Changes in growth patterns and intracellular calcium concentrations in Aspergillus awamori treated with amphotericin B

Growth patterns and intracellular Ca²⁺ concentrations in the mutant strain Aspergillus awamori 66A containing a recombinant aequorin gene were studied in the presence of a permeabilizing fungicidal agent amphotericin B. The cell response, i.e., changes in the growth and development of the fungus (initiation of spore germination, mycelial growth, and intensity of sporulation) was dose-dependent. Low concentrations of amphotericin B (2.5 μM) stimulated spore germination: the number of germinating spores was 2–3 times higher than in the control (without the fungicide). At higher amphotericin concentrations (20 μM) spore germination was inhibited. Amphotericin B had a dose-dependent effect on mycelial growth and sporulation intensity on solid Vogel medium. Intracellular Ca²⁺ concentrations in the presence of amphotericin B were investigated using the luminescence of the photoprotein aequorin. High concentrations of amphotericin B (10 and 20 μM) were shown to cause an instantaneous increase in Ca²⁺ concentrations compared to the control and lower amphotericin concentration (2.5 μM). Ca²⁺ concentrations remained elevated throughout the experiment and correlated with the inhibition of mycelial growth and development.     

EFFECTS OF NITRATE AND PHOSPHATE DISCHARGE FROM A FERTILIZER PRODUCTION PLANT IN A FJORD

Pedersen, A.^1,2, Knutzen, J.², Walday, M.², Molvær, M.² & Johnsen, T.^{2 1}Department of Ecology and Evolutionary Biology, University of Connecticut at Stamford, Stamford, CT. 06901 U.S.A;. ²Norwegian Institute for Water Research, P.O.B. 173 Kjelsås, 0411 Oslo, Norway. “Hydro Agri Glomfjord” a fertilizer producer has been discharging large quantities of ammonium, nitrate and phosphate into Glomfjord (in northern Norway, N 66,48°, E 13,57°) since 1947. The effects of the nutrient load to the Fjord have resulted in classical eutrophication symptoms. Elevated nutrient levels have resulted in frequent plankton blooms and seasonally reduced O₂ levels in the deep-water bodies as well as an eradication of the normal littoral assemblages. The fucoid belt had been replaced by various green algae 6 km from the outlet and outward the fjord. The effect is only seen on the northern side of the Fjord. After some improvement in the discharge loads in the 1980's, the condition in the pelagic column improved with regard to plankton blooms and deep water O₂ concentration. The littoral communities didn't, show any indication of recovery and a pollution indication index based on algal composition, showed even increased eutrophication.     

A mechanistic study on SMOB-ADP1: an NADH:flavin oxidoreductase of the two-component styrene monooxygenase of Acinetobacter baylyi ADP1

Two styrene monooxygenase types, StyA/StyB and StyA1/StyA2B, have been described each consisting of an epoxidase and a reductase. A gene fusion which led to the chimeric reductase StyA2B and the occurrence in different phyla are major differences. Identification of SMOA/SMOB-ADP1 of Acinetobacter baylyi ADP1 may enlighten the gene fusion event since phylogenetic analysis indicated both proteins to be more related to StyA2B than to StyA/StyB. SMOB-ADP1 is classified like StyB and StyA2B as HpaC-like reductase. Substrate affinity and turnover number of the homo-dimer SMOB-ADP1 were determined for NADH (24 µM, 64 s^?1) and FAD (4.4 µM, 56 s^?1). SMOB-ADP1 catalysis follows a random sequential mechanism, and FAD fluorescence is quenched upon binding to SMOB-ADP1 (K _d = 1.8 µM), which clearly distinguishes that reductase from StyB of Pseudomonas. In summary, this study confirmes made assumptions and provides phylogenetic and biochemical data for the differentiation of styrene monooxygenase-related flavin reductases.     

Somatic Hypermutation at A/T-Rich Oligonucleotide Substrates Shows Different Strand Polarities in Ung-Deficient or -Proficient Backgrounds

A/T mutations at immunoglobulin loci are introduced by DNA polymerase η (Polη) during an Msh2/6-dependent repair process which results in A''s being mutated 2-fold more often than T''s. This patch synthesis is initiated by a DNA incision event whose origin is still obscure. We report here the analysis of A/T oligonucleotide mutation substrates inserted at the heavy chain locus, including or not including internal C''s or G''s. Surprisingly, the template composed of only A''s and T''s was highly mutated over its entire 90-bp length, with a 2-fold decrease in mutation from the 5′ to the 3′ end and a constant A/T ratio of 4. These results imply that Polη synthesis was initiated from a break in the 5′-flanking region of the substrate and proceeded over its entire length. The A/T bias was strikingly altered in an Ung^−/− background, which provides the first experimental evidence supporting a concerted action of Ung and Msh2/6 pathways to generate mutations at A/T bases. New analysis of Pms2^−/− animals provided a complementary picture, revealing an A/T mutation ratio of 4. We therefore propose that Ung and Pms2 may exert a mutual backup function for the DNA incision that promotes synthesis by Polη, each with a distinct strand bias.     

不同气候变化情景下2070-2099年中国潜在植被及其敏感性

潜在植被作为当前气候条件、无人类干扰下,所能发育演替形成的最稳定、最成熟的一种顶极植被类型,能够反映立地植被发展的趋势。潜在植被的研究有助于人类了解植被与气候系统的作用机制,可为区域植被恢复工程和生态建设提供参考依据。基于综合顺序分类系统,利用A1B、A2及B1情景下2070-2099年气象数据对中国潜在植被进行了模拟,在不同气候变化情景下分析了未来中国潜在植被的空间分布和潜在植被对不同气候变化的敏感性。结果表明:(1)不同气候变化背景下中国潜在植被分布的规律具有相似性,但潜在植被类在总数和各情景下分布的面积存在差异性;(2)比较发现,中国的气候条件在20世纪和21世纪均不适宜炎热极干热带荒漠类(ⅦA)的发育;(3)中国潜在植被在3种气候变化情景下表现为敏感性的区域占到国土总面积的64.10%,在西北地区、北方地区、南方地区及青藏地区不同自然区敏感性地区所占各区的比例不同,分别为68.20%、70.82%、49.94%及66.59%。     

Evidence for the control of Eosinophilia by the major histocompatibility complex in mice

The genetic control of eosinophilia has been studied in congenic strains of mice. Eosinophilia was induced with cyclophosphamide followed by keyhole limpet hemocyanin in complete Freund's adjuvant. After this treatment, BALB/c mice developed a high eosinophil response, whereas CBA, C57BL and A/J mice developed a low one. The major histocompatibility complex (M-HQ was found to exert a control on eosinophilia, as B 10.D2 mice developed a higher eosinophil response than B10, B10.A, or B10.BR. BALB/c-H-2 ^k mice had a lower response than BALB/c, and A.TL mice had a higher response than A/J or A.TH. If a single gene within the MHC is responsible for these effects, the most likely position for it is in the vicinity of the Tla locus. Splenectomy reduced eosinophilia in BALB/c and A.TL mice, but not in A/J mice,indicating that the spleen is a significant site of eosinophil production in high responder strains.     

Antagonism of 2–5 A-mediated inhibition of protein synthesis in intact cells by 2',5'-(pA)3

In vitro studies have shown that the translational inhibitory activity of 2–5 A can be blocked by the oligoribonucleotide 2',5'-(pA)₃. We have examined the effect of simultaneous introduction of inhibitor and antagonist into intact mouse cells using calcium phosphate coprecipitation. Upon introduction of 10⁻⁴ M 2',5'-(pA)₃ and 10⁻⁶ M 2–5 A, inhibition of protein synthesis was prevented. Efficiency of calcium phosphate precipitation of 2–5 A in the presence or absence of 2',5'-(pA)₃ was comparable. Introduction of 2',5'-(pA)₃ analogs showed that nucleotides which do not bind well to the 2–5 A dependent endonuclease do not prevent 2–5 A inhibitory activity. Thus, 2',5'-(pA)₃ functions as an antagonist of 2–5 A in vivo.     

Application of a Mouse Ligated Peyer’s Patch Intestinal Loop Assay to Evaluate Bacterial Uptake by M cells

The inside of our gut is inhabited with enormous number of commensal bacteria. The mucosal surface of the gastrointestinal tract is continuously exposed to them and occasionally to pathogens. The gut-associated lymphoid tissue (GALT) play a key role for induction of the mucosal immune response to these microbes^{1, 2}. To initiate the mucosal immune response, the mucosal antigens must be transported from the gut lumen across the epithelial barrier into organized lymphoid follicles such as Peyer''s patches. This antigen transcytosis is mediated by specialized epithelial M cells^{3, 4}. M cells are atypical epithelial cells that actively phagocytose macromolecules and microbes. Unlike dendritic cells (DCs) and macrophages, which target antigens to lysosomes for degradation, M cells mainly transcytose the internalized antigens. This vigorous macromolecular transcytosis through M cells delivers antigen to the underlying organized lymphoid follicles and is believed to be essential for initiating antigen-specific mucosal immune responses. However, the molecular mechanisms promoting this antigen uptake by M cells are largely unknown. We have previously reported that glycoprotein 2 (Gp2), specifically expressed on the apical plasma membrane of M cells among enterocytes, serves as a transcytotic receptor for a subset of commensal and pathogenic enterobacteria, including Escherichia coli and Salmonella enterica serovar Typhimurium (S. Typhimurium), by recognizing FimH, a component of type I pili on the bacterial outer membrane ⁵. Here, we present a method for the application of a mouse Peyer''s patch intestinal loop assay to evaluate bacterial uptake by M cells. This method is an improved version of the mouse intestinal loop assay previously described ^{6, 7}. The improved points are as follows: 1. Isoflurane was used as an anesthetic agent. 2. Approximately 1 cm ligated intestinal loop including Peyer''s patch was set up. 3. Bacteria taken up by M cells were fluorescently labeled by fluorescence labeling reagent or by overexpressing fluorescent protein such as green fluorescent protein (GFP). 4. M cells in the follicle-associated epithelium covering Peyer''s patch were detected by whole-mount immunostainig with anti Gp2 antibody. 5. Fluorescent bacterial transcytosis by M cells were observed by confocal microscopic analysis. The mouse Peyer''s patch intestinal loop assay could supply the answer what kind of commensal or pathogenic bacteria transcytosed by M cells, and may lead us to understand the molecular mechanism of how to stimulate mucosal immune system through M cells.     

Hypochlorous acid-modified low-density lipoprotein inactivates the lysosomal protease cathepsin B: protection by ascorbic and lipoic acids

Abstract

Unregulated uptake of oxidized LDL by the scavenger receptor (s) of macrophages is thought to be an early event in atherosclerotic lesion development. Accumulation of oxidized LDL within macrophages may result from resistance of the modified LDL to enzymatic hydrolysis or from direct inactivation of lysosomal enzymes by reactive LDL-associated moieties. Since HOCl-modified LDL has been detected in vivo, the effects of HOCl-modified LDL on the activities of the cysteine protease cathepsin B and the aspartyl protease cathepsin D were investigated. LDL (0.5 mg protein/ml), which had been exposed to HOCl (25–200 µM), caused rapid dose-dependent inactivation of cathepsin B, but not of cathepsin D. Exposure of LDL to HOCl results primarily in the formation of LDL-associated chloramines, and the model chloramine N^-acetyl-lysine chloramine also caused dose-dependent inactivation of cathepsin B. Incubation of HOCl-modified LDL with ascorbic and lipoic acids (25–200 µM) resulted in dose-dependent reduction of LDL-associated chloramines and concomitant protection against cathepsin B inactivation. Thus, the data indicate that HOCl-modified LDL inactivates cathepsin B by a chloramine-dependent mechanism, most likely via oxidation of the enzyme's critical cysteine residue. Furthermore, small molecule antioxidants, such as ascorbic and lipoic acids, may be able to inhibit this potentially proatherogenic process by scavenging LDL-associated chloramines.     

The life history and production of Chaoborus punctipennis (Diptera: Chaoboridae) in Lake Norman,North Carolina,U.S.A.

A study of the life history and production of Chaoborus punctipennis (Say) in Lake Norman, North Carolina, U.S.A. was conducted from February 1978 through January 1979. Four sublittoral (～8 m) and two profundal (～30 m) locations were sampled. Larvae and pupae were collected with a modified Petersen grab and a plankton net, and adults were collected with emergence and light traps. Based on larval, pupal, and adult collections, there appear to be two generations per year — an overwintering spring generation and a summer generation. Annual dry weight standing stock biomass, dry weight production, and P/ B ratio were estimated from each sampling location and depth zone. Production was estimated by the size-frequency method. Standing stock biomass (30.9 mg · m^-2) and production (170.8 mg · m^-2) were highest in the profundal zone. In the sublittoral zone, standing stock biomass and production were 4.7 mg · m^-2, and 29.6 mg · m^-2, respectively. Annual P/ B ratios in the profundal and sublittoral zones were 5.5 and 6.3, respectively.     

Specific inhibitors of methane oxidation in Methylosinus trichosporium

Methane oxidation by washed cell suspensions of Methylosinus trichosporium OB3B was selectively inhibited by 25 compounds, including metal binding components such as carbon monoxide (85% O₂: 15% CO), KCN (10^-6 M), αα′-dipyridyl (10^-4 M), 8-quinolinol (10^-4 M), thiosemicarbazide (10^-5 M), thiourea (10^-5 M), hydroxylamine (10^-4 M), histidine (10^-2 M), British Anti-Lewisite (5x10^-3 M), and miscellaneous known inhibitors of other oxygenases. A role for copper in the methane oxygenase system was suggested by the pattern of inhibition and relief of inhibition by added metal ions.     

α2-Macroglobulin-like protein 1 can conjugate and inhibit proteases through their hydroxyl groups,because of an enhanced reactivity of its thiol ester

Proteins in the α-macroglobulin (αM) superfamily use thiol esters to form covalent conjugation products upon their proteolytic activation. αM protease inhibitors use theirs to conjugate proteases and preferentially react with primary amines (e.g. on lysine side chains), whereas those of αM complement components C3 and C4B have an increased hydroxyl reactivity that is conveyed by a conserved histidine residue and allows conjugation to cell surface glycans. Human α₂-macroglobulin–like protein 1 (A2ML1) is a monomeric protease inhibitor but has the hydroxyl reactivity–conveying histidine residue. Here, we have investigated the role of hydroxyl reactivity in a protease inhibitor by comparing recombinant WT A2ML1 and the A2ML1 H1084N mutant in which this histidine is removed. Both of A2ML1s'' thiol esters were reactive toward the amine substrate glycine, but only WT A2ML1 reacted with the hydroxyl substrate glycerol, demonstrating that His-1084 increases the hydroxyl reactivity of A2ML1''s thiol ester. Although both A2ML1s conjugated and inhibited thermolysin, His-1084 was required for the conjugation and inhibition of acetylated thermolysin, which lacks primary amines. Using MS, we identified an ester bond formed between a thermolysin serine residue and the A2ML1 thiol ester. These results demonstrate that a histidine-enhanced hydroxyl reactivity can contribute to protease inhibition by an αM protein. His-1084 did not improve A2ML1''s protease inhibition at pH 5, indicating that A2ML1''s hydroxyl reactivity is not an adaption to its acidic epidermal environment.     

Crohn's disease of the appendix

Granulomatous inflammation typifying Crohn''s disease was centred within or confined to appendices in six patients, two of whom developed lesions attributable to Crohn''s disease elsewhere in the gut. The remaining four patients have remained symptom-free for periods varying from two to six years. Histological evidence of Crohn''s disease was also present in five of 46 appendices available for re-examination in a survey of 63 cases of Crohn''s enterocolitis. It is adduced that appendiceal involvement in Crohn''s disease is not uncommon.     

Draft Genome Sequence of the Novel Enteric Bacterium Galloisinimonas intestini B14T KCTC 32180, Isolated from the Gut of a Galloisiana Species (Notoptera: Grylloblattidae) Fossil Insect

We report the 3.74-Mb genome sequence of Galloisinimonas intestini B14^T, isolated from the gut of one of the world''s rarest insect species, Galloisiana sp., collected at a Mosan cave, Moonkyung, Gyungsangbook-do, South Korea. Strain B14^T is a novel genus candidate of the family Enterobacteriaceae.     

The Role of Hemoglobin's C-terminal Region: the Work of Eraldo Antonini

Studies on Carboxypeptidase Digests of Human Hemoglobin(Antonini, E., Wyman, J., Zito, R., Rossi-Fanelli, A., and Caputo, A. (1961) J. Biol. Chem. 236, PC60–PC63)The Effect of Oxygenation on the Rate of Digestion of Human Hemoglobins by Carboxypeptidases(Zito, R., Antonini, E., and Wyman, J. (1964) J. Biol. Chem. 239, 1804–1808)Eraldo Antonini (1931–1983) received his degree in Medicine and Surgery from the University of Rome in 1955. He then began studying hemoglobin and myoglobin with Alessandro Rossi-Fanelli at the Institute of Biochemistry of the University of Rome and at the Regina Elena Institute for Cancer Research.Open in a separate windowEraldo Antonini (right) in Caprarola in 1971 for Jeffries Wyman''s (left) birthday party, with Robert W. Noble (back).In 1961, Antonini and Rossi-Fanelli published a paper describing the effect on human hemoglobin''s activity when the C-terminal amino acid residues are removed from the molecule''s α and/or β chains. This paper is reprinted here as a Journal of Biological Chemistry (JBC) Classic. In the study, the scientists used carboxypeptidase A to digest the C-terminal tyrosine and histidine on the molecule''s β chain and carboxypeptidase B to remove the C-terminal lysine, tyrosine, and arginine on the molecule''s α chain. The resulting protein appeared intact but had an increased oxygen affinity, lowered cooperativity, and dramatically reduced Bohr effect.This observation inspired Max Perutz, who wrote: “Several years later, my electron density maps showed that these residues form salt bridges with neighboring subunits in deoxyhaemoglobin which get broken on transition to oxyhaemogloblin. Remembering Antonini''s observation, I realized at once that these bridges must represent the additional bonds between the subunits in the T structure predicted by Monod, Wyman and Changeux''s theory of allostery. Antonini had also demonstrated that the release of Bohr protons is colinear with oxygen uptake. When Kilmartin''s and my work proved that most of the Bohr protons originate from the salt bridges, it became clear to me that oxygen uptake is linked to the rupture of these bridges” (1).In the second JBC Classic reprinted here, Antonini follows up on the first paper by doing a reciprocal experiment in which he looks at differences in digestion rates of oxy- and deoxyhemoglobin, reasoning, “If enzymatic modification can affect conformation and changes of conformation resulting from combination with ligand (oxygen), one might expect that the rate of attack on the hemoglobin by the enzymes should depend on the presence or absence of ligand; this would determine conformation, and conformation, in turn, would control the rate.” Again using carboxypeptidases A and B, he showed that the rate of digestion is different for the oxy- and deoxy- forms of the molecule, indicating a differential accessibility of the C-terminal residues to these enzymes.This work was later extended and perfected by John V. Kilmartin on a suggestion by Perutz, who pointed out the crucial role of the C-terminal residues for the molecular mechanism of cooperativity and the Bohr effect. Kilmartin was able to differentiate the role of the C-terminal histidine from that of tyrosine by preparing and characterizing a modified hemoglobin devoid of histidine.Over the next several years, Antonini continued to study hemoglobin, looking at the properties of the α and β chains, the acid-base equilibria of hemoglobin, the Bohr effect and its dependence on temperature, the oxidation-reduction equilibria, ligand-induced conformational changes in hemoglobin, and the kinetics of the reaction of myoblogins and hemoglobins with ligands. This work culminated in the publication of Hemoglobin and Myoglobin in Their Reactions with Ligands in 1971 (2), which was a landmark in the field.In the 1970s, Antonini expanded his scientific interests and started focusing on electron-transfer metalloproteins (such as cytochrome c oxidase) and on proteolytic enzymes. He eventually became Professor of Molecular Biology at the University of Camerino and was later made Professor of Chemistry and Director of the Institute of Chemistry in the Faculty of Medicine and Surgery at the University of Rome. He also received the Feltrinelli Prize from the Accademia Nazionale dei Lincei in 1974.One of Antonini''s coauthors on the two JBC Classics reprinted here is JBC Classic author Jeffries Wyman (3) who came to Rome in 1961 for a week-long visit and ended up remaining for 25 years and working with Antonini. This collaboration produced a series of outstanding papers and conceptual advancements that have had a long lasting influence on protein chemistry.^1,2     

Glaucocalyxin A and B Regulate Growth and Induce Oxidative Stress in Lettuce (Lactuca sativa L.) Roots

Glaucocalyxin (Gla) A–C are major ent-kaurane diterpenoids isolated from Isodon japonicus var. glaucocalyx (Maxim.) H. W. Li. This study investigated the possible interference of these diterpenoids with root growth and its mechanism of action in lettuce (Lactuca sativa L.) seedlings. Results indicated the dual stimulatory and inhibitory effects of Gla A and B on root growth and their phytotoxic effects on root hair development. The promotion of root growth by lower levels of Gla A and B (20–40 μM) resulted from enhanced cell length and increased mitotic activity. However, higher concentrations (80–200 μM) of Gla A and B had inhibitory effects. In addition, Gla A and B inhibited root hair development of lettuce seedlings in a dose-dependent manner at concentrations between 20 and 200 μM. Exposure of lettuce roots to Gla A and B at 200 μM increased levels of malondialdehyde and the generation of O ₂ ^·? , indicating lipid peroxidation and induction of oxidative stress. Activities of the antioxidant enzymes superoxide dismutase, catalase, and peroxidase were significantly elevated. Reactive oxygen species (ROS) scavengers dihydroxybenzene disulfonic acid (Tiron) and dimethylthiourea at 100 μM could efficiently alleviate the phytotoxicity induced by Gla A and B at 200 μM. These results demonstrated that the deleterious effect of Gla A and B at higher concentrations (80–200 μM) on roots may occur through the imposition of oxidative stress on cell growth and cell division. Due to the lack of an α,β-unsaturated ketone in α-methylenecyclopentanone moiety, Gla C could not induce ROS generation and exhibited no effect on the roots, even at the highest concentration (200 μM). Therefore, the α-methylenecyclopentanone moiety in the ent-kaurene diterpenoids was presented as an essential possible active center for the phytotoxicity.     

Backbone 1H, 13C and 15N resonance assignments of dengue virus NS2B–NS3p in complex with aprotinin

Dengue virus, belongs to Flaviviridae, is an arthropod transmitted virus that threatens millions of people’s lives. As with other flaviviruses, a positive single-stranded 11-kilobases RNA in the dengue virus genome encodes three structural proteins (capsid protein C, membrane protein M, and envelope protein E) and seven non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5). The two component protease NS2B–NS3p is essential for viral replication and is believed to be a potential antiviral drug target. Aprotinin, a native inhibitor, is proved to retard the activity of NS2B–NS3p. The backbone assignments of NS2B–NS3p will be essential for determining the high resolution solution structure of NS2B–NS3p and screening new antiviral drugs. Herein, we report the backbone ¹H, ¹⁵N, ¹³C resonance assignments of the N terminal fragment of NS2B (4.8 kDa) and NS3p (18.5 kDa) in complex with aprotinin (6.5 kDa) by high resolution NMR.     

Development of pyrazole and spiropyrazoline analogs as multifunctional agents for treatment of Alzheimer’s disease

Cholinergic hypothesis of Alzheimer’s disease has been advocated as an essential tool in the last couple of decades for the drug development. Here in, we report de novo fragment growing strategy for the design of novel 3,5-diarylpyrazoles and hit optimization of spiropyrazoline derivatives as acetyl cholinesterase inhibitors. Both type of scaffolds numbering forty compounds were synthesized and evaluated for their potencies against AChE, BuChE and PAMPA. Introduction of lipophilic cyclohexane ring in 3,5-diarylpyrazole analogs led to spiropyrazoline derivatives, which facilitated and improved the potencies. Compound 44 (AChE = 1.937 ± 0.066 µM; BuChE = 1.166 ± 0.088 µM; hAChE = 1.758 ± 0.095 µM; P_e = 9.491 ± 0.34 × 10⁻⁶ cm s¹) showed positive results, which on further optimization led to the development of compound 67 (AChE = 0.464 ± 0.166 µM; BuChE = 0.754 ± 0.121 µM; hAChE = 0.472 ± 0.042 µM; P_e = 13.92 ± 0.022 × 10⁻⁶ cm s¹). Compounds 44 and 67 produced significant displacement of propidium iodide from the peripheral anionic site (PAS) of AChE. They were found to be safer to MC65 cells and decreased metal induced Aβ_1-42 aggregation. Further, in-vivo behavioral studies, on scopolamine induced amnesia model, the compounds resulted in better percentage spontaneous alternation scores and were safe, had no influence on locomotion in tested animal groups at dose of 3 mg/kg. Early pharmacokinetic assessment of optimized hit molecules was supportive for further drug development.     

Loss of the Protease Dimerization Inhibition Activity of Tipranavir (TPV) and Its Association with the Acquisition of Resistance to TPV by HIV-1

Tipranavir (TPV), a protease inhibitor (PI) inhibiting the enzymatic activity and dimerization of HIV-1 protease, exerts potent activity against multi-PI-resistant HIV-1 isolates. When a mixture of 11 multi-PI-resistant (but TPV-sensitive) clinical isolates (HIV_11MIX), which included HIV_B and HIV_C, was selected against TPV, HIV_11MIX rapidly (by 10 passages [HIV_11MIX^P10]) acquired high-level TPV resistance and replicated at high concentrations of TPV. HIV_11MIX^P10 contained various amino acid substitutions, including I54V and V82T. The intermolecular FRET-based HIV-1 expression assay revealed that TPV''s dimerization inhibition activity against cloned HIV_B (cHIV_B) was substantially compromised. The introduction of I54V/V82T into wild-type cHIV_NL4-3 (cHIV_{NL4-3^I54V/V82T}) did not block TPV''s dimerization inhibition or confer TPV resistance. However, the introduction of I54V/V82T into cHIV_B (cHIV_B^I54V/V82T) compromised TPV''s dimerization inhibition and cHIV_B^I54V/V82T proved to be significantly TPV resistant. L24M was responsible for TPV resistance with the cHIV_C genetic background. The introduction of L24M into cHIV_NL4-3 (cHIV_NL4-3^L24M) interfered with TPV''s dimerization inhibition, while L24M increased HIV-1''s susceptibility to TPV with the HIV_NL4-3 genetic background. When selected with TPV, cHIV_{NL4-3^I54V/V82T} most readily developed TPV resistance and acquired E34D, which compromised TPV''s dimerization inhibition with the HIV_NL4-3 genetic background. The present data demonstrate that certain amino acid substitutions compromise TPV''s dimerization inhibition and confer TPV resistance, although the loss of TPV''s dimerization inhibition is not always associated with significantly increased TPV resistance. The findings that TPV''s dimerization inhibition is compromised with one or two amino acid substitutions may explain at least in part why the genetic barrier of TPV against HIV-1''s development of TPV resistance is relatively low compared to that of darunavir.     

Membrane receptors for Vicia graminea anti-N lectin and its binding to native and neuraminidase-treated human erythrocytes

Membrane receptors for Vicia graminea (Vg) lectin on human red cells were analyzed using deoxycholate lysates obtained from ¹²⁵I-erythrocyte membranes incubated with a purified lectin immobilized on Sepharose 4B. The glycoproteins (GP) specifically bound to the gel were eluted and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. Using native erythrocytes the results obtained demonstrate that N red cells have exposed Vg receptors located on GPα (synonym glycophorin A) and GPδ (synonym glycophorin B) whereas on M erythrocytes the Vg receptors are restricted to GPδ. The presence of Vg receptors was also found on the hybrid glycoprotein (made of the N-ter of GPδ and C-ter of GPα) carried by St(a+) erythrocytes. A similar amount of radioactivity was bound to Vg-Sepharose incubated with neuraminidase-treated N or M membranes. The material eluted was tentatively identified as asialo GPα and asialo GPδ, suggesting that numerous receptors have been uncovered mainly on asialo GPα species from M erythrocytes. No glycoprotein component could be identified from the material eluted from Vg Sepharose incubated with native or neuraminidase-treated membrane from a Tn(+) individual. Scatchard plot analysis obtained from binding experiments at equilibrium with M, N, and St(a+) cells revealed the existence of at least two classes of receptors both on native and neuraminidase-treated erythrocytes. Desialylation of the M, N, and St(a+) erythrocytes resulted in an increase in the number of low- and high-affinity binding sites but had no significant effect on the association constants. However, high-affinity binding constants were about six times higher with N (7.07 × 10⁷ and 6.61 × 10⁷m^?1 for native and neuraminidase-treated N cells, respectively) as compared to M erythrocytes (1.13 × 10⁷ and 1.17 × 10⁷m^?1 for native and neuraminidase-treated M cells, respectively) whereas the low-affinity binding constants were similar for all types of cells (in the range of 0.1 to 0.3 × 10⁷m^?1). The number of Vg binding sites increases from 0.085 × 10⁵ to 0.8 × 10⁵ (high affinity) and from 2.10 × 10⁵ to 6.25 × 10⁵ (low affinity) per native and neuraminidase-treated N cell, respectively. On native and neuraminidase-treated M cells the number of Vg receptors increases from 0.011 × 10⁵ to 0.51 × 10⁵ (high affinity) and 0.13 × 10⁵ (low affinity), respectively. The large increase in the number of Vg receptors on neuraminidase-treated M cells is correlated with a large increase in agglutinability. Under similar treatment St(a+) cells behave like N erythrocytes whereas only 0.16 × 10⁵ Vg receptors of low affinity could be detected on neuraminidase-treated Tn erythrocytes. The results demonstrate that sialic acid is not required for binding and favor the view that the binding site of V. graminea lectin accommodates with two types of erythrocyte membrane receptors, one including both a contribution of polypeptide and oligosaccharide chains and a second which involves a simple interaction with sugar sequence Galβ1–3GalNAc available only when sialic acids are removed. The latter disaccharide is recognized by the Arachis hypogea lectin which therefore inhibits further binding of the V. graminea to neuraminidase-treated erythrocytes.