Naturally acquired IgM antibody response to the C-terminal region of the merozoite surface protein 1 of Plasmodium vivax in Korea: use for serodiagnosis of vivax malaria

The antibody levels against the C-terminal region of the merozoite surface protein 1 of Plasmodium vivax (PvMSP1c) were measured in 276 patients with P. vivax malaria (patient group), 320 malaria-na?ve healthy individuals (control group 1), and 70 malaria-na?ve individuals with various disorders (control group 2) using the immunoglobulin M (IgM) capture enzyme-linked immunosorbent assay (ELISA) and the direct sandwich ELISA. To evaluate the antibody response during relapse, 5 relapsed patients were tested using the IgM capture ELISA. The IgM antibodies were negative in 99.7% of control group 1 and in 100% of control group 2; they were positive in 90.6% of the patient group. The total antibody levels were positive in 88.4% of the patient group with the direct sandwich ELISA. The sera from the second malaria episode, i.e., relapsed patients, were 100% positive on the IgM capture ELISA. The results of this study suggest that the IgM capture ELISA may be a useful diagnostic method for P. vivax malaria for both primary infection and relapse.     

Antioxidant and antidiabetic activities of mycelial and fruit-body extracts from <Emphasis Type="Italic">Mycoleptodonoides aitchisonii</Emphasis>

Mycoleptodonoides aitchisonii (Berk.) Maas Geest. is a culinary mushroom that is recognized as both a nutritious food and an excellent source of bioactive compounds. The purpose of this study was to investigate the antioxidant and antidiabetic properties of M. aitchisonii (MA) both in vitro and in vivo. Total oxyradical scavenging capacity (TOSC) assays revealed that fruit-body extracts had higher antioxidant capacity than mycelial extracts, 0.9-fold higher as measured by peroxynitrite (PN) scavenging assay, 3.7-fold higher as measured by peroxyl radical (PR) scavenging assay, and 1.6-fold as measured by hydroxyl radical (HR) scavenging assay, respectively. The assay of Akt phosphorylation, which is inhibited by Interleukin 6 (IL-6) in the signal transduction pathway for diabetes, was employed to evaluate the antidiabetic activity. Fruit-body extracts significantly increased Akt phosphorylation according to the fruit-body extract concentration, with a maximum increment of 77% at a concentration of 100 μg/mL compared to 51.4% decrement caused by IL-6, but there was no effect of mycelial extracts. Treatment with 5% MA fruit-body powder and streptozotocin (STZ) decreased the blood sugar level to 233.8 mg/dL in diabetic mice compared to 333.8 mg/dL after treatment with STZ alone. Additionally, MA treatment lowered total cholesterol (TC), triglyceride (TG), and LDL-cholesterol levels, while it increased the HDL-cholesterol level. All these findings indicate that fruit-body of M. aitchisonii has potential utility in preventing various diseases such as disorders of sugar and lipid metabolism.     

Probing the critical residues for intramolecular fructosyl transfer reaction of a levan fructotransferase

Levan fructotransferase (LFTase) preferentially catalyzes the transfructosylation reaction in addition to levan hydrolysis, whereas other levan-degrading enzymes hydrolyze levan into a levan-oligosaccharide and fructose. Based on sequence comparisons and enzymatic properties, the fructosyl transfer activity of LFTase is proposed to have evolved from levanase. In order to probe the residues that are critical to the intramolecular fructosyl transfer reaction of the Microbacterium sp. AL-210 LFTase, an error-prone PCR mutagenesis process was carried out, and the mutants that led to a shift in activity from transfructosylation towards hydrolysis of levan were screened by the DNS method. After two rounds of mutagenesis, TLC and HPLC analyses of the reaction products by the selected mutants revealed two major products; one is a di-D-fructose- 2,6':6,2'-dianhydride (DFAIV) and the other is a levanbiose. The newly detected levanbiose corresponds to the reaction product from LFTase lacking transferring activity. Two mutants (2-F8 and 2-G9) showed a high yield of levanbiose (38-40%) compared with the wild-type enzyme, and thus behaved as levanases. Sequence analysis of the individual mutants responsible for the enhanced hydrolytic activity indicated that Asn-85 was highly involved in the transfructosylation activity of LFTase.     

SIDECAR POLLEN suggests a plant-specific regulatory network underlying asymmetric microspore division in Arabidopsis

Asymmetric cell division is a universal strategy to generate diverse cell types necessary for patterning and proliferation of all eukaryotes. The development of haploid male gametophytes (pollen grains) in flowering plants is a remarkable example in which division asymmetry governs the functional specialization and germline differentiation essential for double fertilization. The male gametophyte is patterned via two mitotic divisions resulting in three highly differentiated daughter cells at maturity, a vegetative cell and two sperm cells. The first asymmetric division segregates a unique male germ cell from an undetermined haploid microspore and is executed in an elaborate sequence of cellular events. However the molecular mechanisms governing the division asymmetry in microspores are poorly understood. Recently we studied the phenotype of sidecar pollen (scp) mutants in detail, and demonstrated a requirement of SCP for both the correct timing and orientation of microspore division. SCP is a microspore-specific member of the LOB/AS2 domain family (LBD27/ASL29) showing that a plant-specific regulator plays a key role in oriented division of polarized microspores. Identification of SCP will serve as a new platform to further explore the largely unknown molecular networks regulating division asymmetry in microspores that establishes the male germline in flowering plants.Key words: sidecar pollen, microspore division, division asymmetry, male gametophyte development, male germline, LBD/ASL family proteinUnlike animals, flowering plants do not set aside a distinct germline from an early stage of the life cycle. Instead the angiosperm germline or germ cells are only segregated in the male and female gametophytes by a limited number of post-meiotic mitoses.¹ However, in common with their metazoan cousins, angiosperms utilize division asymmetry for cellular patterning and differentiation of their germlines. Through the unique patterning of a ‘cell-within-a-cell’ structure with three highly differentiated cells, the male gametophyte (pollen grains) serves its biological role to deliver two sessile male gametes to the female gametophyte. Two sequential but different modes of mitotic divisions pattern the male gametophyte (Fig. 1).² The first division (of the microspore) is asymmetric giving rise to two completely different daughter cells, a larger vegetative cell that will form the pollen tube and a smaller germ cell that is engulfed within the vegetative cell cytoplasm. The second division (of the germ cell) usually appears symmetric^† and produces a pair of linked sperm cells. Microspores artificially induced to undergo symmetric division using microtubule inhibitors lack the germ cell and fail to form the typical three-celled structure showing that asymmetry in microspore division is critical for patterning of the male gametophyte.⁴Open in a separate windowFigure 1Male gametophyte development in Arabidopsis (upper part) and mutations that block germ cell formation (lower part). (Upper part) Male gametophyte development involves two rounds of mitotic division. Prior to the first division the centrally positioned microspore nucleus migrates towards the radial wall (the future germ cell pole marked with an asterisk). At this eccentric site the polarized microspores undergo oriented mitosis and cytokinesis giving rise to highly unequal daughter cells, a vegetative cell and a germ cell of which the later produces a pair of sperm cells by symmetric division. (Lower part) Mutants that fail to establish a distinct germ cell arising from specific defects are illustrated. Arrows in red indicate the developmental origin of the phenotypic defects in mutants. Note that two daughter nuclei in the mutants are in grey to show that their cell fates have not yet been thoroughly investigated. n, nucleus; Vn, vegetative nucleus; Gn, generative nucleus; Gc, generative (or germ) cell; Sc, sperm cell; WT, wild type; gem1, gemini pollen1; scp, sidecar pollen; tio, two-in-one; hik/tes, hinkel/tetraspore 12a/12b, kinesin-12a/kinesin-12b.     

A monoclonal antibody specific for Candida albicans Als4 demonstrates overlapping localization of Als family proteins on the fungal cell surface and highlights differences between Als localization in vitro and in vivo

The Candida albicans agglutinin-like sequence (ALS) family encodes large cell surface glycoproteins that function in adhesion of the fungus to host and abiotic surfaces. Monoclonal antibodies (mAbs) specific for each Als protein were developed to study Als localization on the C. albicans surface. An anti-Als4 mAb demonstrated that Als4 covers the surface of yeast cells, with a greater abundance of Als4 on cells grown at 30 °C compared to 37 °C. On germ tubes, Als4 is localized in a restricted area proximal to the mother yeast. Immunolabeling with several anti-Als mAbs showed overlapping localization of Als1 and Als4 on yeast cells and Als1, Als3 and Als4 on germ tubes. Overlapping localization of Als proteins was also observed on yeast and hyphae recovered from mouse models of disseminated and oral candidiasis. Differences between Als localization in vivo and in vitro suggested changes in regulation of Als production in the host compared to the culture flask. Characterization with the anti-Als mAbs reveals the simultaneous presence and differences in relative abundance of Als proteins, creating an accurate image of Als representation and localization that can be used to guide conclusions regarding individual and collective Als protein function.     

Substantial overproduction of antibodies by applying osmotic pressure and sodium butyrate

Much of the current cell technology has enabled increased antibody production levels due to judicious nutrient feeding to raise cell densities and design better bioreactors. This study demonstrates that hybridomas can be hyperstimulated to produce higher immunoglobulin (lg) levels by suppressing cell growth and increasing culture longevity through adaptation to higher osmolarity media and addition of sodium butyrate. Prior to adaptation, cells placed in higher osmotic pressures (350 and 400 mOsm) were severely suppressed in growth down to 25% of the control (300 mOsm), although total lg titers achieved were similar to the control, approximately 140 mg/L. After a week of adaptation to 350 and 400 mOsm media, cell growth was not as dramatically suppressed, but considerably higher lg levels were attained at these elevated osmolarities. The highest yield of 265 mg/L was obtained at 350 mOsm compared to 140 mg/L at 300 mOsm, while maximum viable cell numbers dropped from 35 x 10(5) cells/mL to 31 x 10(5) cells/mL and culture longevity was extended by 20 h more than the control. Sodium butyrate, known to enhance protein production in other cell types, was then supplemented at a range of concentrations between 0.01 and 0.4 mM to the 350 mOsm culture to further enhance the lg levels. Butyrate at a concentration of 0.1 mM, in combination with osmotic pressure at 350 mOsm, further elevated the lg levels to 350 mg/L. Concomitantly, maximum viable cell numbers were reduced to 22 x 10(5) cells/mL, but culture longevity was extended by 40 h in the 0.1 mM butyrate supplemented culture compared to the control condition. Specific antibody productivity, q(Mab), continued to stay high during the stationary phase and was further elevated during the decline phase: thus, overall lg levels can be increased by 2.3 times by combining osmotic pressure and butyrate treatment. (c) 1993 John Wiley & Sons, Inc.     

Expression of EuNOD-ARP1 encoding auxin-repressed protein homolog is upregulated by auxin and localized to the fixation zone in root nodules of Elaeagnus umbellata

Root nodule formation is controlled by plant hormones such as auxin. Auxin-repressed protein (ARP) genes have been identified in various plant species but their functions are not clear. We have isolated a full-length cDNA clone (EuNOD-ARP1) showing high sequence homology to previously identified ARP genes from root nodules of Elaeagnus umbellata. Genomic Southern hybridization showed that there are at least four ARP-related genes in the genome of E. umbellata. The cDNA clone encodes a polypeptide of 120 amino acid residues with no signal peptide or organelle-targeting signals, indicating that it is a cytosolic protein. Its cytosolic location was confirmed using Arabidopsis protoplasts expressing a EuNOD-ARP1:smGFP fusion protein. Northern hybridization showed that EuNOD-ARP1 expression was higher in root nodules than in leaves or uninoculated roots. Unlike the ARP genes of strawberry and black locust, which are negatively regulated by exogenous auxin, EuNOD-ARP1 expression is induced by auxin in leaf tissue of E. umbellata. In situ hybridization revealed that EuNOD-ARP1 is mainly expressed in the fixation zone of root nodules.