Cyanogenic glycosides of Malesherbia

Twenty-eight species of Malesherbia were tested and found to be cyanogenic. Analysis of HPLC, GC, NMR and comparisons of R_f values on paper chromatograms showed all to possess tetraphyllin A and B as major cyanogens, with epitetraphyllin B and deidaclin being present occasionally. These data confirm the close relationship of this family with other families which produce structurally related cyanogens, the Passifloraceae, Turneraceae and Flacourtiaceae. In none of these families is tetraphyllin A the major cyanogen, however. The genus Gynopleura, sometimes segregated from Malesherbia, does not differ in this character.     

Tetraphyllin B from Adenia digitata

Tetraphyllin B has been isolated in good yield from the South African plant Adenia digitata. Its structure was established by ¹H and ¹³C NMR.     

Two cyanogenic glucosides,tetraphyllin B and epi-tetraphyllin B,from Adenia volkensii

Tetraphyllin B and its previously unknown epimer have been isolated from the Kenyan plant Adenia volkensii. The structures were established by their NMR spectra and by GLC.     

Tetraphyllin B and epitetraphyillin B sulphates: Novel cyanogenic glucosides from Passiflora caerulea and P. alato-caerulea

An epimeric mixture of tetraphyllin B-4-sulphate and epitetraphyllin B-4-sulphate was isolated from Passiflora caerulea and P. alato-caerulea.     

A mouse model for visualization of GABAB receptors

GABA_B receptors are the G‐protein‐coupled receptors for the neurotransmitter γ‐aminobutyric acid (GABA). Receptor subtypes are based on the subunit isoforms GABA_B1a and GABA_B1b, which combine with GABA_B2 subunits to form heteromeric receptors. Here, we used a modified bacterial artificial chromosome (BAC) containing the GABA_B1 gene to generate transgenic mice expressing GABA_B1a and GABA_B1b subunits fused to the enhanced green fluorescence protein (eGFP). We demonstrate that the GABA_B1‐eGFP fusion proteins reproduce the cellular expression patterns of endogenous GABA_B1 proteins in the brain and in peripheral tissue. Crossing the GABA_B1‐eGFP BAC transgene into the GABA_B1^?/? background restores pre and postsynaptic GABA_B functions, showing that the GABA_B1‐eGFP fusion proteins substitute for the lack of endogenous GABA_B1 proteins. Finally, we demonstrate that the GABA_B1‐eGFP fusion proteins replicate the temporal expression patterns of native GABA_B receptors in cultured neurons. These transgenic mice therefore provide a validated tool for direct visualization of native GABA_B receptors. genesis 47:595–602, 2009. © 2009 Wiley‐Liss, Inc.     

Lophirones B and C Extenuate AFB1‐Mediated Oxidative Onslaught on Cellular Proteins,Lipids, and DNA through Nrf‐2 Expression

The capability of lophirones B and C to extenuate aflatoxin B₁ (AFB₁)‐mediated onslaught on cellular proteins, lipids, and DNA was investigated for 6 weeks. Lophirones B and C significantly (P < 0.05) increase the expression and specific activity of cytoprotective enzymes (glutathione‐S‐trans‐ferase, nioctinamide adenine dicludeotide:quinone oxidoreductase‐1, epoxide hydrolase, and uridyl glucuronosyl transferase). There was significant (P < 0.05) reduction in the level of antioxidant system in AFB₁‐induced hepatocarcinogenesis. Furthermore, lophirones B and C significantly (P < 0.05) attenuated AFB₁‐mediated decrease in the specific activities of antioxidant enzymes. Oxidative stress biomarkers, malondialdehyde, lipid hydroperoxides, conjugated dienes, protein carbonyl, and fragmented DNA were significantly (P < 0.05) elevated in AFB₁‐treated rats. Although lophirones B and C did not significantly (P < 0.05) alter these biomarkers, an AFB₁‐mediated increase in these biomarkers was significantly attenuated. Results obtained showed that lophirones B and C extenuate AFB₁‐mediated onslaught on cellular proteins, lipids, and DNA by enhancing nuclear erythroid–related factor‐2 expression.     

Effect of several analogs of 2,4,6-triphenyldioxane-1,3 on constitutive androstane receptor activation

2,4,6-Triphenyldioxane-1,3 (TPD) is a highly effective species-specific inducer of CYP2В in rats. Several analogs of TPD were synthesized to verify a hypothesis that minor changes in the inducer structure can cause changes in induction abilities (R = H, cisTPD and transTPD; R = N(CH₃)₂, transpDMA; R = NO₂, transpNO₂; R = F, transpF; R = OCH₃, transpMeO). Five of six compounds were able to activate CAR in rat liver. Results of Western-blot and ChIP showed that cisTPD and transTPD, transpDMA, transpNO₂, transpF treatment stimulated nuclear accumulation of CAR and evoked CAR receptor PBREM-binding activity in rat liver. cisTPD, transTPD, transpDMA, transpNO₂ and transpF administration significantly increased total CYP content (1.3–2.5 fold) and the level of PROD (12–20 fold), CYP2B specific activity, whereas transpMeO did not have any effects. Western blot and real-time RT-PCR showed that the increase of PROD in liver is related to the high content of CYP2B proteins and paralleled the increase of CYP2B1 (10–43 fold) and CYP2B2 (8–26 fold) mRNAs. At the same time content of CYP2B proteins and CYP2B1 and CYP2B2 mRNA levels were unchanged in rat liver after transpMeO treatment. The dose–response studies have shown that cisTPD, transpDMA, transpF and transpNO₂ have similar potency, and transTPD is less potent derivative. Moreover, it is likely transTPD act as a partial CAR activator. Thus, our results provide evidence to support the conclusion that the differences of TPD analogs ability to activate CYP2B gene expression can be explained by various interactions with CAR.     

Development of a chromatographic method for the isolation and detection of hygromycin B in biological fluids

An affinity chromatography method was developed for the purification of hygromycin B from biological fluids. Lysozyme and α-lactalbumin were immobilized on an N-hydroxysuccinimide activated agarose support. Hygromycin B solubilized in water was bound by the proteins and subsequently eluted using 10 mM sodium citrate buffer, pH 4.0. Hygromycin B was purified from swine plasma, bovine serum and bovine milk samples using a combination of ion-exchange chromatography for initial clean-up of spiked biological samples followed by affinity chromatography. Thin layer chromatographic analysis of the isolated hygromycin B revealed one band with the same R_F value as the hygromycin B standard.     

Male sterility caused by cytoplasm of Brassica oxyrrhina in B. campestris and B. juncea

Summary Synthetic alloploid Brassica oxyrrhina (2n = 18, OO) x B. campestris (2n = 20, AA) was repeatedly backcrossed with B. campestris to place B. campestris nucleus in the cytoplasm of B. oxyrrhina. Alloplasmic plants, obtained in BC₅ generation, were stably male sterile but mildly chlorotic during initial development. Synthetic alloploid B. oxyrrhina-campestris was also hybridized with B. juncea to transfer B. oxyrrhina cytoplasm. Segregation for green and chlorotic plants was observed in BC₁ and BC₂ generations. By selection, however, normal green male sterile B. juncea was obtained in BC₃. Pollen abortion in both B. campestris and B. juncea is post-meiotic.     

Improved resistance to bacterial soft rot by protoplast fusion between Brassica rapa and B. oleracea

Erwinia soft rot is a destructive disease of Brassica rapa vegetables. Reliable sources of resistance and control methods are limited, so development of highly resistant breeding lines is desirable. Protoplasts from B. rapa and B. oleracea genotypes selected for resistance to soft rot were fused in order to combine different sources of resistance. Twelve somatic hybrids (synthetic B. napus) were obtained and confirmed by morphology, nuclear DNA content, and RAPD analysis. They were normal looking plants that easily set seeds following self-pollination and backcrossing to B. rapa. Assays of detached leaves or seedlings inoculated in a mist-chamber showed that most somatic hybrids had lower disease severity ratings than the B. rapa fusion partner and a commercial variety of B. napus. Some progeny from selfing or backcrossing of somatic hybrids to B. rapa showed much more resistance than either fusion partner. The offspring populations of the somatic hybrids (F₁–S₁ and F₁–BC₁) clearly moved to the resistant direction compared to the parents; the percentage of resistant plants increased from 21% (average of parents) to 36% (F₁–S₁) and 48% (F₁–BC₁). These results suggest that it may be possible to obtain highly resistant B. rapa lines by further backcrossing and selection. Received: June 1999 / Accepted: 29 July 1999     

Plant availability of applied and native boron in soils with diverse properties

Laboratory and greenhouse research was conducted to study effects of soil properties on the availability of native and applied B in 14 Virginia soils. Boron absorption could be described by the Langmuir equation in 12 of the 14 soils, and maximum B adsorption (Vmax) in these 12 soils ranged from 3.3 to 26.5 mg kg⁻¹. A multiple regression equation, −19.3+3.51 pH+0.048 clay content, accounted for 89.6% of the variation in Vmax for the 12 soils. Curvilinear relationships (α=0.01) occurred between B in corn (Zea mays L.) tissue from native B and hot-water soluble B, mannitol exchangeable B, and NH₄-acetate and Mehlich III extractable B. Among these four procedures, mannitol exchangeable B correlated most closely (r=0.923) with B in corn tissue from native B. From 0.4 to 13.5% of the applied B was absorbed by corn plants and translocated to shoots. Curvilinear relationships (α=0.01) occurred between B in corn tissue from applied B and soil clay content, NH₄-oxalate extractable Al and Fe, and acidified NH₂OH·HCl extractable Mn. It is evident from these relationships that soil clay and oxyhydroxides of Al, Fe, and Mn have an affinity to adsorb B in somewhat unavailable forms.     

Inhibition of protein tyrosine phosphatase (PTP1B) and α-glucosidase by geranylated flavonoids from Paulownia tomentosa

Protein tyrosine phosphatase 1B (PTP1B) and α-glucosidase are important targets to treat obesity and diabetes, due to their deep correlation with insulin and leptin signalling, and glucose regulation. The methanol extract of Paulownia tomentosa fruits showed potent inhibition against both enzymes. Purification of this extract led to eight geranylated flavonoids (1–8) displaying dual inhibition of PTP1B and α-glucosidase. The isolated compounds were identified as flavanones (1–5) and dihydroflavonols (6–8). Inhibitory potencies of these compounds varied accordingly, but most of the compounds were highly effective against PTP1B (IC₅₀?=?1.9–8.2?μM) than α-glucosidase (IC₅₀?=?2.2–78.9?μM). Mimulone (1) was the most effective against PTP1B with IC₅₀?=?1.9?μM, whereas 6-geranyl-3,3′,5,5′,7-pentahydroxy-4′-methoxyflavane (8) displayed potent inhibition against α-glucosidase (IC₅₀?=?2.2?μM). All inhibitors showed mixed type Ι inhibition toward PTP1B, and were noncompetitive inhibitors of α-glucosidase. This mixed type behavior against PTP1B was fully demonstrated by showing a decrease in V_max, an increase of K_m, and K_ik/K_iv ratio ranging between 2.66 and 3.69.     

Mice carrying a CD20 gene disruption

 CD20 is a hallmark antigen of B lymphocytes. Its expression is restricted to precursor and mature B cells but it is not expressed on plasma cells. The protein is a membrane-embedded phosphoprotein that appears likely to transverse the membrane four times. Its function is unknown although CD20 has been variously proposed to play a role in B-cell activation, proliferation, and calcium transport. A unique homologue of human CD20 has been described in mouse, which also shows a B-cell-specific pattern of expression. Here we describe the generating of mice carrying a CD20 gene disruption. So far, we have failed to detect any major effect of the gene disruption on the differentiation and function of B lymphocytes as judged by the expression of surface markers, antigen receptor signaling, proliferative responses, or calcium uptake. We did note, however, that the mice homozygous for the gene disruption [generated by intercrossing (129 × C57BL/6)F₁ CD20 ^+/- heterozygotes] showed a substantial depletion of the sub-population of peritoneal B cells that lack expression of the B220 (RA3–6B2) isoform of CD45. The loss of the IgM⁺ 6B2^- peritoneal B cells is not, however, attributable to the CD20 gene disruption itself. Rather, it segregates with a polymorphic difference between the 129 and C57BL/6 strains that is linked to the CD20 locus which, intriguingly, is itself close to the CD5 gene. This demonstrates that caution must be exercised when comparing the phenotypes of F₂ litter-mates generated from crosses between 129 embryonic stem-cell-derived chimeras and mice of other strains. Received: 23 December 1997 / Revised: 27 January 1998     

Exploring QSAR for CYP11B2 binding affinity and CYP11B2/CYP11B1 selectivity of diverse functional compounds using GFA and G/PLS techniques

A data set of a series of 132 structurally diverse compounds with cytochrome 11B2 and 11B1 (CYP11B2 and CYP11B1) enzyme inhibitory activities was subjected to molecular shape analysis to explore contributions of shape features as well as electronic, structural, and physicochemical parameters toward enzyme inhibitory activities, in search of appropriate molecular scaffolds with optimum substitutions for highly potent CYP11B2 inhibitors. Genetic function approximation (GFA) and genetic partial least squares (G/PLS) were used as chemometric tools for modeling, and the derived equations were of acceptable statistical quality considering both internal and external validation parameters (Q²: 0.514–0.659, R²_pred: 0.510–0.734). The G/PLS models with spline option for CYP11B2 and CYP11B1 inhibition and selectivity modeling appeared to be the best models based on r_m²_(overall) criterion. The study indicates the importance of the pyridinylnaphthalene and pyridylmethylene-indane scaffolds with less polar and electrophilic substituents for optimum CYP11B2 inhibitory activity and CYP11B2/CYP11B1 selectivity.     

Effects of four constant temperatures on the development of two Bradysia (Diptera: Sciaridae) species

In this study, the effects of temperature on the growth, development, survival, fecundity and other population parameters of two local Bradysia species B. odoriphaga and B. impatiens were studied at four constant temperatures (25, 28, 31 and 34°C). The results show that 25°C is the optimum temperature for the growth and development of B. odoriphaga, while 28°C is more favourable for B. impatiens. The temperature of 31°C restricted the growth and development, while the temperature of 34°C inhibited the eggs hatching in both species, resulting in no egg survival and no subsequent development. High temperatures (>28°C) prolonged the 4th larval stage duration, mean generation time (T) and population doubling time (Dt) of both species. The high temperature of 31°C greatly shortened the female longevity, weakened the oviposition and reduced the survival of both species. Moreover, the life table parameters R₀, r_m and λ were also suppressed by this high temperature. However, the high temperature of 31°C had little impact on the egg survival, pupal weight and male longevity. In addition, at 31°C, the values of R₀, r_m and λ of B. odoriphaga were higher than those of B. impatiens, suggesting that B. odoriphaga is more tolerant to high temperature than B. impatiens. The differences between two Bradydsia species seem determined genetically. Our findings are important for better understanding their biological characteristics at a certain constant temperature and demonstrate the possibility to control and manage those two Bradysia species by increasing ambient temperature.     

Age-related decline in osteoblastogenesis and 1α-hydroxylase/CYP27B1 in human mesenchymal stem cells: stimulation by parathyroid hormone

With aging, there is a decline in bone mass and in osteoblast differentiation of human mesenchymal stem cells (hMSCs) in vitro. Osteoblastogenesis can be stimulated with 1,25‐dihydroxyvitamin D₃ [1,25(OH)₂D₃] and, in some hMSCs, by the precursor 25‐hydroxyvitamin D₃ (25OHD₃). CYP27B1/1α‐hydroxylase activates 25OHD₃ and, to a variable degree, hMSCs express CYP27B1. In this study, we tested the hypotheses (i) that age affects responsiveness to 25OHD₃ and expression/activity of CYP27B1 in hMSCs and (ii) that parathyroid hormone (PTH) upregulates CYP27B1 in hMSCs, as it does in renal cells. There were age‐related declines in osteoblastogenesis (n = 8, P = 0.0286) and in CYP27B1 gene expression (n = 27, r = ?0.498; P = 0.008) in hMSCs. Unlike hMSCs from young subjects (≤50 years), hMSCs from older subjects (≥55 years) were resistant to 25OHD₃ stimulation of osteoblastogenesis. PTH1‐34 (100 nm ) provided hMSCs with responsiveness to 25OHD₃ (P = 0.0313, Wilcoxon matched pairs test) and with two episodes of increased 1,25(OH)₂D₃ synthesis, of cAMP response element binding protein (CREB) activation, and of CYP27B1 upregulation. Both increases in CYP27B1 expression by PTH were obliterated by CREB‐siRNA or KG‐501 (which specifically inhibits the downstream binding of activated CREB). Only the second period of CREB signaling was diminished by AG1024, an inhibitor of insulin‐like growth factor‐I receptor kinase. Thus, PTH stimulated hMSCs from elders with responsiveness to 25OHD₃ by upregulating expression/activity of CYP27B1 and did so through CREB and IGF‐I pathways.     

1,25-Dihydroxyvitamin D3 suppresses inflammation-induced expression of plasminogen activator inhibitor-1 by blocking nuclear factor-κB activation

Plasminogen activator inhibitor (PAI)-1 is a major fibrinolytic inhibitor. High PAI-1 is associated with increased renal and cardiovascular disease risk. Previous studies demonstrated PAI-1 down-regulation by 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃), but the molecular mechanism remains unknown. Here we show that exposure of mouse embryonic fibroblasts to TNFα or LPS led to a marked induction of PAI-1, which was blunted by 1,25(OH)₂D₃, NF-κB inhibitor or p65 siRNA, suggesting the involvement of NF-κB in 1,25(OH)₂D₃-induced repression. In mouse Pai-1 promoter a putative cis-κB element was identified at −299. EMSA and ChIP assays showed that TNF-α increased p50/p65 binding to this κB site, which was disrupted by 1,25(OH)₂D₃. Luciferase reporter assays showed that PAI-1 promoter activity was induced by TNFα or LPS, and the induction was blocked by 1,25(OH)₂D₃. Mutation of the κB site blunted TNFα, LPS or 1,25(OH)₂D₃ effects. 1,25(OH)₂D₃ blocked IκBα degradation and arrested p50/p65 nuclear translocation. In mice LPS stimulated PAI-1 expression in the heart and macrophages, and the stimulation was blunted by pre-treatment with a vitamin D analog. Together these data demonstrate that 1,25(OH)₂D₃ down-regulates PAI-1 by blocking NF-κB activation. Inhibition of PAI-1 production may contribute to the reno- and cardio-protective effects of vitamin D.     

Determination of amphotericin B in human serum by liquid chromatography

A rapid reversed-phase high-performance liquid chromatographic method with a 30-mm long column is described for assaying amphotericin B in serum. After deproteinization of serum samples with methanol, the supernatant was injected onto a reversed-phase C₁₈ column, using 2.5 mM Na₂EDTA-acetonitrile (70:30, v/v) as the mobile phase. Amphotericin B was eluted at 1.5 min. Calibration plot of the peak area against concentration was linear from 0.05 to 25 μg/ml (C.V. of 3%). Within-day and day-to-day imprecision (C.V.) ranged between 1.33% and 3.61%. The application was evaluated in 55 serum samples from patients treated with amphotericin B.     

Resistance to Leptosphaeria maculans is conserved in a specific region of the Brassica B genome

 Offspring from asymmetric hybrids between Brassica napus and the three B-genome species Brassica nigra, Brassica juncea and Brassica carinata were analysed for the presence of B-genome markers and resistance to the fungus Leptosphaeria maculans, the causal agent of blackleg disease. Twenty five plants from each species combination were analysed in the first backcross (BC₁) generation, 30 plants in BC₂ and 60 plants in BC₃. The plants were analysed by 46 RFLP markers detecting 85 loci dispersed throughout the B. nigra genome. The plants with additional B. carinata DNA had a decrease in the presence of RFLP markers ranging from 59% in BC₁ to 36% in BC₂ and down to 11% in BC₃. Similar results were obtained in the lines with additional DNA from B. juncea where the 60% presence of RFLP markers in BC₁ was reduced to 33% in BC₂ and to 10% in BC₃. However presence of the markers were significantly lower in the B. nigra-derived material where BC₁ had 46%, BC₂ 25% and BC₃ 8%. Since at least two loci could be detected on each end of the eight linkage groups of the B genome, the degree of symmetry was estimated. After one back-cross between 0.5 and 1.25% intact chromosomes were retained, whereas in BC₂ this frequency was 0.21% for all three B-genome donor species. The maintenance of half-chromosomes ranged from 2.63% to 5.38% in BC₁ and between 0.73% and 1.15% in BC₂. No chromosome arms were found in any of the BC₃ plants. In total, four co-segregating markers for cotyledon and adult-leaf resistance to L. maculans were found which detected six loci located on linkage groups 2, 5 and 8. When the results from the three donor species were compared, one triplicate region in the B genome had preserved the resistance loci in all three species. Received: 19 January 1999 / Accepted: 30 January 1999