Effect of N-Methyl Substituents in 2-Methoxycarbonylphenylsulfamides on Kinetics and Mechanism of Formation of (1H)-2,1,3-Benzothiadiazin-4(3H)-One-2,2-Dioxides

The cyclization reactions of N-methyl-N’-(2-methoxycarbonylphenyl)sulfamide (1a), N-methyl-N-(2-methoxycarbonylphenyl)-sulfamide (2a), and 2-methoxycarbonylphenylsulfamide (3a) were studied in aqueous amine buffers (butylamine, ethanolamine, morpholine, glycinamide). The dependences observed between the rate constants and buffer concentrations show that the reactions are subject to base catalysis in all the three cases, the decomposition of the tetrahedral intermediate being rate limiting. The ratio of the relative rate constants of the base catalyzed cyclizations reactions of the three derivatives is 1a: 2a: 3a = 1: 20000: 100. The logarithm of rate constants of the base catalyzed cyclization reactions was plotted against the pK_a values of conjugated acids of the individual amines used as the buffers in the cyclization of compound 1a, and the value of the Brönsted coefficient obtained was about 0.1, which means that the proton transfer from the intermediate to the basic buffer component is thermodynamically favorable. The intermediate is a much weaker base, and the reaction is controlled by diffusion. The slope of an analogous dependence for compound 2a gradually decreases from values near to 0.5 to values near to zero, which means that the intermediate formed from compound 2a (pK_a ≈ 9.3) has a pK_a value comparable with that of the acid buffer component.     

Image analysis for the automated estimation of clonal growth and its application to the growth of smooth muscle cells

Abstract. Image analysis was used for the automated measurement of colony frequency ( f ) and colony diameter ( d ) in cultures of smooth muscle cells, Initial studies with the inverted microscope showed that number of cells ( N ) in a colony varied directly with d : log N = 1.98 log d - 3.469 Image analysis generated the complement of a cumulative distribution for f as a function of d . The number of cells in each segment of the distribution function was calculated by multiplying f and the average N for the segment. These data were displayed as a cumulative distribution function. The total number of colonies ( f_T ) and the total number of cells ( N_T ) were used to calculate the average colony size ( N_A ). Population doublings (PD) were then expressed as log₂ N_A . Image analysis confirmed previous studies in which colonies were sized and counted with an inverted microscope. Thus, image analysis is a rapid and automated technique for the measurement of clonal growth.     

Age-related alterations in plasma membrane glycoprotein content and scheduled or unscheduled DNA synthesis.

WI-38 cells of various ages and SV40-transformed WI-38 cells were examined for differences in plasma membrane composition of glycoproteins and DNA synthesis. Sialic acid per milligram of protein content of the membranes of WI-38 cells decreased with passage of time in culture. Other glycoprotein fractions and alkaline phosphatase activity disappeared in the WI-38 cells with passage of time in culture (Phase III). Studies of DNA repair correlated with changes observed in the plasma membrane glycoprotein content of WI-38 cells over a passage of time in culture were also reported. Both the extent and rate of ultraviolet-induced unscheduled DNA synthesis remained relatively constant during the passage of the WI-38 cells until late phase III. At that time the extent of unscheduled DNA synthesis was measurably reduced. The number of cells in a population of phase III cells able to perform semiconservative DNA synthesis diminished with age in culture but not to an extent capable of explaining the observed changes seen in membrane composition of semiconservative DNA synthesis during passage of the cells in culture. Cells with an extended lifespan SV40-transformed WI-38 (VA 13.2 RA) cells, did not vary in membrane composition, semiconservative DNA synthesis, or unscheduled DNA synthesis over 200 serial subpassages of the cells in culture.     

Polysaccharides derived from Balanophora polyandra significantly suppressed the proliferation of ovarian cancer cells through P53‐mediated pathway

Ovarian cancer (OC) is ranked the first among the cancers threatening women's health. It attracts tremendous attention of cancer researchers because of its extremely high mortality rate. Recent studies have indicated that traditional herbal medicines (THMs) can play a pivotal role in cancer prevention and treatment. THMs are gaining popularity as a source of anti‐cancer agents. The plant of Balanophora polyandra, which has been used as a traditional herbal medicine, has been known for exhibiting potential haemostatic, analgesic, anti‐inflammatory and anti‐cancer properties. However, few studies on inhibitory effect of B. polyandra on OC have been performed. In the present study, we found that B. polyandra polysaccharides (BPP) induced cell cycle arrest at S phase, triggered apoptosis and inhibited migration and invasion of OC cells. Furthermore, we also found that there was a potential and close relationship between BPP and P53‐mediated pathway. Overall, these findings suggest that BPP can be a potential therapeutic agent for the treatment of OC.     

Sirtuin 5 regulates the proliferation,invasion and migration of prostate cancer cells through acetyl‐CoA acetyltransferase 1

Sirtuin 5 (SIRT5) is a NAD⁺‐dependent class III protein deacetylase, and its role in prostate cancer has not yet been reported. Therefore, to explore the diagnosis and treatment of prostate cancer, we investigated the effect of SIRT5 on prostate cancer. Sirtuin 5 was assessed by immunohistochemistry in 57 normal and cancerous prostate tissues. We found that the tissue expression levels of SIRT5 in patients with Gleason scores ≥7 were significantly different from those in patients with Gleason scores <7 (P < .05, R > 0). Further, mass spectrometry and pathway screening experiments showed that SIRT5 regulated the activity of the mitogen‐activated protein kinase (MAPK) pathway, which in turn modulated the expression of MMP9 and cyclin D1. Being a substrate of SIRT5, acetyl‐CoA acetyltransferase 1 (ACAT1) was regulated by SIRT5. SIRT5 also regulated MAPK pathway activity through ACAT1. These results revealed that SIRT5 promoted the activity of the MAPK pathway through ACAT1, increasing the ability of prostate cancer cells to proliferate, migrate and invade. Overall, these results indicate that SIRT5 expression is closely associated with prostate cancer progression. Understanding the underlying mechanism may provide new targets and methods for the diagnosis and treatment of the disease.     

Dissecting the roles of Cse1 and Nup2 in classical NLS‐cargo release in vivo

The importin α/β transport machinery mediates the nuclear import of cargo proteins that bear a classical nuclear localization sequence (cNLS). These cargo proteins are linked to the major nuclear protein import factor, importin‐β, by the importin‐α adapter, after which cargo/carrier complexes enter the nucleus through nuclear pores. In the nucleus, cargo is released by the action of RanGTP and the nuclear pore protein Nup2, after which the importins are recycled to the cytoplasm for further transport cycles. The nuclear export of importin‐α is mediated by Cse1/CAS. Here, we exploit structures of functionally important complexes to identify residues that are critical for these interactions and provide insight into how cycles of protein import and recycling of importin‐α occur in vivo using a Saccharomyces cerevisiae model. We examine how these molecular interactions impact protein localization, cargo import, function and complex formation. We show that reversing the charge of key residues in importin‐α (Arg44) or Cse1 (Asp220) results in loss of function of the respective proteins and impairs complex formation both in vitro and in vivo. To extend these results, we show that basic residues in the Nup2 N‐terminus are required for both Nup2 interaction with importin‐α and Nup2 function. These results provide a more comprehensive mechanistic model of how Cse1, RanGTP and Nup2 function in concert to mediate cNLS‐cargo release in the nucleus.     

Fungal functional ecology: bringing a trait‐based approach to plant‐associated fungi

Fungi play many essential roles in ecosystems. They facilitate plant access to nutrients and water, serve as decay agents that cycle carbon and nutrients through the soil, water and atmosphere, and are major regulators of macro‐organismal populations. Although technological advances are improving the detection and identification of fungi, there still exist key gaps in our ecological knowledge of this kingdom, especially related to function . Trait‐based approaches have been instrumental in strengthening our understanding of plant functional ecology and, as such, provide excellent models for deepening our understanding of fungal functional ecology in ways that complement insights gained from traditional and ‐omics‐based techniques. In this review, we synthesize current knowledge of fungal functional ecology, taxonomy and systematics and introduce a novel database of fungal functional traits (Fun^Fun). Fun^Fun is built to interface with other databases to explore and predict how fungal functional diversity varies by taxonomy, guild, and other evolutionary or ecological grouping variables. To highlight how a quantitative trait‐based approach can provide new insights, we describe multiple targeted examples and end by suggesting next steps in the rapidly growing field of fungal functional ecology.     

Can a benthic diatom community complement chemical analyses and discriminate between disturbed and undisturbed saline wetland habitats? A study of seven soda pans in Serbia

Soda pans are protected in the European Union as Natura 2000 areas in the category of “Pannonic saline steppes and marshes”. There are at least six soda pans of reference status in Serbia and only a half have strict legal protection. The number of similar, but disturbed (modified) habitats that could be reconstructed is still unknown. We conducted sampling in five natural and two disturbed soda pans aiming to compare a difference in physical and chemical water properties and benthic diatom communities. In addition, we tried to apply recently developed Diatom Index for Soda Pans (DISP) and Trait-based index (TBI) to test the applicability of taxonomic and trait-based approaches in ecological status assessment of soda pans in the southern part of the Carpathian Basin. In contrast to natural soda pans, lower pH and nutrients levels were recorded in disturbed pans. A total of 86 taxa of benthic diatoms, including 11 new for diatom flora of Serbia, were identified. A lower species richness and Shannon index was recorded in natural soda pans. Nitzschia austriaca, N. supralitorea, Navicula veneta and N. wiesneri were dominant diatoms in soda pans of the reference status. Both diatom indices were in average lower in disturbed pans, and this difference was statistically significant for DISP index. We concluded that two indices, supplemented with indicator values for some local diatom species, can be a promising tool for soda pan conservation in this part of the Carpathian Basin.

     

Consequences of MnSOD interactions with nitric oxide: Nitric oxide dismutation and the generation of peroxynitrite and hydrogen peroxide

The present study demonstrates that manganese superoxide dismutase (MnSOD) (Escherichia coli), binds nitric oxide (^√NO) and stimulates its decay under both anaerobic and aerobic conditions. The results indicate that previously observed MnSOD-catalyzed ^√NO disproportionation (dismutation) into nitrosonium (NO⁺) and nitroxyl (NO^? ) species under anaerobic conditions is also operative in the presence of molecular oxygen. Upon sustained aerobic exposure to ^√NO, MnSOD-derived NO^? species initiate the formation of peroxynitrite (ONOO^? ) leading to enzyme tyrosine nitration, oxidation and (partial) inactivation. The results suggest that both ONOO^? decomposition and ONOO^? -dependent tyrosine residue nitration and oxidation are enhanced by metal centre-mediated catalysis. We show that the generation of ONOO^? is accompanied by the formation of substantial amounts of H₂O₂. MnSOD is a critical mitochondrial antioxidant enzyme, which has been found to undergo tyrosine nitration and inactivation in various pathologies associated with the overproduction of ^√NO. The results of the present study can account for the molecular specificity of MnSOD nitration in vivo. The interaction of ^√NO with MnSOD may represent a novel mechanism by which MnSOD protects the cell from deleterious effects associated with overproduction of ^√NO.