Asp residues of the Glu-Glu-Asp-Asp pore filter contribute to ion permeation and selectivity of the Cav3.2 T-type channel

Voltage-activated Ca²⁺ channels are membrane protein machinery performing selective permeation of external calcium ions. The main Ca²⁺ selective filters of all high-voltage-activated Ca²⁺ channel isoforms are commonly composed of four Glu residues (EEEE), while those of low-voltage-activated T-type Ca²⁺ channel isoforms are made up of two Glu and two Asp residues (EEDD). We here investigate how the Asp residues at the pore loops of domains III and IV affect biophysical properties of the Ca_v3.2 channel. Electrophysiological characterization of the pore mutant channels in which the pore Asp residue(s) were replaced with Glu, showed that both Asp residues critically control the biophysical properties of Ca_v3.2, including relative permeability between Ba²⁺ and Ca²⁺, anomalous mole fraction effect (AMFE), voltage dependency of channel activation, Cd²⁺ blocking sensitivity, and pH effects, in distinctive ways.     

Vector-based RNAi,a novel tool for isoform-specific knock-down of VEGF and anti-angiogenesis gene therapy of cancer

Vascular endothelial growth factor (VEGF) carries out multifaceted functions in tumor development, and it exists as at least five isoforms with distinct biologic activities and clinical implications. Several strategies have been developed to block VEGF for cancer therapy; however, the approach to target-specific VEGF isoform(s) has not been explored to date. In the present study, we show that DNA vector-based RNA interference (RNAi), in which RNAi sequences targeting murine VEGF isoforms are inserted downstream of an RNA polymerase III promoter, has potential applications in isoform-specific "knock-down" of VEGF. Large molecular weight VEGF isoforms were specifically reduced in vitro in the presence of isoform-specific RNAi constructs. Additionally, H1 promoter may be superior to U6 promoter when used for vector-based RNAi of VEGF isoforms. This strategy provides a novel tool to study the function of various VEGF isoforms and may contribute to VEGF isoform-specific treatment in cancer.     

Synthesis and exploration of 2-morpholino-4-phenylthiazol-5-yl acrylamide derivatives for their effects against carbonic anhydrase I,II, IX and XII isoforms as a non-sulfonamide class of inhibitors

Novel series of 2-morpholino-4-phenylthiazol-5-yl acrylamide derivatives (8a–s) have been synthesized and explored as a non-sulfonamide class of carbonic anhydrase (CA, EC 4.2.1.1) inhibitors. The newly synthesized molecules were evaluated for their CA inhibitory potency against four isoforms: the cytosolic isozyme hCA I, II as well as trans-membrane tumor associated isoform hCA IX and hCA XII taking acetazolamide (AAZ) as standard drug. The results revealed that most of the compounds showed good activity against hCA II, IX, and XII whereas none of them were active against hCA I (K_i >100 μM). It is observed that the physiologically most important cytosolic isoform hCA II was inhibited by these molecules in the range of K_i 9.3–77.7 μM. It is also found the both the transmembrane isoforms hCA IX and XII were also inhibited with K_is ranging between 54.7–96.7 μM and 4.6–8.8 μM, respectively. The binding modes of the active compounds within the catalytic pockets of hCA II, IX and XII were evaluated by docking studies. This new non-sulfonamide class of selective inhibitors of hCA II, IX and XII over the hCA I isoform may be used for further understanding the physiological roles of some of these isoforms in various pathologies.     

Cancer: Towards a general theory of the target

General theories (GT) are reductionist explications of apparently independent facts. Here, in reviewing the literature, I develop a GT to simplify the cluttered landscape of cancer therapy targets by revealing they cluster parsimoniously according to only a few underlying principles. The first principle is that targets can be only exploited by either or both of two fundamentally different approaches: causality‐inhibition, and ‘acausal’ recognition of some marker or signature. Nonetheless, each approach must achieve both of two separate goals, efficacy (reduction in cancer burden) and selectivity (sparing of normal cells); if the mechanisms are known, this provides a definition of rational treatment. The second principle is target fragmentation, whereby the target may perform up to three categoric functions (cytoreduction, modulation, cytoprotection), potentially mediated by physically different target molecules, even on different cell types, or circulating freely. This GT remains incomplete until the minimal requirements for cure, or alternatively, proof that cure is impossible, become predictable.     

Phagotrophic phytoflagellates in microbial food webs

Phagotrophy by pigmented flagellates is known from the literature but has recently been rediscovered in the context of microbial food webs. Particle ingestion rates were found to be equivalent for pigmented and nonpigmented microflagellates in both field and laboratory studies. Ingestion rates of the chrysophytes Ochromonas danica, O. minuta, and Poterioochromonas malhamensis, the dinoflagellate Peridinium inconspicuum, and the cryptophytes Cryptomonas ovata and C. erosa were compared with those of two nonpigmented Monas species using 0.57 μm polystyrene beads as a food source. Ingestion rates were 0.31 to 3.17 beads/cell/h and filtration rates were 10⁻⁷ to 10⁻⁸ ml/cell/h with no detectable difference between pigmented and nonpigmented forms. Ingestion rates in unpigmented Monas species showed a linear increase with increasing particle concentration from 1.9 × 10⁶ to 1.6 × 10⁷ beads/ml. Light and DOC levels in the range of those encountered by phytoflagellates in the field also influenced laboratory measurements of bead ingestion by Poterioochromonas malhamensis. Ingestion rates decreased and photosynthesis increased over the natural PAR light range from 0 to 1800 microeinsteins/s/m². At 40 microeinsteins/s/m² maximum ingestion rates and high rates of photosynthesis occurred simultaneously. Ingestion rates decreased above 4 mgC/l supplied as glucose. DOC levels commonly occurring in Lake Oglethorpe range from 3.5 to 10.0 mgC/l. These studies suggest that mixotrophy, the trophic utilization of particulate food and dissolved organic matter as well as photosynthetically fixed organic matter, is a balanced process that can be regulated by environmental conditions. In field studies during a chrysophyte bloom, phytoflagellate grazing exceeded heterotrophic microflagellate grazing and constituted up to 55% of the bactivory of all microflagellates, ciliates, rotifers, and crustaceans combined. Neither bacterial abundance, light nor temperature were good predicters of grazing rates for the phagotrophic phytoflagellate association as a whole during this unstratified period. Phagotrophs are often most abundant at the metalimnetic plate during stratification.     

Lysine 77 is a Key Residue in Aggregation of Equinatoxin II, a Pore-forming Toxin from Sea Anemone Actinia equina

Among eighteen point mutants of equinatoxin II produced in E. coli, containing a single cystein substitution at variable position, EqtIIK77C was chosen for its peculiar properties. It was almost 100 times less hemolytic than the wild-type, but its hemolytic activity could be restored by chemical modification of the thiol group, provided that a positive charge was reintroduced. This indicates that a positive charge at this position is necessary for toxin activity. The mutant formed larger pores as compared to the wild type, but displayed the same cation selectivity. The pores reverted to normal size upon reintroduction of the positive charge. The conformation of EqtIIK77C and its binding to lipid membranes, either vesicles or red blood cells, was almost normal. However the kinetics of calcein release from lipid vesicles was substantially slower than that of the wild-type. Taken together with the different size of the pore formed, this is an indication that mutation of Lys77 → Cys influences the normal development of the aggregate which is required for assembling the functional pore. Received: 18 May 1999/Revised: 18 September 1999     

Influence of pore residues on permeation properties in the Kv2.1 potassium channel. Evidence for a selective functional interaction of K+ with the outer vestibule

The Kv2.1 potassium channel contains a lysine in the outer vestibule (position 356) that markedly reduces open channel sensitivity to changes in external [K(+)]. To investigate the mechanism underlying this effect, we examined the influence of this outer vestibule lysine on three measures of K(+) and Na(+) permeation. Permeability ratio measurements, measurements of the lowest [K(+)] required for interaction with the selectivity filter, and measurements of macroscopic K(+) and Na(+) conductance, were all consistent with the same conclusion: that the outer vestibule lysine in Kv2.1 interferes with the ability of K(+) to enter or exit the extracellular side of the selectivity filter. In contrast to its influence on K(+) permeation properties, Lys 356 appeared to be without effect on Na(+) permeation. This suggests that Lys 356 limited K(+) flux by interfering with a selective K(+) binding site. Combined with permeation studies, results from additional mutagenesis near the external entrance to the selectivity filter indicated that this site was located external to, and independent from, the selectivity filter. Protonation of a naturally occurring histidine in the same outer vestibule location in the Kv1.5 potassium channel produced similar effects on K(+) permeation properties. Together, these results indicate that a selective, functional K(+) binding site (e.g., local energy minimum) exists in the outer vestibule of voltage-gated K(+) channels. We suggest that this site is the location of K(+) hydration/dehydration postulated to exist based on the structural studies of KcsA. Finally, neutralization of position 356 enhanced outward K(+) current magnitude, but did not influence the ability of internal K(+) to enter the pore. These data indicate that in Kv2.1, exit of K(+) from the selectivity filter, rather than entry of internal K(+) into the channel, limits outward current magnitude. We discuss the implications of these findings in relation to the structural basis of channel conductance in different K(+) channels.     

长山群岛海域真核微藻粒级结构及扇贝摄食选择性

采用高通量测序-分子鉴定分级技术于2019年对长山群岛全海域真核微藻粒级结构进行了研究。结果发现,春季以中（47%）、小粒级（41%）为主,夏季以小（39%）、大粒级（38%）为主,秋季以大粒级（60%）为主,春、夏、秋季小、中、大粒级微藻比例为42：47：11、39：23：38、22：18：60。小粒级微藻优势种为细小微胞藻（Micromonas pusilla）、融合微胞藻（Micromonas commoda）和金牛微球藻（Ostreococcus tauri）,中粒级微藻优势种为剧毒卡尔藻（Karlodinium veneficum）、大粒级微藻优势种为柔弱几内亚藻（Guinardia delicatula）、平野亚历山大藻（Alexandrium hiranoi）、多纹膝沟藻（Gonyaulax polygramma）,综合整个真核微藻群落,春季由中粒径的剧毒卡尔藻占据优势（23.9%）,夏季由大粒径的平野亚历山大藻占据优势（29.4%）,秋季由大粒径的多纹膝沟藻占据优势（66.8%）,有毒甲藻在该海域中占有绝对优势,贝毒累积风险较高,小粒径微藻中金牛微球藻和抑食金球藻曾在渤海引发褐潮,潜在威胁该海域贝类养殖业。虾夷扇贝对小粒级和大粒级微藻的选择性较低,对中粒级微藻的选择性较高,尤其对水体中优势种剧毒卡尔藻一直表现出主动选择。光学需氧量、无机氮、溶解氧、石油类及部分重金属Cd、As、Hg影响着整个长山群岛海域真核微藻粒级结构时空演变。     

寄主植物对荔枝蒂蛀虫产卵的引诱作用

试验观察荔枝蒂蛀虫Conopomorpha sinensisBradley对寄主植物品种、器官及器官部位的产卵选择性规律。结果表明,与老梢相比,荔枝蒂蛀虫明显倾向于选择嫩梢产卵;4个荔枝品种果枝中以妃子笑上落卵数最多,在淮枝、糯米糍荔枝果实上较少,桂味荔枝上最少;不同的荔枝品种果实气味引诱雌虫产卵的能力不同,其中妃子笑引诱力最强,淮枝、糯米糍引诱力较低,桂味最低;在同一荔枝果实不同部位的落卵量,以果皮上、下两部分较大,全果明显低于果皮上部,而荔枝肉和荔枝核上没有落卵。