Selection of a carbohydrate-binding domain with a helix-loop-helix structure

We obtained a novel carbohydrate-binding peptide having a helix-loop-helix scaffold from a random peptide library. The helix-loop-helix peptide library randomized at five amino acid residues was displayed on the major coat protein of a filamentous phage. Affinity selection with a ganglioside, Galbeta1-3GalNAcbeta1-4(Neu5Acalpha2-3)Galbeta1-4Glcbeta1-1'Cer (GM1), gave positive phage clones. Surface plasmon resonance spectroscopy showed that a corresponding 35-mer synthetic peptide had high affinity for GM1 with a dissociation constant of 0.24 microM. This peptide preferentially binds to GM1 rather than asialo GM1 and GM2, suggesting that a terminal galactose and sialic acid are required for the binding as for cholera toxin. Circular dichroism spectroscopic studies indicated that a helical structure is important for the affinity and specificity. Furthermore, alanine scanning at randomized positions showed that arginine and phenylalanine play an especially important role in the recognition of carbohydrates. Such a de novo helix-loop-helix peptide would be available for the design of carbohydrate-binding proteins.     

Involvement of aldolase A in X-ray resistance of human HeLa and UV(r)-1 cells

To find novel proteins involved in radio-resistance of human cells, we searched for nuclear proteins, whose expression levels alter after X-ray irradiation in HeLa cells, using agarose fluorescent two-dimensional differential gel electrophoresis following mass spectrometry. We identified 6 proteins, whose levels were increased in nuclei 24 h after irradiation at 5 Gy, including aldolase A. Nuclear aldolase A levels increased twofold after the irradiation, however, total aldolase A levels did not change. When the expression of aldolase A was suppressed by its specific siRNA, sensitization of the suppressed cells to X-ray-induced cell death was observed. In addition, UV^r-1 cells with higher aldolase A expression exhibited lower sensitivity to X-ray-induced cell death than the parental RSa cells with lower aldolase A expression. These results suggest that aldolase A may play a role in the radio-response of human cells, probably in nuclei, in addition to its glycolytic role in the cytosol.     

Active oxygen participation in chlorophyll destruction and lipid peroxidation in SO2-fumigated leaves of spinach

Chlorophyll a and carotenoids of spinach began to be destroyed2 to 3 hr after fumigation with 2 ppm SO₂ under light, whereaschlorophyll b was undamaged during 8 hr of exposure to SO₂.Pheophytin a was not affected by the fumigation. When disks excised from leaves fumigated with SO₂ at 2 ppm for2 hr were illuminated, chlorophyll a and carotenoids were brokendown, while they were not destroyed in darkness. The destructionof these pigments was suppressed under nitrogen. Chlorophylla destruction was inhibited by l,2-dihydroxybenzene-3,5-disulfonate(tiron), hydro-quinone and ascorbate, but not by l,4-diazabicyclo-[2,2,2]-octane(DABCO), methio-nine, histidine, benzoate and formate. Chlorophylla destruction was inhibited by phenazine methosulfate but stimulatedby methyl viologen. Addition of superoxide dismutase (SOD) tothe homogenate of SO₂-fumigated leaves inhibited the chlorophylla destruction. The activity of endogenous SOD was reduced to40% by 2-hr fumigation before the loss of chlorophyll was observed.These results suggest that chlorophyll a destruction by SO₂was due to superoxide radicals (O₂^–). Moreover, malondialdehyde (MDA), a product of lipid peroxidation,was formed in SO₂-fumigated leaves. MDA formation was inhibitedby tiron, hydroquinone and DABCO but not by benzoate and formate.MDA formation was increased by D₂O. These results suggest thatlipid peroxidation in SO₂-fumigated leaves was due to singletoxygen ¹O₂ produced from O_2^–. (Received May 15, 1980; )     

Genetic Background Influences P/Q-type Ca2+ Channel α1A Subunit mRNA Expression in Olfactory Bulb and Reproductive Ability of N-type Ca2+ Channel α1B Subunit-Deficient Mice

The Ca²⁺ channel α_1B subunit is a pore-forming component capable of generating N-type Ca²⁺ channel activity. Although the N-type Ca²⁺ channel plays a role in a variety of neuronal functions, α_1B-deficient mice did not show apparent behavioral abnormality. In a previous study, we observed a compensatory increase of mRNA expression of the P/Q-type Ca²⁺ channel α_1A subunit gene in olfactory bulb of α_1B-deficient mice with a CBA × C57BL/6 background; these mice showed a normal reproductive ability. In this study, we found that the mRNA expression level of the α_1A subunit was the same in olfactory bulb of wild, heterozygous, and homozygous α_1B-deficient mice with a CBA/JN background, and the homozygous male mice produced no offspring. These results suggest that the genetic background influences α_1A subunit mRNA expression and reproductive ability in α_1B-deficient mice.     

Novel functional biodegradable polymer II: fibroblast growth factor-2 activities of poly(gamma-glutamic acid)-sulfonate

Basic fibroblast growth factor (FGF-2) mitogenic activities of sulfonated poly(gamma-glutamic acid) (gamma-PGA-S) were investigated with chlorate-treated L929 fibroblast culture tests. When 72% of the carboxyl groups in gamma-PGA were sulfonated (gamma-PGA-S72), cell numbers reached a maximum. The activity of gamma-PGA-S72 was higher than that of gamma-PGA and synthetic heparinoids and was almost comparable to that of heparin. Cytotoxicity of gamma-PGA-S72 was not observed, regardless of the degree of sulfonation. FGF-2-protective effects of gamma-PGA-S72 against acid and thermal inactivation were also evaluated, and gamma-PGA-S72 showed higher FGF-2-protective effects in comparison to nonsulfonated gamma-PGA. The steric structures of various sulfonated gamma-PGA-Ss were analyzed by molecular modeling (molecular orbital method (MOPAC)) and indicated that gamma-PGA-Ss are helical in vacuo. Results from MOPAC and the molecular mechanics method (MM2) demonstrated that electrostatic interactions can take place between sulfonic and carboxyl groups of gamma-PGA-S and basic amino acid residues in FGF-2. gamma-PGA-S72 can interact with FGF-2 strongly.     

An Optimized Pentaplex PCR for Detecting DNA Mismatch Repair-Deficient Colorectal Cancers

Purpose

Microsatellite instability (MSI) is used to screen colorectal cancers (CRC) for Lynch Syndrome, and to predict outcome and response to treatment. The current technique for measuring MSI requires DNA from normal and neoplastic tissues, and fails to identify tumors with specific DNA mismatch repair (MMR) defects. We tested a panel of five quasi-monomorphic mononucleotide repeat markers amplified in a single multiplex PCR reaction (pentaplex PCR) to detect MSI.

Experimental Design

We investigated a cohort of 213 CRC patients, comprised of 114 MMR-deficient and 99 MMR-proficient tumors. Immunohistochemical (IHC) analysis evaluated the expression of MLH1, MSH2, PMS2 and MSH6. MSI status was defined by differences in the quasi-monomorphic variation range (QMVR) from a pool of normal DNA samples, and measuring differences in allele lengths in tumor DNA.

Results

Amplification of 426 normal alleles allowed optimization of the QMVR at each marker, and eliminated the requirement for matched reference DNA to define MSI in each sample. Using ≥2/5 unstable markers as the criteria for MSI resulted in a sensitivity of 95.6% (95% CI = 90.1–98.1%) and a positive predictive value of 100% (95% CI = 96.6%–100%). Detection of MSH6-deficiency was limited using all techniques. Data analysis with a three-marker panel (BAT26, NR21 and NR27) was comparable in sensitivity (97.4%) and positive predictive value (96.5%) to the five marker panel. Both approaches were superior to the standard approach to measuring MSI.

Conclusions

An optimized pentaplex (or triplex) PCR offers a facile, robust, very inexpensive, highly sensitive, and specific assay for the identification of MSI in CRC.     

The association between the phytoplankton, Rhopalosolen species (Chlorophyta; Chlorophyceae), and Anopheles gambiae sensu lato (Diptera: Culicidae) larval abundance in western Kenya

Algae are important food resources of the larvae of the African malaria vectors, Anopheles gambiae Giles and Anopheles arabiensis Patton (Anopheles gambiae sensu lato), and other zooplankton, but empirical evidence remains meager about the agal flora in ephemeral water bodies. The animals present in natural aquatic habitats in western Kenya were sampled from July to November 2002 to study abiotic and biotic environmental factors determining A. gambiae sl larval abundance. The five highest concentrations of third and fourth instars and pupae (hereafter referred to as old-stage larvae) were sampled in conjunction with the unicellular epizoic green algae, Rhopalosolen species (Chlorophyta; Chlorophyceae). Canonical correspondence analysis revealed that the presence of Rhopalosolen species was the most important determinant of the animal assemblage. The density of old-stage A. gambiae sl larvae was positively correlated with the presence of Rhopalosolen species, but the density of first and second instars of A. gambiae sl was not. The water bodies with Rhopalosolen sp. yielded larger mosquitoes in spite of the higher density of larvae. We demonstrated that the productivity of water bodies in terms of the larvae of malaria vectors can differ in magnitude depending on the agal flora. We discuss phytoplankton as a regulator of mosquito larval populations.     

Articulospora sp. produces Art1, an inhibitor of bacterial histidine kinase

A two-component system (TCS) comprising a histidine kinase (HK) sensor and a response regulator (RR) plays important roles in regulating the virulence of many pathogenic bacteria. We used a new screening method to isolate novel inhibitor Art1 against bacterial sensory HK from an acetone extract of solid cultures of Articulospora sp., an aquatic hypomycete. Art1 inhibited the ATP-dependent autophosphorylation of recombinant glutathione S-transferase-fusion protein SasA, a cyanobacterial HK, with an IC50 value of 9.5 microg/ml.     

Enzymatic properties of cellobiose 2-epimerase from <Emphasis Type="Italic">Ruminococcus albus</Emphasis> and the synthesis of rare oligosaccharides by the enzyme

The gene for cellobiose 2-epimerase (CE) from Ruminococcus albus NE1 was overexpressed in Escherichia coli cells. The recombinant CE was purified to homogeneity by a simple purification procedure with a high yield of 88%, and the molecular mass was 43.1 kDa on sodium dodecyl sulfate polyacrylamide gel electrophoresis and 44.0 kDa on gel chromatography. It exhibited optimal activity around at 30 degrees C and pH 7.5, and the enzyme activity was inhibited by Al3+, Fe3+, Co2+, Cu2+, Zn2+, Pb2+, Ag+, N-bromosuccinimide, iodoacetate, and 4-chloromercuribenzoate. In addition to cello-oligosaccharides, the enzyme was found to effectively 2-epimerize lactose to yield 4-O-beta-D-galactopyranosyl-D-mannose (epilactose), which occurs in cow milk as a rare oligosaccharide. The Km and kcat/Km values toward lactose were 33 mM and 1.6 s(-1) mM(-1), and those toward cellobiose were 13.8 mM and 4.6 s(-1) mM(-1), respectively. N-Acetyl-D-glucosamine, uridine 5'-diphosphate-glucose, D-glucose 6-phosphate, maltose, sophorose, laminaribiose, and gentiobiose were inert as substrates for the recombinant CE. We demonstrated that epilactose was resistant to rat intestinal enzymes, utilized by human adult bifidobacteria, and stimulated the tight junction permeability in Caco-2 cells. These results strongly suggest that this rare disaccharide is promising for use as a prebiotic.