New steroidal aromatase inhibitors: Suppression of estrogen-dependent breast cancer cell proliferation and induction of cell death

Background  

Aromatase, the cytochrome P-450 enzyme (CYP19) responsible for estrogen biosynthesis, is an important target for the treatment of estrogen-dependent breast cancer. In fact, the use of synthetic aromatase inhibitors (AI), which induce suppression of estrogen synthesis, has shown to be an effective alternative to the classical tamoxifen for the treatment of postmenopausal patients with ER-positive breast cancer. New AIs obtained, in our laboratory, by modification of the A and D-rings of the natural substrate of aromatase, compounds 3a and 4a, showed previously to efficiently suppress aromatase activity in placental microsomes. In the present study we have investigated the effects of these compounds on cell proliferation, cell cycle progression and induction of cell death using the estrogen-dependent human breast cancer cell line stably transfected with the aromatase gene, MCF-7 aro cells.     

Mutations on the Switch III region and the alpha3 helix of Galpha16 differentially affect receptor coupling and regulation of downstream effectors

Background

Gα₁₆ can activate phospholipase Cβ (PLCβ) directly like Gα_q. It also couples to tetratricopeptide repeat 1 (TPR1) which is linked to Ras activation. It is unknown whether PLCβ and TPR1 interact with the same regions on Gα₁₆. Previous studies on Gα_q have defined two minimal clusters of amino acids that are essential for the coupling to PLCβ. Cognate residues in Gα₁₆ might also be essential for interacting with PLCβ, and possibly contribute to TPR1 interaction and other signaling events.

Results

Alanine mutations were introduced to the two amino acid clusters (246–248 and 259–260) in the switch III region and α3 helix of Gα₁₆. Regulations of PLCβ and STAT3 were partially weakened by each cluster mutant. A mutant harboring mutations at both clusters generally produced stronger suppressions. Activation of Jun N-terminal kinase (JNK) by Gα₁₆ was completely abolished by mutating either clusters. Contrastingly, phosphorylations of extracellular signal-regulated kinase (ERK) and nuclear factor κB (NF-κB) were not significantly affected by these mutations. The interactions between the mutants and PLCβ2 and TPR1 were also reduced in co-immunoprecipitation assays. Coupling between G₁₆ and different categories of receptors was impaired by the mutations, with the effect of switch III mutations being more pronounced than those in the α3 helix. Mutations of both clusters almost completely abolished the receptor coupling and prevent receptor-induced Gβγ release.

Conclusion

The integrity of the switch III region and α3 helix of Gα₁₆ is critical for the activation of PLCβ, STAT3, and JNK but not ERK or NF-κB. Binding of Gα₁₆ to PLCβ2 or TPR1 was reduced by the mutations of either cluster. The same region could also differentially affect the effectiveness of receptor coupling to G₁₆. The studied region was shown to bear multiple functionally important roles of G₁₆.     

Influence of yeast immobilization on fermentation and aldehyde reduction during the production of alcohol-free beer

Production of alcohol-free beer by limited fermentation is optimally performed in a packed-bed reactor. This highly controllable system combines short contact times between yeast and wort with the reduction of off-flavors to concentrations below threshold values. In the present study, the influence of immobilization of yeast to DEAE-cellulose on sugar fermentation and aldehyde reduction was monitored. Immobilized cells showed higher activities of hexokinase and pyruvate decarboxylase compared to cells grown in batch culture. In addition, a higher glucose flux was observed, with enhanced excretion of main fermentation products, indicating a reduction in the flux of sugar used for biomass production. ADH activity was higher in immobilized cells compared to that in suspended cells. However, during prolonged production a decrease was observed in NAD-specific ADH activity, whereas NADP-specific activity increased in the immobilized cells. The shifts in enzyme activities and glucose flux correlate with a higher in vivo reduction capacity of the immobilized cells.     

Identification of methylation markers for the prediction of nodal metastasis in oral and oropharyngeal squamous cell carcinoma

Hypermethylation is an important mechanism for the dynamic regulation of gene expression, necessary for metastasizing tumour cells. Our aim is to identify methylation tumour markers that have a predictive value for the presence of regional lymph node metastases in patients with oral and oropharyngeal squamous cell carcinoma (OOSCC). Significantly differentially expressed genes were retrieved from four reported microarray expression profiles comparing pN0 and pN+ head-neck tumours, and one expression array identifying functionally hypermethylated genes. Additional metastasis-associated genes were included from the literature. Thus genes were selected that influence the development of nodal metastases and might be regulated by methylation. Methylation-specific PCR (MSP) primers were designed and tested on 8 head-neck squamous cell carcinoma cell lines and technically validated on 10 formalin-fixed paraffin-embedded (FFPE) OOSCC cases. Predictive value was assessed in a clinical series of 70 FFPE OOSCC with pathologically determined nodal status. Five out of 28 methylation markers (OCLN, CDKN2A, MGMT, MLH1 and DAPK1) were frequently differentially methylated in OOSCC. Of these, MGMT methylation was associated with pN0 status (P = 0.02) and with lower immunoexpression (P = 0.02). DAPK1 methylation was associated with pN+ status (P = 0.008) but did not associate with protein expression. In conclusion, out of 28 candidate genes, two (7%) showed a predictive value for the pN status. Both genes, DAPK1 and MGMT, have predictive value for nodal metastasis in a clinical group of OOSCC. Therefore DNA methylation markers are capable of contributing to diagnosis and treatment selection in OOSCC. To efficiently identify additional new methylation markers, genome-wide methods are needed.     

Physiological and biochemical characterization of NERICA‐L‐44: a novel source of heat tolerance at the vegetative and reproductive stages in rice

The predicted increase in the frequency and magnitude of extreme heat spikes under future climate can reduce rice yields significantly. Rice sensitivity to high temperatures during the reproductive stage is well documented while the same during the vegetative stage is more speculative. Hence, to identify and characterize novel heat‐tolerant donors for both the vegetative and reproductive stages, 71 rice accessions, including approximately 75% New Rice for Africa (NERICAs), were phenotyped across field experiments during summer seasons in Delhi, India, and in a controlled environment study at International Rice Research Institute , Philippines. NERICA‐L‐44 (NL‐44) recorded high seedling survival (52%) and superior growth and greater reproductive success exposed to 42.2°C (sd ± 2.3) under field conditions. NL‐44 and the heat‐tolerant check N22 consistently displayed lower membrane damage and higher antioxidant enzymes activity across leaves and spikelets. NL‐44 recorded 50–60% spikelet fertility, while N22 recorded 67–79% under controlled environment temperature of 38°C (sd ±1.17), although both had about 87% fertility under extremely hot field conditions. N22 and NL‐44, exposed to heat stress (38°C), had similar pollen germination percent and number of pollen tubes reaching the ovary. NL‐44 maintained low hydrogen peroxide production and non‐photochemical quenching (NPQ) with high photosynthesis while N22 avoided photosystem II damage through high NPQ under high‐temperature stress. NL‐44 with its reproductive stage resilience to extreme heat stress, better antioxidant scavenging ability in both vegetative tissue and spikelets and superior yield and grain quality is identified as a novel donor for increasing heat tolerance at both the vegetative and reproductive stages in rice.     

Oncostatin-M inhibits luteinizing hormone stimulated Leydig cell progenitor formation in vitro

Background  

The initial steps of stem Leydig cell differentiation into steroid producing progenitor cells are thought to take place independent of luteinizing hormone (LH), under the influence of locally produced factors such as leukaemia inhibitory factor (LIF), platelet derived growth factor A and stem cell factor. For the formation of a normal sized Leydig cell population in the adult testis, the presence of LH appears to be essential.