Induction of in vitro flowering in Dendrobium Madame Thong-In (Orchidaceae) seedlings is associated with increase in endogenous N(6)-(Delta (2)-isopentenyl)-adenine (iP) and N (6)-(Delta (2)-isopentenyl)-adenosine (iPA) levels

We analysed the endogenous cytokinin levels of Dendrobium Madame Thong-In seedlings grown in vitro during vegetative and flowering-inductive periods. HPLC was used to fractionate the extracts and radioimmunoassay (RIA) was used for assay of zeatin (Z), dihydrozeatin (DZ), N(6)-(Delta(2)-isopentenyl)-adenine (iP) and their derivatives. Coconut water used in experiments was found to contain high level (>136 pmol ml(-1)) of zeatin riboside (ZR). Protocorms and seedlings cultured in medium with coconut water were found to contain 0.5-3.9 pmol g(-1) FW of the cytokinins analysed. Seedlings (1.0-1.5 cm) cultured in flowering-inductive liquid medium containing 6-benzyladenine (BA, 4.4 muM) and coconut water (CW, 15%) contained up to 200 and 133 pmol g(-1) FW of iP and iPA, respectively. These levels were significantly higher than all other cytokinins analysed in seedlings of the same stage and were about 80- to 150-folds higher than seedlings cultured in non-inductive medium. During the transitional (vegetative to reproductive) stage, the endogenous levels of iP (178 pmol g(-1) FW) and iPA (63 pmol g(-1) FW) were also significantly higher than cytokinins in the zeatine (Z) and dihydrozeatin (DZ) families in the same seedlings. Seedlings that grew on inductive medium but remained vegetative contained lower levels of iPA. The importance of the profiles of iP and its derivatives in induction of in vitro flowering of D. Madame Thong-In is discussed.     

Role of tfdC(I)D(I)E(I)F(I) and tfdD(II)C(II)E(II)F(II) gene modules in catabolism of 3-chlorobenzoate by Ralstonia eutropha JMP134(pJP4)

The enzymes chlorocatechol-1,2-dioxygenase, chloromuconate cycloisomerase, dienelactone hydrolase, and maleylacetate reductase allow Ralstonia eutropha JMP134(pJP4) to degrade chlorocatechols formed during growth in 2,4-dichlorophenoxyacetate or 3-chlorobenzoate (3-CB). There are two gene modules located in plasmid pJP4, tfdC(I)D(I)E(I)F(I) (module I) and tfdD(II)C(II)E(II)F(II) (module II), putatively encoding these enzymes. To assess the role of both tfd modules in the degradation of chloroaromatics, each module was cloned into the medium-copy-number plasmid vector pBBR1MCS-2 under the control of the tfdR regulatory gene. These constructs were introduced into R. eutropha JMP222 (a JMP134 derivative lacking pJP4) and Pseudomonas putida KT2442, two strains able to transform 3-CB into chlorocatechols. Specific activities in cell extracts of chlorocatechol-1,2-dioxygenase (tfdC), chloromuconate cycloisomerase (tfdD), and dienelactone hydrolase (tfdE) were 2 to 50 times higher for microorganisms containing module I compared to those containing module II. In contrast, a significantly (50-fold) higher activity of maleylacetate reductase (tfdF) was observed in cell extracts of microorganisms containing module II compared to module I. The R. eutropha JMP222 derivative containing tfdR-tfdC(I)D(I)E(I)F(I) grew four times faster in liquid cultures with 3-CB as a sole carbon and energy source than in cultures containing tfdR-tfdD(II)C(II)E(II)F(II). In the case of P. putida KT2442, only the derivative containing module I was able to grow in liquid cultures of 3-CB. These results indicate that efficient degradation of 3-CB by R. eutropha JMP134(pJP4) requires the two tfd modules such that TfdCDE is likely supplied primarily by module I, while TfdF is likely supplied by module II.     

fra(1) (p11), fra(1) (q22) and r(1) (p11q22) in a retarded girl.

A mentally retarded girl with a 46,XX/47, XX+r(1) (p11q22q22p11)/47, XX+r(1) (p11q22) fra(1) (p31) fra(1) (p11) fra(1) (q22) karyotype who inherited the fragile sites from the normal mother was studied. The conicidence of fra(1) (p11) and fra(1) (q22) with the ring chromosome breakpoints strongly suggests a cause-effect relationship. This finding agrees with other reported associations between fragile sites and structural chromosome abnormalities and constitutes the fourth reported of a de novo structurally abnormal chromosome as a consequence of presumed in vivo fragile sites instability. Although risk figures for chromosome anomalies and cancer associated with fragile sites are lacking, carriers of fra (1) (p11) may have a higher risk for abnormalities of chromosome 1 in somatic and gonadal cells than the general population.     

Characterization of O(6)-methylguanine-DNA-methyltransferase (O(6)-MGMT) activity in Xiphophorus fishes

We utilized a custom-synthesized double-strand oligonucleotide containing a single O(6)-methylguanine (O(6)-MG) residue within a restriction endonuclease recognition site to determine O(6)-methylguanine-DNA-methyltransferase (O(6)-MGMT) activity in various tissue extracts prepared from Xiphophorus fish. The results suggest Xiphophorus fish O(6)-MGMT activity has many of the same characteristics as Escherichia coli and mammalian O(6)-MGMT's including rapid reaction kinetics consistent with stoichiometric removal of methyl groups, but exhibits a temperature optimum of 23 degrees C.Results from protein extract activity assays indicate O(6)-MGMT activity patterns among four Xiphophorus tissues followed the order: brain> or =testes>gill> or =liver. In mammals, O(6)-MGMT activity is high in liver, while activity in brain is minimal (i.e. approximately 9% of liver); however, we report that in the Xiphophorus fishes examined, brain tissue extracts exhibited much higher (approximately six-fold) O(6)-MGMT activity levels than liver. Comparison of O(6)-MGMT activity between Xiphophorus species employed in tumor induction experiments did not indicate significant differences in ability to clear the pre-mutagenic O(6)-MG from the oligonucleotide substrate.     

Linkage of plasminogen (PLG) and apolipoprotein(a) (LPA) in baboons.

Four allelic forms of serum plasminogen (PLG) were detected in baboons (Papio hamadryas Linneaus 1758) by isoelectric focusing and were determined to be inherited as autosomal codominant traits. Linkage analysis of data from 179 progeny and their parents revealed that PLG is tightly linked (lod score = 30.20) to the gene encoding apolipoprotein(a) (LPA), as in humans. No recombinant individuals were identified. This is the first linkage detected between PLG and LPA in any species other than humans and is the first genetic linkage identified in a nonhuman primate species by family studies.     

Spider prey (Araneae) of Trypoxylon (Trypargilum) rogenhoferi (Hymenoptera: Sphecidae) in southeastern Brazil

Fifty five nests and 216 cells of Trypoxylon (Trypargilum) rogenhoferi were obtained from trap-nests (cut bamboo canes) in Santa Carlota Farm (Itaoca Section = IS and Santana Section = SS), Cajuru and on the S?o Paulo University Campus, Ribeir?o Preto (= RP), both in the State of S?o Paulo, Brazil (Sept. 1993-Oct. 1995). The prey (spiders) of 40 cells from IS, 58 from SS and 39 from RP were identified. The greatest nesting frequency occurred during the hot and wet season (September to April). T. rogenhoferi preyed upon individuals of five spider families, with Araneidae (orb-weaver spiders) being the most frequent (99.6%). Alpaida aff. negro (58%) was the most frequently collected species in IS, followed by A. alto (24.8%); in SS (59.6%) and RP (64.7%) the most frequent species was A. veniliae, followed in SS by A. aff. negro (14.9%) and in RP by A. leucogramma (13.5%). The size of reproductive niches, H' = 1.25 (IS), H' = 1.30 (SS) and H' = 1.29 (RP) were not significantly different. There was a positive correlation between the reproductive niche width (H') and evenness.     

The alpha(4) integrin subunit Tyr(187) has a key role in alpha(4)beta(7)-dependent cell adhesion

The integrin alpha(4)beta(7) is the cell adhesion receptor for the mucosal vascular addressin MAdCAM-1, and this interaction is dominant in lymphocyte homing to Peyer's patch high endothelial venules, and plays key roles in lymphocyte recruitment at sites of inflammation. To identify alpha(4) subunit amino acids important for alpha(4)beta(7)/MAdCAM-1 interaction, we expressed mutant alpha(4) and wild type beta(7) chains in K562 cells and analyzed the effect of the mutations on cell adhesion to a soluble MAdCAM-1 (sMAdCAM-1-Ig). Transfectants expressing mutated alpha(4) at Tyr(187) displayed a substantial decrease in adhesion to this ligand, which was associated with a reduced alpha(4)beta(7)/sMAdCAM-1-Ig interaction, as determined by soluble binding assays. Addition of Mn(2+) to the adhesion assays did not restore the impaired adhesion. Mutations at alpha(4) Gln(152)Asp(153) also affected transfectant adhesion to sMAdCAM-1-Ig, but did not involve an alteration of alpha(4)beta(7)/MAdCAM-1 binding, and adhesion was restored by Mn(2+). Instead, mutations at alpha(4) Asn(123)Glu(124) did not affect this adhesion. Mutation of alpha(4) Tyr(187) abolished alpha(4)beta(7)-mediated cell adhesion to CS-1/fibronectin, an additional ligand for alpha(4)beta(7), while alpha(4) Gln(152)Asp(153) transfectant mutants showed a reduced adhesion. These results identify alpha(4) Tyr(187) as a key residue during receptor alpha(4)beta(7)/ligand interactions, indicating that it plays important roles in alpha(4)beta(7)-mediated leukocyte adhesion, and provide a potential target for therapeutic intervention in several inflammatory pathologies.     

Collagen-like triple helix formation of synthetic (Pro-Pro-Gly)10 analogues: (4(S)-hydroxyprolyl-4(R)-hydroxyprolyl-Gly)10, (4(R)-hydroxyprolyl-4(R)-hydroxyprolyl-Gly)10 and (4(S)-fluoroprolyl-4(R)-fluoroprolyl-Gly)10.

For the rational design of a stable collagen triple helix according to the conventional rule that the pyrrolidine puckerings of Pro, 4-hydroxyproline (Hyp) and 4-fluoroproline (fPro) should be down at the X-position and up at the Y-position in the X-Y-Gly repeated sequence for enhancing the triple helix propensities of collagen model peptides, a series of peptides were prepared in which X- and Y-positions were altogether occupied by Hyp(R), Hyp(S), fPro(R) or fPro(S). Contrary to our presumption that inducing the X-Y residues to adopt a down-up conformation would result in an increase in the thermal stability of peptides, the triple helices of (Hyp(S)-Hyp(R)-Gly)(10) and (fPro(S)-fPro(R)-Gly)(10) were less stable than those of (Pro-Hyp(R)-Gly)(10) and (Pro-fPro(R)-Gly)(10), respectively. As reported by B?chinger's and Zagari's groups, (Hyp(R)-Hyp(R)-Gly)(10) which could have an up-up conformation unfavorable for the triple helix, formed a triple helix that has a high thermal stability close to that of (Pro-Hyp(R)-Gly)(10). These results clearly show that the empirical rule based on the conformational preference of pyrrolidine ring at each of X and Y residues should not be regarded as still valid, at least for predicting the stability of collagen models in which both X and Y residues have electronegative groups at the 4-position.     

Tetrameres (Tetrameres) grusi (Shumakovich, 1946) (Nematoda: Tetrameridae) in Eurasian cranes (Grus grus) in central Iran

The proventriculi of 11 Eurasian cranes (Grus grus) from central Iran were examined for the existence of parasitic helminths. Preliminary reports suggested that the death of these birds was related to untimely cold weather. Nine proventriculi (82%) were heavily infected by the nematode Tetrameres grusi. Glandular structure of the infected proventriculi was replaced by epithelial atrophy but significant inflammatory reactions were not observed in any of the infected organs. In serious infections, the nematode produced vast structural and functional changes, causing organ dysfunction and glandular necrosis. The coincidence of heavy helminth infection at times of environmental stress may lead to debilitation, wasting, and perhaps mortality in migratory cranes.     

Bis(6-ethylpicolinato)oxovanadium(IV) complex with normoglycemic activity in KK-A(y) mice.

A novel bis(6-ethylpicolinato)(H(2)O)oxovanadium(IV) complex (VO(6epa)(2) x (H(2)O)) was prepared and its structure was revealed by X-ray analysis (space group Pc(#7), a=10.838(2), b=11.148(5), c=16.642(3) A, and Z=2). Because VO(6epa)(2) x (H(2)O) exhibited higher in vitro insulinomimetic activity compared to that of vanadyl sulfate in terms of inhibition of free fatty acid (FFA) release from isolated rat adipocytes in the presence of epinephrine, its in vivo effect on whether the complex has a blood glucose normalizing effect was examined in KK-A(y) mice, a model animal of type 2 diabetes mellitus. VO(6epa)(2) x (H(2)O) was found to normalize the high blood glucose levels of KK-A(y) mice when given intraperitoneally at doses of 49 micromol/kg body weight for the first 4 days and then 39 micromol/kg body weight for 10 days. In addition, VO(6epa)(2) x (H(2)O) improved glucose tolerance ability as examined by the oral glucose test and seemed to have little toxicity in terms of serum parameters. VO(6epa)(2) x (H(2)O) showed higher normoglycemic activity than bis(6-methylpicolinato)oxovanadium(IV) (VO(6mpa)(2)) at the same dose. These results indicated that greater enhancement of the blood glucose normalizing effect in KK-A(y) mice by ethyl substitution compared to methyl substitution may be due to its being more strongly lipophilic.     

Response to mercury (II) ions in Methylococcus capsulatus (Bath)

The mercury (II) ion is toxic and is usually detoxified in Bacteria by reduction to elemental mercury, which is less toxic. This is catalysed by an NAD(P)H-dependent mercuric reductase (EC 1.16.1.1). Here, we present strong evidence that Methylococcus capsulatus (Bath) - a methanotrophic member of the Gammaproteobacteria - uses this enzyme to detoxify mercury. In radiorespirometry studies, it was found that cells exposed to mercury dissimilated 100% of [(14) C]-methane provided to generate reducing equivalents to fuel mercury (II) reduction, rather than the mix of assimilation and dissimilation found in control incubations. The detoxification system is constitutively expressed with a specific activity of 352 (±18) nmol NADH oxidized min(-1) (mg protein)(-1) . Putative mercuric reductase genes were predicted in the M.?capsulatus (Bath) genome and found in mRNA microarray studies. The MerA-derived polypeptide showed high identity (>?80%) with MerA sequences from the Betaproteobacteria.     

Characterization of divalent cation localization in the minor groove of the A(n)T(n) and T(n)A(n) DNA sequence elements by (1)H NMR spectroscopy and manganese(II)

The localization of Mn(2+) in A-tract DNA has been studied by (1)H NMR spectroscopy using a series of self-complementary dodecamer oligonucleotides that contain the sequence motifs A(n)(n) and T(n)A(n), where n = 2, 3, or 4. Mn(2+) localization in the minor groove is observed for all the sequences that have been studied, with the position and degree of localization being highly sequence-dependent. The site most favored for Mn(2+) localization in the minor groove is near the 5'-most ApA step for both the T(n)A(n) and the A(n)T(n) series. For the T(n)A(n) series, this results in two closely spaced symmetry-related Mn(2+) localization sites near the center of each duplex, while for the A(n)T(n) series, the two symmetry-related sites are separated by as much as one half-helical turn. The degree of Mn(2+) localization in the minor groove of the T(n)A(n) series decreases substantially as the AT sequence element is shortened from T(4)A(4) to T(2)A(2). The A(n)T(n) series also exhibits length-dependent Mn(2+) localization; however, the degree of minor groove occupancy by Mn(2+) is significantly less than that observed for the T(n)A(n) series. For both A(n)T(n) and T(n)A(n) sequences, the 3'-most AH2 resonance is the least broadened of the AH2 resonances. This is consistent with the observation that the minor groove of A-tract DNA narrows in the 5' to 3' direction, apparently becoming too narrow after two base pairs for the entry of a fully hydrated divalent cation. The results that are reported illustrate the delicate interplay that exists between DNA nucleotide sequence, minor groove width, and divalent cation localization. The proposed role of cation localization in helical axis bending by A-tracts is also discussed.     

Internal conductance to CO(2) diffusion and C(18)OO discrimination in C(3) leaves

(18)O discrimination in CO(2) stems from the oxygen exchange between (18)O-enriched water and CO(2) in the chloroplast, a process catalyzed by carbonic anhydrase (CA). A proportion of this (18)O-labeled CO(2) escapes back to the atmosphere, resulting in an effective discrimination against C(18)OO during photosynthesis (Delta(18)O). By constraining the delta(18)O of chloroplast water (delta(e)) by analysis of transpired water and the extent of CO(2)-H(2)O isotopic equilibrium (theta(eq)) by measurements of CA activity (theta(eq) = 0.75-1.0 for tobacco, soybean, and oak), we could apply measured Delta(18)O in a leaf cuvette attached to a mass spectrometer to derive the CO(2) concentration at the physical limit of CA activity, i.e. the chloroplast surface (c(cs)). From the CO(2) drawdown sequence between stomatal cavities from gas exchange (c(i)), from Delta(18)O (c(cs)), and at Rubisco sites from Delta(13)C (c(c)), the internal CO(2) conductance (g(i)) was partitioned into cell wall (g(w)) and chloroplast (g(ch)) components. The results indicated that g(ch) is variable (0.42-1.13 mol m(-2) s(-1)) and proportional to CA activity. We suggest that the influence of CA activity on the CO(2) assimilation rate should be important mainly in plants with low internal conductances.     

Comparison of (13)C(alpha)H and (15)NH backbone dynamics in protein GB1

This study presents a site-resolved experimental view of backbone C(alpha)H and NH internal motions in the 56-residue immunoglobulin-binding domain of streptococcal protein G, GB1. Using (13)C(alpha)H and (15)NH NMR relaxation data [T(1), T(2), and NOE] acquired at three resonance frequencies ((1)H frequencies of 500, 600, and 800 MHz), spectral density functions were calculated as F(omega) = 2omegaJ(omega) to provide a model-independent way to visualize and analyze internal motional correlation time distributions for backbone groups in GB1. Line broadening in F(omega) curves indicates the presence of nanosecond time scale internal motions (0.8 to 5 nsec) for all C(alpha)H and NH groups. Deconvolution of F(omega) curves effectively separates overall tumbling and internal motional correlation time distributions to yield more accurate order parameters than determined by using standard model free approaches. Compared to NH groups, C(alpha)H internal motions are more broadly distributed on the nanosecond time scale, and larger C(alpha)H order parameters are related to correlated bond rotations for C(alpha)H fluctuations. Motional parameters for NH groups are more structurally correlated, with NH order parameters, for example, being larger for residues in more structured regions of beta-sheet and helix and generally smaller for residues in the loop and turns. This is most likely related to the observation that NH order parameters are correlated to hydrogen bonding. This study contributes to the general understanding of protein dynamics and exemplifies an alternative and easier way to analyze NMR relaxation data.     

(AGCTG) (AGCTG) (AGCTG) (GGGTG) as the primordial sequence of intergenic spacers: the role in immunoglobulin class switch

The means employed for immunoglobulin heavy chain class switch appears to be no different from that by which meiotic intergenic crossing-overs at accomplished. As with other intergenic spacers, the 5' noncoding sequence of each Ig CH (immunoglobulin heavy chain constant region) gene apparently undergoes unconstrained sequence changes due to randomly sustained base substitutions, deletions, and duplications. Yet, there remains sufficient regional sequence homology between the Ig Cmu 5' noncoding sequence and those of its somatic recombination partners, e.g., Ig C gamma 1, Ig C gamma 2b, Ig C alpha, because each of these 5' noncoding sequences is made of multiple copies in various stages of degeneracy of one primordial 20 base pair-long sequence: (AGCTG) (AGCTG) (AGCTG) (GGGTG).     

The role of the CPNKEKEC sequence in the beta(2) subunit I domain in regulation of integrin alpha(L)beta(2) (LFA-1)

The alpha(L) I (inserted or interactive) domain of integrin alpha(L)beta(2) undergoes conformational changes upon activation. Recent studies show that the isolated, activated alpha(L) I domain is sufficient for strong ligand binding, suggesting the beta(2) subunit to be only indirectly involved. It has been unclear whether the activity of the alpha(L) I domain is regulated by the beta(2) subunit. In this study, we demonstrate that swapping the disulfide-linked CPNKEKEC sequence (residues 169-176) in the beta(2) I domain with a corresponding beta(3) sequence, or mutating Lys(174) to Thr, constitutively activates alpha(L)beta(2) binding to ICAM-1. These mutants do not require Mn(2+) for ICAM-1 binding and are insensitive to the inhibitory effect of Ca(2+). We have also localized a component of the mAb 24 epitope (a reporter of beta(2) integrin activation) in the CPNKEKEC sequence. Glu(173) and Glu(175) of the beta(2) I domain are identified as critical for mAb 24 binding. Because the epitope is highly expressed upon beta(2) integrin activation, it is likely that the CPNKEKEC sequence is exposed or undergoes conformational changes upon activation. Deletion of the alpha(L) I domain did not eliminate the mAb 24 epitope. This confirms that the alpha(L) I domain is not critical for mAb 24 binding, and indicates that mAb 24 detects a change expressed in part in the beta(2) subunit I domain. These results suggest that the CPNKEKEC sequence of the beta(2) I domain is involved in regulating the alpha(L) I domain.     

H(2)O(2) opens BK(Ca) channels via the PLA(2)-arachidonic acid signaling cascade in coronary artery smooth muscle

H(2)O(2) is a reactive oxygen species that contracts or relaxes vascular smooth muscle, but the molecular basis of these effects remains obscure. We previously demonstrated that H(2)O(2) opens the large-conductance, calcium- and voltage-activated (BK(Ca)) potassium channel of coronary myocytes (2) and now report physiological and biochemical evidence that the effect of H(2)O(2) on coronary smooth muscle involves the phospholipase A(2) (PLA(2))/arachidonic acid (AA) signaling cascades. H(2)O(2) stimulation of BK(Ca) channel activity was inhibited by arachidonyl trifluoromethyl ketone, an inhibitor of cytosolic PLA(2). Furthermore, H(2)O(2) stimulated release of [(3)H]AA from coronary myocytes, and exogenous AA mimicked the effect of H(2)O(2) on BK(Ca) channels. Inhibitors of protein kinase C activity attenuated the effect of H(2)O(2) on BK(Ca) channels, [(3)H]AA release, or intact coronary arteries. In addition, the effect of H(2)O(2) or AA on BK(Ca) channels was inhibited by blockers of lipoxygenase metabolism. In contrast, inhibitors of cyclooxygenase or cytochrome P-450 had no effect. We propose that H(2)O(2) relaxes coronary arteries by stimulating BK(Ca) channels via the PLA(2)/AA signaling cascade and that lipoxygenase metabolites mediate this response.     

Role of benzimidazole (Bid) in the delta-opioid agonist pseudopeptide H-Dmt-Tic-NH-CH(2)-Bid (UFP-502)

H-Dmt-Tic-NH-CH(2)-Bid (UFP-502) was the first delta-opioid agonist prepared from the Dmt-Tic pharmacophore. It showed interesting pharmacological properties, such as stimulation of mRNA BDNF expression and antidepression. To evaluate the importance of 1H-benzimidazol-2-yl (Bid) in the induction of delta-agonism, it was substituted by similar heterocycles: The substitution of NH(1) by O or S transforms the reference delta-agonist into delta-antagonists. Phenyl ring of benzimidazole is not important for delta-agonism; in fact 1H-imidazole-2-yl retains delta-agonist activity.