Substituted aminobenzimidazole pyrimidines as cyclin-dependent kinase inhibitors

A series of aminobenzimidazole-substituted pyrimidines were synthesized and evaluated for biochemical activity against CDK1. A high-speed parallel synthesis approach enabled the identification of a potent lead series having improved potency in the CDK1 assay (IC(50)<10nM). Cell cycle analysis showed that the compounds induced a G2/M block. Docking studies were carried out with a CDK1 homology model, and provide a rationale for the observed activities.     

Variation in seed protein and isoenzyme patterns in Cucurbita cultivars

The genetic variability in the seed proteins and the enzyme alcohol dehydrogenase (ADH) in representative species of the genus Cucurbita was studied. The banding patterns were obtained by means of vertical block electrophoresis in polyacrylamide gel. A specific protein components and ADH isoenzymes were established in the polymorphic banding patterns which can be applied individually or in combination as potential biochemical markers for breeding purposes.     

Inhibition of sulfur mustard-increased protease activity by niacinamide,N-acetyl-L-cysteine or dexamethasone

The pathologic mechanisms underlying sulfur mustard-induced skin vesication remain undefined. Papirmeister et al. (1985) have postulated a biochemical mechanism for sulfur mustard-induced cutaneous injury involving DNA alkylation, metabolic disruption, and enhanced proteolysic activity. We have previously utilized a chromogenic peptide substrate assay to establish that human peripheral blood lymphocytes exposed to sulfur mustard exhibited enhanced proteolytic activity. In this study, compounds known to alter the biochemical events associated with sulfur mustard exposure or to reduce protease activity were tested for their ability to block the sulfur mustard-increased proteolysis. Treatment of cells with niacinamide, N-acetyl-L-cysteine, or dexamethasone resulted in a decrease of sulfur mustard-increased protease activity. Complete inhibition of sulfur mustard-increased proteolysis was achieved by using protease inhibitors (antipain, leupeptin, and 4-(2-aminoethyl)-benzenesulfonylfluoride). These data suggest that therapeutic intervention in the biochemical pathways that culminate in protease activation or direct inhibition of proteolysis might serve as an approach to the treatment of sulfur mustard-induced pathology.Abbreviations APMSF 4-(2-aminoethyl)-benzenesulfonylfluoride, HCI - CPSPA Chromogenic Peptide Substrate Protease Assay - EDTA ethylenediaminetetraacetic acid - HD sulfur mustard - PBL human peripheral blood lymphocytes - pNA p-nitroaniline     

Adaptive tolerance and clonal anergy are distinct biochemical states

Adaptive tolerance is a process by which T cells become desensitized when Ag stimulation persists following an initial immune response in vivo. To examine the biochemical changes in TCR signaling present in this state, we used a mouse model in which Rag2(-/-) TCR-transgenic CD4(+) T cells were transferred into CD3epsilon(-/-) recipients expressing their cognate Ag. Compared with naive T cells, adaptively tolerant T cells had normal levels of TCR and slightly increased levels of CD4. Following activation with anti-TCR and anti-CD4 mAbs, the predominant signaling block in the tolerant cells was at the level of Zap70 kinase activity, which was decreased 75% in vitro. Phosphorylations of the Zap70 substrates (linker of activated T cells and phospholipase Cgamma1 were also profoundly diminished. This proximal defect impacted mostly on the calcium/NFAT and NF-kappaB pathways, with only a modest decrease in ERK1/2 phosphorylation. This state was contrasted with T cell clonal anergy in which the RAS/MAPK pathway was preferentially impaired and there was much less inhibition of Zap70 kinase activity. Both hyporesponsive states manifested a block in IkappaB degradation. These results demonstrate that T cell adaptive tolerance and clonal anergy are distinct biochemical states, possibly providing T cells with two molecular mechanisms to curtail responsiveness in different biological circumstances.     

Parasitoid wasp sting: a cocktail of GABA, taurine, and beta-alanine opens chloride channels for central synaptic block and transient paralysis of a cockroach host

The wasp Ampulex compressa injects venom directly into the prothoracic ganglion of its cockroach host to induce a transient paralysis of the front legs. To identify the biochemical basis for this paralysis, we separated venom components according to molecular size and tested fractions for inhibition of synaptic transmission at the cockroach cercal-giant synapse. Only fractions in the low molecular weight range (<2 kDa) caused synaptic block. Dabsylation of venom components and analysis by HPLC and MALDI-TOF-MS revealed high levels of GABA (25 mM), and its receptor agonists beta-alanine (18 mM), and taurine (9 mM) in the active fractions. Each component produces transient block of synaptic transmission at the cercal-giant synapse and block of efferent motor output from the prothoracic ganglion, which mimics effects produced by injection of whole venom. Whole venom evokes picrotoxin-sensitive chloride currents in cockroach central neurons, consistent with a GABAergic action. Together these data demonstrate that Ampulex utilizes GABAergic chloride channel activation as a strategy for central synaptic block to induce transient and focal leg paralysis in its host.     

Mapping of chromosome loci determined manifestation of morphological and biochemical traits of quality in <Emphasis Type="Italic">Brassica rapa</Emphasis> L. crops

Mapping chromosomal loci responsible for seven morphological and five biochemical traits of quality in populations of doubled haploid lines of leafy, rooted, and oilseed crops of Brassica rapa L. species was performed for the first time. In total, 140 quantitative trait loci (QTLs) were mapped determining manifestation of the studied morphological and biochemical economically important traits of quality under field and greenhouse conditions; the control of the same traits under different growth conditions or simultaneous control of several traits by one locus were noted. Molecular markers genetically linked with the selected QTLs were detected. The block genomic structure of the genetic components (chromosomal loci and linkage groups) determining manifestation of morphological and biochemical traits of quality that make B. rapa plants nutritionally valuable is discussed for the first time. The conclusion is drawn that the revealed QTLs and identified molecular markers may be of interest for the forthcoming study of genetic control by them of economically important characters and for the marker assisted selection in B. rapa.     

Adenosine kinase deficiency disrupts the methionine cycle and causes hypermethioninemia, encephalopathy, and abnormal liver function

Four inborn errors of metabolism (IEMs) are known to cause hypermethioninemia by directly interfering with the methionine cycle. Hypermethioninemia is occasionally discovered incidentally, but it is often disregarded as an unspecific finding, particularly if liver disease is involved. In many individuals the hypermethioninemia resolves without further deterioration, but it can also represent an early sign of a severe, progressive neurodevelopmental disorder. Further investigation of unclear hypermethioninemia is therefore important. We studied two siblings affected by severe developmental delay and liver dysfunction. Biochemical analysis revealed increased plasma levels of methionine, S-adenosylmethionine (AdoMet), and S-adenosylhomocysteine (AdoHcy) but normal or mildly elevated homocysteine (Hcy) levels, indicating a block in the methionine cycle. We excluded S-adenosylhomocysteine hydrolase (SAHH) deficiency, which causes a similar biochemical phenotype, by using genetic and biochemical techniques and hypothesized that there was a functional block in the SAHH enzyme as a result of a recessive mutation in a different gene. Using exome sequencing, we identified a homozygous c.902C>A (p.Ala301Glu) missense mutation in the adenosine kinase gene (ADK), the function of which fits perfectly with this hypothesis. Increased urinary adenosine excretion confirmed ADK deficiency in the siblings. Four additional individuals from two unrelated families with a similar presentation were identified and shown to have a homozygous c.653A>C (p.Asp218Ala) and c.38G>A (p.Gly13Glu) mutation, respectively, in the same gene. All three missense mutations were deleterious, as shown by activity measurements on recombinant enzymes. ADK deficiency is a previously undescribed, severe IEM shedding light on a functional link between the methionine cycle and adenosine metabolism.     

Cell Cycle Synchronization of the Murine EO771 Cell Line Using Double Thymidine Block Treatment

This study shows that double thymidine block treatment efficiently arrests the EO771 cells in the S-phase without altering cell growth or survival. A long-term analysis of cell behavior, using 5(6)-carboxyfluorescein diacetate N-succinimidyl ester (CFSE) staining, show synchronization to be stable and consistent over time. The EO771 cell line is a medullary breast-adenocarcinoma cell line isolated from a spontaneous murine mammary tumor, and can be used to generate murine tumor implantation models. Different biological (serum or amino acid deprivation), physical (elutriation, mitotic shake-off), or chemical (colchicine, nocodazole, thymidine) treatments are widely used for cell synchronization. Of the different methods tested, the double thymidine block is the most efficient for synchronization of murine EO771 cells if a large quantity of highly synchronized cells is recommended to study functional and biochemical events occurring in specific points of cell cycle progression.     

Changes of cyanide content and linamarase activity in wounded cassava roots

When cassava (Manihot esculenta Crantz) root was cut into blocks and incubated under laboratory conditions, the blocks showed more widespread and more even symptoms of physiological deterioration than those under natural conditions. Thus, the tissue block system has potential for biochemical studies of natural deterioration of cassava root. The changes in cyanide content and linamarase (linamarin β-d-glucoside glucohydrolase; EC 3.2.1.21) activity in various tissues during physiological deterioration were investigated. Total cyanide content increased in all parts of block tissue after 3-day incubation. The degree of increase in cyanide was most pronounced in white parenchymal tissue, 2 to 3 millimeters thick, next to the cortex (A-part tissue), where no physiological symptoms appeared. On the other hand, linamarase activity was decreased in all parts of block tissue after a 3-day incubation. A time course analysis of A-part tissue indicated a clear reciprocal relationship between changes in total cyanide and linamarase activity; total cyanide increased, while linamarase activity decreased. Free cyanide constituted a very small portion of the total cyanide and did not change markedly.     

The effect of inhibitors of DNA synthesis on 40-kilodalton polypeptide translation in rat L6 myoblasts

Messenger RNA coding for a polypeptide of 40 kilodaltons (P40) was translated in proliferating rat L6 myoblasts but not in the terminally differentiated myotubes. The relationship between DNA synthesis, differentiation, and P40 mRNA translation was studied. Aphidicolin, a reversible inhibitor of DNA synthesis, was shown to block DNA synthesis in proliferating myoblasts without allowing these cells to differentiate. A second inhibitor, cytosine arabinoside, when added to dividing myoblasts also prevented differentiation. In the absence of biochemical differentiation P40 mRNA remained in the translated state. Translational repression of this mRNA was, therefore, linked to the biochemical differentiation of rat L6 myoblasts.     

Anti-tubulin immunofluorescence studies of recovery from vinblastine cytotoxicity in Chinese hamster cells

Antitubulin immunofluorescent staining was used to examine the relationship among crystal formation, mitotic arrest, and recovery potential in vinblastine-treated Chinese hamster cells. Although vinblastine caused a mitotic block at concentrations as low as 5 x 10(-9) M, it induced tubulin crystal formation only at concentrations higher than 10(-6) M. At these higher concentrations, cells took 48-72 h to recover after return to normal medium. This extended period of time was apparently needed for breakdown of the crystals and regeneration of normal cytoplasmic microtubules. At concentrations less than 10(-6) M, although the mitotic block was still effective, no crystals were present. Possibly because of this lack of crystal formation, the cells recovered rapidly, generating cytoplasmic microtubules within 30 min, and beginning to undergo mitosis within 60 min. These findings tend to support biochemical evidence that tubulin binds to vinblastine at two types of binding site: a high affinity, low capacity site, responsible for tubulin disaggregation; and a low affinity site, responsible for protofilament splaying.     

Metabolism,cellular actions,and cytotoxicity of selenomethionine in cultured cells

Selenomethionine metabolism and the biochemical basis for its cytotoxicity were analyzed in cultured human and murine lymphoid cells. The metabolic pathways were also addressed, using purified mammalian enzymes and crude tissue extracts. Selenomethionine was found to be effectively metabolized to S-adenosylmethionine analog, and that analog was further metabolized in transmethylation reactions and in polyamine synthesis, similarly to the corresponding sulphur metabolites of methionine. Selenomethionine did not block these pathways, nor was there a specific block on the synthesis of DNA, RNA, or proteins when added to the culture medium. Selenomethionine showed cytotoxicity at above 40 microM levels. Yet, low selenomethionine levels (10 microM) could replace methionine and support cell growth in the absence of methionine. Selenomethionine toxicity took place concomitantly with changes in S-adenosylmethionine pools. D-form was less cytotoxic than L-form. Methionine concentration modified the cytotoxicity. Together, this indicates that selenomethionine uptake and enzymic metabolism are involved in the cytotoxicity in a yet unknown way.     

Can differences in microbial abundances help explain enhanced N2O emissions in a permanent grassland under elevated atmospheric CO2?

Long‐term effects of elevated atmospheric CO₂ on the ammonia‐oxidizing and denitrifying bacteria in a grassland soil were investigated to test whether a shift in abundance of these N‐cycling microorganisms was responsible for enhanced N₂O emissions under elevated atmospheric CO₂. Soil samples (7.5 cm increments to 45 cm depth) were collected in 2008 from the University of Giessen Free Air Carbon dioxide Enrichment (GiFACE), a permanent grassland exposed to moderately elevated atmospheric CO₂ (+20%) since 1998. GiFACE plots lay on a soil moisture gradient because of gradually changing depth to the underlying water table and labeled as the DRY block (furthest from water table), MED block (intermediate to water table), and WET block (nearest to water table). Mean N₂O emissions measured since 1998 have been significantly higher under elevated CO₂. This study sought to identify microbial and biochemical parameters that might explain higher N₂O emissions under elevated CO₂. Soil biochemical parameters [extractable organic carbon (EOC), dissolved organic nitrogen (DON), NH₄⁺, NO₃^?], and abundances of genes encoding the key enzymes involved in ammonia oxidation (amoA) and denitrification (nirK, nirS, nosZ) depended more on soil depth and block (underlying soil moisture gradient) than on elevated CO₂. Ammonia oxidation and denitrification gene abundances, relative abundances (ratios) of nirS to nirK, of nosZ to both nirS and to nirK, and of the measured soil biochemical properties DON and NO₃^? tended to be lower in elevated CO₂ plots as compared with ambient plots in the MED and WET blocks while the DRY block exhibited an opposite trend. High N₂O emissions under elevated CO₂ in the MED and WET blocks correlated with lower nosZ to nirK ratios, suggesting that increased N₂O emissions under elevated CO₂ might be caused by a higher proportion of N₂O‐producing rather than N₂O consuming (N₂ producing) denitrifiers.     

Study of individual Pacinian corpuscle mechanoreceptors following application of colchicine to a nerve]

Colchicine application to the cat caudal mesenteric nerve containing sensory fibers for single mechanoreceptors (Pacinian corpuscles) causes degeneration of the axis cast of the nerve endings. Ultrastructural changes in the receptors showed no difference from the axonal degeneration after the nerve section but the rate of degeneration was considerably slower. Ultrastructural, electrophysiological, and biochemical changes occurring in the Pacinian corpuscles were not the result of direct action of colchicine, but appeared to be realized through the nerve by the axoplasmic transport block. It is suggested that the receptor's structure is under the sensory neuron neurotrophic control.     

A dominant-negative approach that prevents diphthamide formation confers resistance to Pseudomonas exotoxin A and diphtheria toxin

Diphtheria toxin (DT), Pseudomonas aeruginosa Exotoxin A (ETA) and cholix toxin from Vibrio cholerae share the same mechanism of toxicity; these enzymes ADP-rybosylate elongation factor-2 (EF-2) on a modified histidine residue called diphthamide, leading to a block in protein synthesis. Mutant Chinese hamster ovary cells that are defective in the formation of diphthamide have no distinct phenotype except their resistance to DT and ETA. These observations led us to predict that a strategy that prevents the formation of diphthamide to confer DT and ETA resistance is likely to be safe. It is well documented that Dph1 and Dph2 are involved in the first biochemical step of diphthamide formation and that these two proteins interact with each other. We hypothesized that we could block diphthamide formation with a dominant negative mutant of either Dph1 or Dph2. We report in this study the first cellular-targeted strategy that protects against DT and ETA toxicity. We have generated Dph2(C-), a dominant-negative mutant of Dph2, that could block very efficiently the formation of diphthamide. Cells expressing Dph2(C-) were 1000-fold more resistant to DT than parental cells, and a similar protection against Pseudomonas exotoxin A was also obtained. The targeting of a cellular component with this approach should have a reduced risk of generating resistance as it is commonly seen with antibiotic treatments.     

Cannabinoid receptors CB1 and CB2 form functional heteromers in brain

Exploring the role of cannabinoid CB(2) receptors in the brain, we present evidence of CB(2) receptor molecular and functional interaction with cannabinoid CB(1) receptors. Using biophysical and biochemical approaches, we discovered that CB(2) receptors can form heteromers with CB(1) receptors in transfected neuronal cells and in rat brain pineal gland, nucleus accumbens, and globus pallidus. Within CB(1)-CB(2) receptor heteromers expressed in a neuronal cell model, agonist co-activation of CB(1) and CB(2) receptors resulted in a negative cross-talk in Akt phosphorylation and neurite outgrowth. Moreover, one specific characteristic of CB(1)-CB(2) receptor heteromers consists of both the ability of CB(1) receptor antagonists to block the effect of CB(2) receptor agonists and, conversely, the ability of CB(2) receptor antagonists to block the effect of CB(1) receptor agonists, showing a bidirectional cross-antagonism phenomenon. Taken together, these data illuminate the mechanism by which CB(2) receptors can negatively modulate CB(1) receptor function.     

Morphine inhibits cyclic AMP-dependent protein kinase and ornithine decarboxylase activities in neuroblastoma X glioma hybrid cells.

The effect of morphine on neuroblastoma x glioma hybrid cells is not limited to the inhibition of adenylate cyclase activity. It is demonstrated that in morphine-treated cells there is a marked reduction in the activity of both cyclic AMP-dependent protein kinase and ornithine decarboxylase (ODC) in response to stimulation by PGE₁. The effect of morphine is to block a cascade of events which may be crucial for the normal biochemical processes in these cells.     

Lesion bypass activities of human DNA polymerase mu

DNA polymerase mu (Polmu) is a newly discovered member of the polymerase X family with unknown cellular function. The understanding of Polmu function should be facilitated by an understanding of its biochemical activities. By using purified human Polmu for biochemical analyses, we discovered the lesion bypass activities of this polymerase in response to several types of DNA damage. When it encountered a template 8-oxoguanine, abasic site, or 1,N(6)-ethenoadenine, purified human Polmu efficiently bypassed the lesion. Even bulky DNA adducts such as N-2-acetylaminofluorene-adducted guanine, (+)- and (-)-trans-anti-benzo[a]pyrene-N(2)-dG were unable to block the polymerase activity of human Polmu. Bypass of these simple base damage and bulky adducts was predominantly achieved by human Polmu through a deletion mechanism. The Polmu specificity of nucleotide incorporation indicates that the deletion resulted from primer realignment before translesion synthesis. Purified human Polmu also effectively bypassed a template cis-syn TT dimer. However, this bypass was achieved in a mainly error-free manner with AA incorporation opposite the TT dimer. These results provide new insights into the biochemistry of human Polmu and show that efficient translesion synthesis activity is not strictly confined to the Y family polymerases.     

Parasitoid wasp sting: A cocktail of GABA,taurine, and β‐alanine opens chloride channels for central synaptic block and transient paralysis of a cockroach host

The wasp Ampulex compressa injects venom directly into the prothoracic ganglion of its cockroach host to induce a transient paralysis of the front legs. To identify the biochemical basis for this paralysis, we separated venom components according to molecular size and tested fractions for inhibition of synaptic transmission at the cockroach cercal‐giant synapse. Only fractions in the low molecular weight range (<2 kDa) caused synaptic block. Dabsylation of venom components and analysis by HPLC and MALDI‐TOF‐MS revealed high levels of GABA (25 mM), and its receptor agonists β‐alanine (18 mM), and taurine (9 mM) in the active fractions. Each component produces transient block of synaptic transmission at the cercal‐giant synapse and block of efferent motor output from the prothoracic ganglion, which mimics effects produced by injection of whole venom. Whole venom evokes picrotoxin‐sensitive chloride currents in cockroach central neurons, consistent with a GABAergic action. Together these data demonstrate that Ampulex utilizes GABAergic chloride channel activation as a strategy for central synaptic block to induce transient and focal leg paralysis in its host. © 2006 Wiley Periodicals, Inc. © 2006 Wiley Periodicals, Inc. J Neurobiol, 2006