Syntheses of 2,4,6-trisubstituted triazines as antimalarial agents

A series of 2,4,6-trisubstituted-1,3,5-triazines (2a-s) were synthesized and evaluated for their in vitro antimalarial activity against P. falciparum. Out of the 19 compounds synthesized eight compounds showed MIC in the range of 1-2 microg/mL. These compounds are in vitro several times more active than cycloguanil.     

Solid support synthesis of 6-aryl-2-substituted pyrimidin-4-yl phenols as anti-infective agents

A library of 30 trisubstituted pyrimidines were synthesized and evaluated for their in vitro antimalarial and antitubercular activity. Out of the 30 compounds synthesized, 23 compounds have shown in vitro antimalarial activity against Plasmodium falciparum in the range of 0.25-2 microg/mL and 16 compounds have shown antitubercular activity against Mycobacterium tuberculosis H37Ra, at a concentration of 25 microg/mL.     

Autoantibodies against oxidized LDL are associated with severe chest pain attacks in patients with coronary heart disease

Autoantibodies against oxidized low-density lipoprotein (oxLDL) predict the progression of atherosclerosis. Several studies have shown that oxLDL is present in atherosclerotic lesions and that several factors present in active atherosclerotic plaques can oxidatively modify LDL. Oxidation of LDL induces production of autoantibodies against oxLDL (oxLDLab) that can be measured using an EIA test. Our aim was to see whether oxLDLab are associated with severe chest pain attacks in coronary heart disease (CHD) patients. Patients having two- or three-vessel CHD, as assessed by coronary angiography, and their siblings were recruited into the study (n = 568, mean age 55.8 years, range 29.3–83.2 years). Nondiabetic patients having a history of severe chest pain attacks had significantly higher oxLDLab levels (0.611 ± 0.56) than those who did not have a history of severe chest pain attacks (0.487 ± 0.40) (p = 0.027), even though age, cholesterol level, body mass index, and blood pressure were similar in both groups. However, no difference was found in oxLDLab levels in diabetic patients with or without a history of severe chest pain attacks. Increased levels of oxLDL autoantibodies are associated with severe chest pain attacks in nondiabetic patients with CHD.     

Effects of acid sphingomyelinase deficiency on male germ cell development and programmed cell death

Deficiency of acid sphingomyelinase (ASM), an enzyme responsible for producing a pro-apoptotic second messenger ceramide, has previously been shown to promote the survival of fetal mouse oocytes in vivo and to protect oocytes from chemotherapy-induced apoptosis in vitro. Here we investigated the effects of ASM deficiency on testicular germ cell development and on the ability of germ cells to undergo apoptosis. At the age of 20 weeks, ASM knock-out (ASMKO) sperm concentrations were comparable with wild-type (WT) sperm concentrations, whereas sperm motility was seriously affected. ASMKO testes contained significantly elevated levels of sphingomyelin at the age of 8 weeks as detected by high-performance, thin-layer chromatography. Electron microscopy revealed that the testes started to accumulate pathological vesicles in Sertoli cells and in the interstitium at the age of 21 days. Irradiation of WT and ASMKO mice did not elevate intratesticular ceramide levels at 16 h after irradiation. In situ end labeling of apoptotic cells also showed a similar degree of cell death in both groups. After a 21-day recovery period, the numbers of primary spermatocytes and spermatogonia at G2 as well as spermatids were essentially the same in the WT and ASMKO testes, as detected by flow cytometry. In serum-free cultures both ASMKO and WT germ cells showed a significant increase in the level of ceramide, as well as massive apoptosis. In conclusion, ASM is required for maintenance of normal sphingomyelin levels in the testis and for normal sperm motility, but not for testicular ceramide production or for the ability of the germ cells to undergo apoptosis.     

General Description of Partly Meromictic Hypertrophic Lake Verevi,its Ecological Status,Changes during the Past Eight Decades,and Restoration Problems

The present study describes generally the ecosystem of Lake Verevi while more detailed approaches are presented in the same issue. The main task of the article is to estimate long-term changes and find the best method for the restoration of good ecological status. Lake Verevi (surface 12.6 ha, mean depth 3.6 m, maximum depth 11 m, drainage area 1.1 km², water exchange 0.63-times per year) is a hypertrophic hard-water lake located in town Elva (6400 inhabitants). Long-term complex limnological investigations have taken place since 1929. The lake has been contaminated by irregular discharge of urban wastewaters from oxidation ponds since 1978, flood from streets, and infiltrated waters from the surrounding farms. The so-called spring meromixis occurred due to extremely warm springs in recent years. The index value of buffer capacity of Lake Verevi calculated from natural conditions is on the medium level. Water properties were analysed according to the requirements of the EU Water Framework Directive. According to the classification, water quality as a long-term average of surface layers is moderate-good, but the water quality of bottom layers is bad. Values in deeper layers usually exceed 20–30 times the calculated reference values by Vighi and Chiaudani’s model. Naturally, at the beginning of the 20th century the limnological type of the lake was moderately eutrophic. During the 1980s and 1990s the ecosystem was out of balance by abiotic characteristics as well as by plankton indicators. Rapid fluctuations of species composition and abundance can be found in recent years. Seasonal variations are considerable and species composition differs remarkably also in the water column. The dominating macrophyte species vary from year to year. Since the annual amount of precipitation from the atmosphere onto the lake surface is several times higher, the impact of swimmers could be considered irrelevant. Some restoration methods were discussed. The first step, stopping external pollution, was completed by damming the inlet. Drainage (siphoning) of the hypolimnetic water is discussed. Secondary pollution occurs because Fe:P values are below the threshold. The authors propose to use phosphorus precipitation and hypolimnetic aeration instead of siphoning.     

Optical Properties and Light Climate in Lake Verevi

The optical properties and light climate during the ice-free period in the highly stratified Lake Verevi (Estonia) have been studied together with other lakes in same region since 1994. The upper water layer above the thermocline belongs to class “moderate” by optical classification of Estonian lakes but can turn “turbid” (concentration of chlorophyll a up to 73 mg m⁻³ and total suspended matter up to 13.2 g m⁻³) during late summer blooms. In the blue part of the spectrum, light is mainly attenuated by dissolved organic matter and in red part notably scattering but also absorption by phytoplanktonic pigments effect the spectral distribution of underwater light. Consequently, the underwater light is of greenish-yellow color (550–650 nm). Rapid change in optical properties occurs with an increase of all optically active substances close to thermocline (2.5–6 m). Optical measurements are often hampered beneath this layer so that modeling of the depth distribution of the diffuse attenuation coefficient is an useful compliment to field measurements. K_d,PAR ranges from 0.8 to 2.9 m⁻¹ in the surface layer, and model results suggest that it may be up to 5.8 m⁻¹ in the optically dense layer. This forms a barrier for light penetration into the hypolimnion.     

X-ray structure of human acid-beta-glucosidase covalently bound to conduritol-B-epoxide. Implications for Gaucher disease

Gaucher disease is an inherited metabolic disorder caused by mutations in the lysosomal enzyme acid-beta-glucosidase (GlcCerase). We recently determined the x-ray structure of GlcCerase to 2.0 A resolution (Dvir, H., Harel, M., McCarthy, A. A., Toker, L., Silman, I., Futerman, A. H., and Sussman, J. L. (2003) EMBO Rep.4, 704-709) and have now solved the structure of Glc-Cerase conjugated with an irreversible inhibitor, conduritol-B-epoxide (CBE). The crystal structure reveals that binding of CBE to the active site does not induce a global conformational change in GlcCerase and confirms that Glu340 is the catalytic nucleophile. However, only one of two alternative conformations of a pair of flexible loops (residues 345-349 and 394-399) located at the entrance to the active site in native GlcCerase is observed in the GlcCerase-CBE structure, a conformation in which the active site is accessible to CBE. Analysis of the dynamics of these two alternative conformations suggests that the two loops act as a lid at the entrance to the active site. This possibility is supported by a cluster of mutations in loop 394-399 that cause Gaucher disease by reducing catalytic activity. Moreover, in silico mutational analysis demonstrates that all these mutations stabilize the conformation that limits access to the active site, thus providing a mechanistic explanation of how mutations in this loop result in Gaucher disease.     

CD21/CD19 coreceptor signaling promotes B cell survival during primary immune responses

The adaptive immune response is tightly regulated to limit responding cells in an Ag-specific manner. On B cells, coreceptors CD21/CD19 modulate the strength of BCR signals, potentially influencing cell fate. The importance of the CD95 pathway was examined in response of B cells to moderate affinity Ag using an adoptive transfer model of lysozyme-specific Ig transgenic (HEL immunoglobulin transgene (MD4) strain) B cells. Although adoptively transferred Cr2+/+ MD4 B cells are activated and persist within splenic follicles of duck egg lysozyme-immunized mice, Cr2-/- MD4 B cells do not. In contrast, Cr2-/- MD4 lpr B cells persist after transfer, suggesting that lack of CD21/CD35 signaling results in CD95-mediated elimination. Cr2 deficiency did not affect CD95 levels, but cellular FLIP (c-FLIP) protein and mRNA levels were reduced 2-fold compared with levels in Cr2+/+ MD4 B cells. In vitro culture with Cr2+/+ MD4 B cells demonstrated that equimolar amounts of rHEL-C3d3 were more effective than hen egg lysozyme alone in up-regulating c-FLIP levels and for protection against CD95-mediated apoptosis. Collectively, this study implies a mechanism for regulating B cell survival in vivo whereby the strength of BCR signaling (including coreceptor) determines c-FLIP levels and protection from CD95-induced death.     

Factor X activator from Vipera lebetina venom is synthesized from different genes

Vipera lebetina venom contains specific coagulant Factor X activator (VLFXA) that cleaves the Arg52-Ile53 bond in the heavy chain of human factor X. VLFXA is a glycoprotein that is composed of a heavy chain (HC) and two light chains (LC) linked by disulfide bonds. The complete amino acid sequences of the three chains of the factor X activator from V. lebetina snake venom are deduced from the nucleotide sequences of cDNAs encoding these chains. The full-length cDNA (2347 bp) sequence of the HC encodes an open reading frame (ORF) of 612 amino acids that includes signal peptide, propeptide and mature metalloproteinase with disintegrin-like and cysteine-rich domains. The light chain LC1 contains 123 and LC2 135 amino acid residues. Both light chains belong to the class of C-type lectin-like proteins. The N-termini of VLFXA chains and inner sequences of peptide fragments detected by liquid chromatography-electrospray ionization tandem mass spectrometry (LC MS/MS) from protein sequence are 100% identical to the sequences deduced from the cDNA. The molecular masses of tryptic fragments of VLFXA chains analyzed by matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) also confirm the protein sequences deduced from the cDNAs. These are the first cloned factor X activator heavy and light chains. We demonstrate that the heavy and light chains are synthesized from different genes.     

Amino acid starvation induced autophagic cell death in PC-12 cells: Evidence for activation of caspase-3 but not calpain-1

While the apoptotic and necrotic cell death pathways have been well studied, there lacks a comprehensive understanding of the molecular events involving autophagic cell death. We examined the potential roles of the apoptosis-linked caspase-3 and the necrosis/apoptosis-linked calpain-1 after autophagy induction under prolonged amino acid (AA) starvation conditions in PC-12 cells. Autophagy induction was observed as early as three hours following amino acid withdrawal. Cell death, measured by lactate dehydrogenase (LDH) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays occurred within 24 h following starvation and was accompanied by an upregulation in caspase-3 activity but not calpain-1. The cell death that occurred following AA starvation was significantly alleviated by treatment with the autophagy inhibitor 3-methyl adenine but not with the broad spectrum caspase inhibitors. Thus, this study demonstrates that 3-methyladenine-sensitive autophagic cell death due to AA starvation in PC-12 cells is mechanistically and biochemically similar to, yet distinct from, classic caspase dependent apoptosis. Shankar Sadasivan and Anu Waghray have contributed equally to this work.