Anticancer effects of the novel 1alpha, 25-dihydroxyvitamin D3 hybrid analog QW1624F2-2 in human neuroblastoma

Vitamin D3 analogs are potential anti-cancer agents with theoretically wide therapeutic index, but there have been limited studies directed towards human neuroblastoma. The antiproliferative ability of the novel vitamin D3 hybrid analog QW-1624F2-2 (QW, 1-hydroxymethyl-16-ene-24, 24-F2-26, 27-bishomo-25-hydroxyvitamin D3) was examined in two human neuroblastoma-derived cell-lines. Analog QW inhibited cell-cycle progression of IMR5 cells with accumulation in G1 phase. QW induced the differentiation of CHP134 as evidenced by increased neurite length. These effects were accompanied by decreased expression of MYCN in both the cell-lines treated with QW. Furthermore, QW inhibited the migration of CHP134 cells in matrigel invasion assays, indicating its anti-invasive ability. In athymic nude mice, we found that QW was less calcemic than EB1089 (1alpha, 25-dihydroxy-22, 24-diene-24, 26,27-trishomovitamin D3). Systemic administration of QW in a mouse xenotransplantation model revealed that it is more effective than EB1089 in suppressing the growth of CHP134 flank tumors. In summary, the low-calcemic hybrid analog QW showed significant anti-tumor activity in vivo and thus exhibits potential as a novel cancer therapeutic.     

Nucleotide Binding States of Subunit A of the A-ATP Synthase and the Implication of P-Loop Switch in Evolution

The crystal structures of the nucleotide-empty (A_E), 5′-adenylyl-β,γ-imidodiphosphate (A_PNP)-bound, and ADP (A_DP)-bound forms of the catalytic A subunit of the energy producer A₁A_O ATP synthase from Pyrococcus horikoshii OT3 have been solved at 2.47 Å and 2.4 Å resolutions. The structures provide novel features of nucleotide binding and depict the residues involved in the catalysis of the A subunit. In the A_E form, the phosphate analog SO₄²⁻ binds, via a water molecule, to the phosphate binding loop (P-loop) residue Ser238, which is also involved in the phosphate binding of ADP and 5′-adenylyl-β,γ-imidodiphosphate. Together with amino acids Gly234 and Phe236, the serine residue stabilizes the arched P-loop conformation of subunit A, as shown by the 2.4-Å structure of the mutant protein S238A in which the P-loop flips into a relaxed state, comparable to the one in catalytic β subunits of F₁F_O ATP synthases. Superposition of the existing P-loop structures of ATPases emphasizes the unique P-loop in subunit A, which is also discussed in the light of an evolutionary P-loop switch in related A₁A_O ATP synthases, F₁F_O ATP synthases, and vacuolar ATPases and implicates diverse catalytic mechanisms inside these biological motors.     

Assessing the impacts of climate change and land transformation on Banksia in the South West Australian Floristic Region

Aim To determine the potential combined effects of climate change and land transformation on the modelled geographic ranges of Banksia. Location Mediterranean climate South West Australian Floristic Region (SWAFR). Methods We used the species distribution modelling software Maxent to relate current environmental conditions to occurrence data for 18 Banksia species, and subsequently made spatial predictions using two simple dispersal scenarios (zero and universal), for three climate‐severity scenarios at 2070, taking the impacts of land transformation on species’ ranges into account. The species were chosen to reflect the biogeography of Banksia in the SWAFR. Results Climate‐severity scenario, dispersal scenario, biogeographic distribution and land transformation all influenced the direction and magnitude of the modelled range change responses for the 18 species. The predominant response of species to all climate change scenarios was range contraction, with exceptions for some northern and widespread species. Including land transformation in estimates of modelled geographic range size for the three climate‐severity scenarios generally resulted in smaller gains and larger declines in species ranges across both dispersal scenarios. Including land transformation and assuming zero dispersal resulted, as expected, in the greatest declines in projected range size across all species. Increasing climate change severity greatly increased the risk of decline in the 18 Banksia species, indicating the critical role of mitigating future emissions. Main conclusions The combined effects of climate change and land transformation may have significant adverse impacts on endemic Proteaceae in the SWAFR, especially under high emissions scenarios and if, as expected, natural migration is limiting. Although these results need cautious interpretation in light of the many assumptions underlying the techniques used, the impacts identified warrant a clear focus on monitoring across species ranges to detect early signs of change, and experiments that determine physiological thresholds for species in order to validate and refine the models.     

Biodegradation of chrysene by the bacterial strains isolated from oily sludge

The biodegradation studies were conducted to test the ability of the bacterial strains (Chry2 and Chry3) isolated from the oily sludge obtained from Gujarat refinery, India, for utilization of chrysene in the liquid medium. Biodegradation of the compound was confirmed using gas chromatography and the percent degradation was calculated to be 15.0 and 17% by Chry2 and Chry3, respectively. The biodegradation results were supported by increase in viable cell count and dry biomass, in the presence of chrysene as the sole carbon source. Both the cultures produced biosurfactant which was indicated by the reduction in surface tension of the growth medium. Presence of catechol 2, 3-dioxygenase gene in Chry3 indicated its potential for degradation of PAHs through meta cleavage degradation pathway. Both the strains were found to possess catechol 1,2-dioxygenase and catechol 2,3-dioxygenase enzyme activities. Based on morphological and biochemical tests, the cultures were tentatively identified as Bacillus sp. (Chry2) and Pseudomonas sp. (Chry3).     

Differential activity of matrix metalloproteinases (MMPs) during photoperiod induced uterine regression and recrudescence in Siberian hamsters (Phodopus sungorus)

Siberian hamsters adapt to seasonal changes by reducing their reproductive function during short days (SD). SD exposure reduces uterine mass and reproductive capacity, but underlying cellular mechanisms remain unknown. Because matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) are important in uterine development, parturition, and postpartum remodeling, their expression in uterine tissue from Siberian hamsters undergoing photoperiod-mediated reproductive regression and recrudescence was investigated. Female hamsters were exposed to long day (LD, 16L:8D, controls) or SD (8L:16D) for 3-12 weeks (regression); a second group was exposed to SD or LD for 14 weeks and then transferred to LD for 0-8 weeks (recrudescence). Hamsters were euthanized, uteri collected, and homogenates analyzed by gelatin zymography or Western blotting for MMP and TIMP protein levels. Uterine weight decreased (67-75%) at SD weeks 12-14 and increased post-LD transfer (PT) reaching LD values by PT week 2. MMP-2, but not MMP-9 activity was reduced by SD week 12 or 14 but increased to LD levels at PT week 2. MMP-3 expression increased at SD week 9 compared to other SD and LD groups. MMP-14 and -13 protein levels decreased at SD week 3 but returned to LD levels by SD week 6. During recrudescence, MMP-3 (PT weeks 0-2), MMP-13 (PT week 4), and MMP-14 (PT weeks 2, 4) protein levels were higher than LD. TIMP-1 and 2 were present at low levels. Significant and differential variations in uterine MMP activity/expression during photoperiod-induced regression and recrudescence were observed. These changes likely reflect increases in tissue remodeling during both the adaptation to SD and the restoration of reproductive function.     

Learning approaches of undergraduate medical students to physiology in a non-PBL- and partially PBL-oriented curriculum

Melaka Manipal Medical College (Manipal Campus; Manipal, Karnataka, India) conducts the Bachelor of Medicine and Bachelor of Surgery program, for which the admission intakes are during the months of March and September. The present study was undertaken to study the differences in learning approaches to physiology of undergraduate medical students in a partially problem-based learning (PBL)- and non-PBL-oriented curriculum. PBL was introduced as a curricular reform for the September 2006 batch of students (partially PBL group), whereas it was not incorporated for the March 2006 batch of students (non-PBL group). Learning approaches to physiology of both groups of students were compared using the short inventory of approaches to learning. Mean scores for deep and strategic approaches were found to be significantly higher for the partially PBL group compared with the non-PBL group. The results of the present study support the earlier observation that PBL promotes a deep approach to learning.     

Inhibition of Cdc7/Dbf4 kinase activity affects specific phosphorylation sites on MCM2 in cancer cells

The Cdc7/Dbf4 kinase is required for initiation of DNA replication and also plays a role in checkpoint function in response to replication stress. Exactly how Cdc7/Dbf4 mediates those activities remains to be elucidated. Cdc7/Dbf4 physically interacts with and phosphorylates the minichromosome maintenance complex (MCM), such as MCM2, MCM4 and MCM6. Cdc7/Dbf4 activity is required for association of Cdc45 followed by recruitment of DNA polymerase on the chromatin. Using high resolution mass spectrometry, we identified six phosphorylation sites on MCM2, two of them have not been described before. We provide evidence that Cdc7/Dbf4 mediates phosphorylation on serine 108 and serine 40 on human MCM2 in vitro and in vivo in cancer cells in the absence of DNA damage. Antibodies specific to pS108 or pS40 confirmed the sites and established useful read-outs for inhibition of Cdc7/Dbf4. This report demonstrates the utility of an in vitro to in vivo workflow utilizing immunoprecipitation and mass spectrometry to map phosphorylation sites on endogenous kinase substrates. The approach can be readily generalized to identify target modulation read-outs for other potential kinase cancer targets.