Anti-neoplastic activity of two flavone isomers derived from Gnaphalium elegans and Achyrocline bogotensis

Over 4000 flavonoids have been identified so far and among these, many are known to have antitumor activities. The basis of the relationships between chemical structures, type and position of substituent groups and the effects these compounds exert specifically on cancer cells are not completely elucidated. Here we report the differential cytotoxic effects of two flavone isomers on human cancer cells from breast (MCF7, SK-BR-3), colon (Caco-2, HCT116), pancreas (MIA PaCa, Panc 28), and prostate (PC3, LNCaP) that vary in differentiation status and tumorigenic potential. These flavones are derived from plants of the family Asteraceae, genera Gnaphalium and Achyrocline reputed to have anti-cancer properties. Our studies indicate that 5,7-dihydroxy-3,6,8-trimethoxy-2-phenyl-4H-chromen-4-one (5,7-dihydroxy-3,6,8-trimethoxy flavone) displays potent activity against more differentiated carcinomas of the colon (Caco-2), and pancreas (Panc28), whereas 3,5-dihydroxy-6,7,8-trimethoxy-2-phenyl-4H-chromen-4-one (3,5-dihydroxy-6,7,8-trimethoxy flavone) cytototoxic action is observed on poorly differentiated carcinomas of the colon (HCT116), pancreas (Mia PaCa), and breast (SK-BR3). Both flavones induced cell death (>50%) as proven by MTT cell viability assay in these cancer cell lines, all of which are regarded as highly tumorigenic. At the concentrations studied (5-80 μM), neither flavone demonstrated activity against the less tumorigenic cell lines, breast cancer MCF-7 cells, androgen-responsive LNCaP human prostate cancer line, and androgen-unresponsive PC3 prostate cancer cells. 5,7-dihydroxy-3,6,8-trimethoxy-2-phenyl-4H-chromen-4-one (5,7-dihydroxy-3,6,8-trimethoxy flavone) displays activity against more differentiated carcinomas of the colon and pancreas, but minimal cytotoxicity on poorly differentiated carcinomas of these organs. On the contrary, 3,5-dihydroxy-6,7,8-trimethoxy-2-phenyl-4H-chromen-4-one (3,5-dihydroxy-6,7,8-trimethoxy flavone) is highly cytotoxic to poorly differentiated carcinomas of the colon, pancreas, and breast with minimal activity against more differentiated carcinomas of the same organs. These differential effects suggest activation of distinct apoptotic pathways. In conclusion, the specific chemical properties of these two flavone isomers dictate mechanistic properties which may be relevant when evaluating biological responses to flavones.     

Status of pollinators and their efficiency in coffee fruit set in a fragmented landscape mosaic in South India

The region of Kodagu, South India, comprises a fragmented landscape with a high density of remnant forest patches dispersed within a mosaic dominated by shaded coffee agro-forests. We evaluated the role of self, wind and insect pollination to Coffea canephora production in this landscape. The giant Asian honeybee, Apis dorsata, which nests in remnant forests, was the main pollinator of coffee (accounting for 58% of the floral visitors). The proportion of flowers that developed into fruits was highest when hand cross-pollinated (44%), followed by open- (insect and wind combined; 33%) and wind- (22.1%) pollination treatments. Pollination by bees therefore increases fruit production by 50% over that achieved by wind. Self-pollination (1.7%) and no pollination (1%) treatments produced very low fruit set, emphasizing the importance of cross-pollination in C. canephora. Unlike measures of pollination success, initial fruit set (five weeks after flowering) proved an unreliable proxy for final fruit set. Size of adjoining forest fragments (mostly 0.3–20 ha, with a few exceeding 200 ha) positively influenced pollinator visitation to coffee flowers, but distance to such fragments had no influence on pollination. This study demonstrates the importance of cross-pollination for crop production in C. canephora, the important contribution that pollinating insects make to coffee production, and the benefits of relatively large forest fragments within the landscape mosaic to support insect pollinators of coffee. A comparison of pollinator composition to that of 100 years ago indicated that coffee pollination services remained intact despite changes in pollinator community composition.     

IFN-gamma induces the erosion of preexisting CD8 T cell memory during infection with a heterologous intracellular bacterium

Memory T cells are critical for the control of intracellular pathogens and require few signals for maintenance; however, erosion of established preexisting memory CD8(+) T cells has been shown to occur during infection with heterologous viral infections. We evaluated whether this also occurs during infection with various intracellular bacteria and what mechanisms may be involved. We demonstrate that erosion of established memory is also induced during infection of mice with various intracellular bacteria, such as Listeria monocytogenes, Salmonella typhimurium, and Mycobacterium bovis (bacillus Calmette-Guérin). The extent of erosion of established CD8(+) T cell memory was dependent on the virulence of the heterologous pathogen, not persistence. Furthermore, when antibiotics were used to comprehensively eliminate the heterologous pathogen, the numbers of memory CD8(+) T cells were not restored, indicating that erosion of preexisting memory CD8(+) T cells was irreversible. Irrespective of the initial numbers of memory CD8(+) T cells, challenge with the heterologous pathogen resulted in a similar extent of erosion of memory CD8(+) T cells, suggesting that cellular competition was not responsible for erosion. After challenge with the heterologous pathogen, effector memory CD8(+) T cells were rapidly eliminated. More importantly, erosion of preexisting memory CD8(+) T cells was abrogated in the absence of IFN-gamma. These studies help reveal the paradoxical role of IFN-gamma. Although IFN-gamma promotes the control of intracellular bacterial replication during primary infection, this comes at the expense of erosion of preexisting memory CD8(+) T cells in the wake of infection with heterologous pathogens.     

Antimicrobial activity of saponin fractions of the leaves of Gymnema sylvestre and Eclipta prostrata

The antimicrobial activity of saponin fractions from the leaves of Gymnema sylvestre and Eclipta prostrata was evaluated against pathogenic bacteria and fungi in an in vitro condition. A series of concentrations of crude and pure saponin fractions were tested for antimicrobial activity by zone of inhibition method. The pure saponin fractions were found to be more effective against tested bacterial pathogens when compared to crude saponin fractions. The minimum inhibitory concentration (MIC) exhibited by the pure saponin fraction of G. sylvestre was found to be in the range of 600–1,200 mg/l against bacterial strains and 1,400 mg/l for fungal isolates. In the case of E. prostrata, the range was 1,000–1,200 mg/l for bacteria and 1,400 mg/l for fungal isolates. The susceptibility of bacterial pathogens for saponin fractions was in the order of P. aeruginosa, E. coli, S. typhi, K. pneumoniae, P. mirablis, S. aureus and for fungal pathogens A. fumigatus followed by A. niger and A. flavus. Whereas, A. niger was more susceptible to inhibition by E. prostrata saponin fractions, followed by A. flavus and A. fumigatus. The antimicrobial potential of saponin fractions was compared with antibiotics, Chloramphenicol and Amphotericin-B with respect to bacteria and fungi. The present study suggests that the saponin fractions G. sylvestre and E. prostrata possess significant antibacterial and antifungal activity. Our results further suggest that saponins of G. sylvestre and E. prostrata can be used as a potential fungicide against pathogenic fungi.     

The action of MBL-associated serine protease 1 (MASP1) on factor XIII and fibrinogen

The complement system is an important recognition and effector mechanism of the innate immune system that upon activation leads to the elimination of foreign bodies. It can be activated through three pathways of which the lectin pathway is one. The lectin pathway relies on the binding of mannan-binding lectin (MBL) or the ficolins and the subsequent activation of the MBL-associated serine proteases (MASPs), namely, MASP1, 2 and 3 which all form complexes with both MBL and the ficolins. Major substrates have only been identified for MASP2 i.e. C4 and C2. For MASP1 only a few protein substrates which are cleaved at a low rate have been identified while none are known for MASP3. Since chromogenic substrate screenings have shown that MASP1 has thrombin-like activity, we wanted to investigate the catalytic potential of MASP1 towards two major proteins involved in the clotting process, fibrinogen and factor XIII, and compare the activity directly with that of thrombin. We found that rMASP1 and thrombin cleave factor XIII A-chain and the fibrinogen beta-chain at identical sites, but differ in cleavage of the fibrinogen alpha-chain. The thrombin turnover rate of factor XIII is approximately 650 times faster than that of rMASP1 at 37 degrees C, pH 7.4. rMASP1 cleavage of fibrinogen leads to the release of the proinflammatory peptide fibrinopeptide B. Thus rMASP1 has similar, but not identical specificity to thrombin and its catalytic activity for factor XIII and fibrinogen cleavage is much lower than that of thrombin. Nevertheless, rMASP1 can drive the formation of cross-linked fibrinogen. Since MASP1 is activated on contact of MBL or the ficolins with microorganisms, fibrinogen and factor XIII may be involved in the elimination of invading pathogens.     

An Empirical Correlation between Secondary Structure Content and Averaged Chemical Shifts in Proteins

It is shown that the averaged chemical shift (ACS) of a particular nucleus in the protein backbone empirically correlates well to its secondary structure content (SSC). Chemical shift values of more than 200 proteins obtained from the Biological Magnetic Resonance Bank are used to calculate ACS values, and the SSC is estimated from the corresponding three-dimensional coordinates obtained from the Protein Data Bank. ACS values of ¹H_α show the highest correlation to helical and sheet structure content (correlation coefficient of 0.80 and 0.75, respectively); ¹H_N exhibits less reliability (0.65 for both sheet and helix), whereas such correlations are poor for the heteronuclei. SSC estimated using this correlation shows a good agreement with the conventional chemical shift index-based approach for a set of proteins that only have chemical shift information but no NMR or x-ray determined three-dimensional structure. These results suggest that even chemical shifts averaged over the entire protein retain significant information about the secondary structure. Thus, the correlation between ACS and SSC can be used to estimate secondary structure content and to monitor large-scale secondary structural changes in protein, as in folding studies.     

Cardiac spinal deafferentation reduces the susceptibility to sustained ventricular tachycardia in conscious rats

The response to myocardial ischemia is complex and involves the cardio-cardiac sympathetic reflex. Specifically, cardiac spinal (sympathetic) afferents are excited by ischemic metabolites and elicit an excitatory sympathetic reflex, which plays a major role in the genesis of ventricular arrhythmias. For example, brief myocardial ischemia leads to ATP release, which activates cardiac spinal afferents through stimulation of P2 receptors. Clinical work with patients and preclinical work with animals document that disruption of this reflex protects against ischemia-induced ventricular arrhythmias. However, the role of afferent signals in the initiation of sustained ventricular tachycardia has not been investigated. Therefore, we tested the hypothesis that cardiac spinal deafferentation reduces the susceptibility to sustained ventricular tachycardia in adult (12-15 wk of age), conscious, male Sprague-Dawley rats. To test this hypothesis, the susceptibility to ventricular tachyarrhythmias produced by occlusion of the left main coronary artery was determined in two groups of conscious rats: 1) deafferentation (bilateral excision of the T1-T5 dorsal root ganglia) and 2) control (sham deafferentation). The ventricular arrhythmia threshold (VAT) was defined as the time from coronary occlusion to sustained ventricular tachycardia resulting in a reduction in arterial pressure. Results document a significantly higher VAT in the deafferentation group (7.0 ± 0.7 min) relative to control (4.3 ± 0.3 min) rats. The decreased susceptibility to tachyarrhythmias with deafferentation was associated with a reduced cardiac metabolic demand (lower rate-pressure product and ST segment elevation) during ischemia.     

Cholesterol signaling at the endoplasmic reticulum occurs in npc1(-/-) but not in npc1(-/-), LDLR(-/-) mice

It remains controversial whether deficiency of the Niemann-Pick C1 (npc1) protein results in altered cholesterol signaling at the endoplasmic reticulum (ER). In this report, we have measured the processed, nuclear form of sterol regulatory element binding protein (SREBP)-1 in livers of npc1 wild-type, heterozygous, and homozygous deficient mice, alone, and in combination with deficiencies of the low density lipoprotein receptor (LDLR) or the multiple drug resistant (mdr)1a, P-glycoprotein. Cleavage of SREBPs to activated forms normally occurs when the ER is deficient in cholesterol. A large decrease in processed SREBP-1 was evident in fasted npc1(-/-) mice and npc1(-/-), mdr1a(-/-) mice, with no decrease evident in npc1(-/-), LDLR(-/-) mice. These results suggest that the increase in cellular cholesterol which occurs in npc1(-/-) and in npc1(-/-), mdr1a(-/-) mice includes the sites responsible for cholesterol signaling, while the similar increase in cholesterol found in npc1(-/-), LDLR(-/-) mice does not.     

Benzopyrans as selective estrogen receptor beta agonists (SERBAs). Part 2: structure-activity relationship studies on the benzopyran scaffold

Benzopyrans are selective estrogen receptor (ER) beta agonists (SERBAs), which bind the ER subtypes alpha and beta in opposite orientations. Here we describe structure-activity relationship studies that led to the discovery of bezopyran 5b. X-ray crystal structures of 5b and a non-selective analog 5c in ERalpha help explain the observed selectivity of the benzopyran platform.