Two-dimensional NMR studies of Kazal proteinase inhibitors. 2. Sequence-specific assignments and secondary structure of reactive site modified turkey ovomucoid third domain

The solution structure of modified turkey ovomucoid third domain (OMTKY3*) was investigated by high-resolution proton NMR techniques. OMTKY3* was obtained by enzymatic hydrolysis of the scissile reactive site peptide bond (Leu18-Glu19) in turkey ovomucoid third domain (OMTKY3). All of the backbone proton resonances were assigned to sequence-specific residues except the NH's of Leu1 and Glu19, which were not observed. Over 80% of the side-chain protons also were assigned. The secondary structure of OMTKY3*, as determined from assigned NOESY cross-peaks and identification of slowly exchanging amide protons, contains antiparallel beta-sheet consisting of three strands (residues 21-25, 28-32, and 49-54), one alpha-helix (residues 33-44), and one reverse turn (residues 26-28). This secondary structure closely resembles that of OMTKY3 in solution [Robertson, A. D., Westler, W. M., & Markley, J. L. (1988) Biochemistry (preceding paper in this issue)]. On the other hand, changes in the tertiary structure of the protein near to and remote from the cleavage site are indicated by differences in the chemical shifts of numerous backbone protons of OMTKY3 and OMTKY3*.     

Chitosan sponges as tissue engineering scaffolds for bone formation

Rat calvarial osteoblasts were grown in porous chitosan sponges fabricated by freeze drying. The prepared chitosan sponges had a porous structure with a 100-200 microm pore diameter, which allowed cell proliferation. Cell density, alkaline phosphatase activity and calcium deposition were monitored for up to 56 d culture. Cell numbers were 4 x 10(6) (day 1), 11 x 10(6) (day 28) and 12 x 10(6) (day 56) per g sponge. Calcium depositions were 9 (day 1), 40 (day 28) and 48 (day 56) microg per sponge. Histological results corroborated that bone formation within the sponges had occurred. These results show that chitosan sponges can be used as effective scaffolding materials for tissue engineered bone formation in vitro.     

Isolation of novel human fetal brain cDNAs mapped to human chromosome bands, 1q25 and 8q24.1

We isolated chromosome band-specific human fetal brain cDNAs by the microdissection mediated cDNA capture method, and localized these cDNA using in situ hybridization histochemistry with developing rat brain sections. Uni-Amp cDNAs were prepared from an 18-week old human fetal brain, and hybridized to human metaphase chromosomes. Eight Uni-Amp cDNAs, hybridized to chromosome band 1q25 or 8q24.1, were recovered by microdissection and PCR amplification with Uni-Amp primers. Among these cDNAs, two novel genes (FB113 of 8q24.1 and FB134 of 1q25) showed a temporospatially interesting expression pattern in the developing rat brains. The expression of FB113 was under dynamic regulation in the developing granule cells of cerebellum and dentate gyrus. FB134 showed a nervous tissue specific expression pattern and an exclusively prominent expression in the developing presubiculum and parasubiculum. By the fluorescence in situ hybridization using human genomic DNAs, FB113 and FB134 were mapped back to the human chromosome bands 8q24.1 and 1q25, respectively. These results indicate that combined application of the microdissection mediated cDNA capture method and in situ hybridization histochemistry can be used for the isolation of chromosomal band-specific genes related to brain development or human genetic diseases.     

Protein recovery using two-phase systems

The technology and the main economic aspects of extractive recovery of intracellular enzymes and other biological active proteins are reviewed briefly. It will be seen that the method has high potential and is already used industrially.     

Modulation of Sweet Taste by Umami Compounds via Sweet Taste Receptor Subunit hT1R2

Although the five basic taste qualities—sweet, sour, bitter, salty and umami—can be recognized by the respective gustatory system, interactions between these taste qualities are often experienced when food is consumed. Specifically, the umami taste has been investigated in terms of whether it enhances or reduces the other taste modalities. These studies, however, are based on individual perception and not on a molecular level. In this study we investigated umami-sweet taste interactions using umami compounds including monosodium glutamate (MSG), 5’-mononucleotides and glutamyl-dipeptides, glutamate-glutamate (Glu-Glu) and glutamate-aspartic acid (Glu-Asp), in human sweet taste receptor hT1R2/hT1R3-expressing cells. The sensitivity of sucrose to hT1R2/hT1R3 was significantly attenuated by MSG and umami active peptides but not by umami active nucleotides. Inhibition of sweet receptor activation by MSG and glutamyl peptides is obvious when sweet receptors are activated by sweeteners that target the extracellular domain (ECD) of T1R2, such as sucrose and acesulfame K, but not by cyclamate, which interact with the T1R3 transmembrane domain (TMD). Application of umami compounds with lactisole, inhibitory drugs that target T1R3, exerted a more severe inhibitory effect. The inhibition was also observed with F778A sweet receptor mutant, which have the defect in function of T1R3 TMD. These results suggest that umami peptides affect sweet taste receptors and this interaction prevents sweet receptor agonists from binding to the T1R2 ECD in an allosteric manner, not to the T1R3. This is the first report to define the interaction between umami and sweet taste receptors.     

Five hTRPA1 Agonists Found in Indigenous Korean Mint,Agastache rugosa

Transient receptor potential ankyrin1 (TRPA1) and transient receptor potential vanilloid 1 (TRPV1) are members of the TRP superfamily of structurally related, nonselective cation channels and mediators of several signaling pathways. Previously, we identified methyl syringate as an hTRPA1 agonist with efficacy against gastric emptying. The aim of this study was to find hTRPA1 and/or hTRPV1 activators in Agastache rugosa (Fisch. et Meyer) O. Kuntze (A.rugosa), commonly known as Korean mint to improve hTRPA1-related phenomena. An extract of the stem and leaves of A.rugosa (Labiatae) selectively activated hTRPA1 and hTRPV1. We next investigated the effects of commercially available compounds found in A.rugosa (acacetin, 4-allylanisole, p-anisaldehyde, apigenin 7-glucoside, L-carveol, β-caryophyllene, trans-p-methoxycinnamaldehyde, methyl eugenol, pachypodol, and rosmarinic acid) on cultured hTRPA1- and hTRPV1-expressing cells. Of the ten compounds, L-carveol, trans-p-methoxycinnamaldehyde, methyl eugenol, 4-allylanisole, and p-anisaldehyde selectively activated hTRPA1, with EC50 values of 189.1±26.8, 29.8±14.9, 160.2±21.9, 1535±315.7, and 546.5±73.0 μM, respectively. The activities of these compounds were effectively inhibited by the hTRPA1 antagonists, ruthenium red and HC-030031. Although the five active compounds showed weaker calcium responses than allyl isothiocyanate (EC₅₀=7.2±1.4 μM), our results suggest that these compounds from the stem and leaves of A.rugosa are specific and selective agonists of hTRPA1.     

Genetic variance modifies apoptosis susceptibility in mature oocytes via alterations in DNA repair capacity and mitochondrial ultrastructure

Although the identification of specific genes that regulate apoptosis has been a topic of intense study, little is known of the role that background genetic variance plays in modulating cell death. Using germ cells from inbred mouse strains, we found that apoptosis in mature (metaphase II) oocytes is affected by genetic background through at least two different mechanisms. The first, manifested in AKR/J mice, results in genomic instability. This is reflected by numerous DNA double-strand breaks in freshly isolated oocytes, causing a high apoptosis susceptibility and impaired embryonic development following fertilization. Microinjection of Rad51 reduces DNA damage, suppresses apoptosis and improves embryonic development. The second, manifested in FVB mice, results in dramatic dimorphisms in mitochondrial ultrastructure. This is correlated with cytochrome c release and a high apoptosis susceptibility, the latter of which is suppressed by pyruvate treatment, Smac/DIABLO deficiency, or microinjection of 'normal' mitochondria. Therefore, background genetic variance can profoundly affect apoptosis in female germ cells by disrupting both genomic DNA and mitochondrial integrity.     

Inferring biological functions and associated transcriptional regulators using gene set expression coherence analysis

Background  

Gene clustering has been widely used to group genes with similar expression pattern in microarray data analysis. Subsequent enrichment analysis using predefined gene sets can provide clues on which functional themes or regulatory sequence motifs are associated with individual gene clusters. In spite of the potential utility, gene clustering and enrichment analysis have been used in separate platforms, thus, the development of integrative algorithm linking both methods is highly challenging.     

CorA affects tolerance of Escherichia coli and Salmonella enterica serovar Typhimurium to the lactoperoxidase enzyme system but not to other forms of oxidative stress

The enzyme lactoperoxidase is part of the innate immune system in vertebrates and owes its antimicrobial activity to the formation of oxidative reaction products from various substrates. In a previous study, we have reported that, with thiocyanate as a substrate, the lactoperoxidase system elicits a distinct stress response in Escherichia coli MG1655. This response is different from but partly overlapping with the stress responses to hydrogen peroxide and to superoxide. In the current work, we constructed knockouts in 10 lactoperoxidase system-inducible genes to investigate their role in the tolerance of E. coli MG1655 to this antimicrobial system. Five mutations resulted in a slightly increased sensitivity, but one mutation (corA) caused hypersensitivity to the lactoperoxidase system. This hypersensitive phenotype was specific to the lactoperoxidase system, since neither the sensitivity to hydrogen peroxide nor to the superoxide generator plumbagin was affected in the corA mutant. Salmonella enterica serovar Typhimurium corA had a similar phenotype. Although corA encodes an Mg2+ transporter and at least three other inducible open reading frames belonged to the Mg2+ regulon, repression of the Mg stimulon by Mg2+ did not change the lactoperoxidase sensitivity of either the wild-type or corA mutant. Prior exposure to 0.3 mM Ni2+, which is also transported by CorA, strongly sensitized MG1655 but not the corA mutant to the lactoperoxidase system. Furthermore, this Ni2+-dependent sensitization was suppressed by the CorA-specific inhibitor Co(III) hexaammine. These results indicate that CorA affects the lactoperoxidase sensitivity of E. coli by modulating the cytoplasmic concentrations of transition metals that enhance the toxicity of the lactoperoxidase system.