proteins, pRb expression was reduced and it became hypophosphorylated in SNU-1103 during serum starvation. Instead, p107 and p130 were expressed at consistently high levels in SNU-1103 during serum starvation. In conclusion, compared with an EBV-negative BJAB cell line, multiple cell cycle regulatory proteins were abnormally or inversely expressed in SNU-1103 during serum starvation.     

PARASITISM OF PHOTOSYNTHETIC DINOFLAGELLATES BY THREE STRAINS OF AMOEBOPHRYA (DINOPHYTA): PARASITE SURVIVAL,INFECTIVITY, GENERATION TIME,AND HOST SPECIFICITY1

Amoebophrya ceratii (Koeppen) Cachon is an obligate parasite of dinoflagellates and may represent a species complex. However, little is known about the biology and host range of different strains of Amoebophrya Cachon. Here, we determined parasite generation time and dinospore infectivity, survival, and ability to infect nonprimary hosts for strains of Amoebophrya from Akashiwo sanguinea (Hirasaka) G. Hansen et Moestrup, Gymnodinium instriatum (Freudenthal et Lee) Coats comb. nov., and Karlodinium micrum (Leadbeater et Dodge) J. Larsen. Akashiwo sanguinea was readily infected, with parasite prevalence reaching 100% in dinospore:host inoculations above a 10:1 ratio. Parasitism also approached 100% in G. instriatum, but only when inoculations exceeded a 40:1 ratio. Karlodinium micrum appeared partially resistant to infection, as parasite prevalence saturated at 92%. Parasite generation time differed markedly among Amoebophrya strains. Survival and infectivity of dinospores decreased over time, with strains from G. instriatum and A. sanguinea unable to initiate infections after 2 and 5 days, respectively. By contrast, dinospores from Amoebophrya parasitizing K. micrum remained infective for up to 11 days. Akashiwo sanguinea and G. instriatum were not infected when exposed to dinospores from nonprimary Amoebophrya strains. Karlodinium micrum, however, was attacked by dinospores of Amoebophrya from the other two host species, but infections failed to reach maturity. Observed differences in host–parasite biology support the hypothesis that Amoebophrya ceratii represents a complex of host‐specific species. Results also suggest that Amoebophrya strains have evolved somewhat divergent survival strategies that may encompass sexuality, heterotrophy during the “free‐living” dinospore stage, and dormancy.