Enhancing venetoclax activity in acute myeloid leukemia by co-targeting MCL1.

Targeted therapies are frequently combined with standard cytotoxic drugs to enhance clinical response. Targeting the B-cell lymphoma 2 (BCL-2) family of proteins is an attractive option to combat chemoresistance in leukemia. Preclinical and clinical studies indicate modest single-agent activity with selective BCL-2 inhibitors (for example, venetoclax). We show that venetoclax synergizes with cytarabine and idarubicin to increase antileukemic efficacy in a TP53-dependent manner. Although TP53 deficiency impaired sensitivity to combined venetoclax and chemotherapy, higher-dose idarubicin was able to suppress MCL1 and induce cell death independently of TP53. Consistent with an MCL1-specific effect, cell death from high-dose idarubicin was dependent on pro-apoptotic Bak. Combining higher-dose idarubicin with venetoclax was able to partially overcome resistance in Bak-deficient cells. Using inducible vectors and venetoclax to differentially target anti-apoptotic BCL-2 family members, BCL-2 and MCL1 emerged as critical and complementary proteins regulating cell survival in acute myeloid leukemia. Dual targeting of BCL-2 and MCL1, but not either alone, prolonged survival of leukemia-bearing mice. In conclusion, our findings support the further investigation of venetoclax in combination with standard chemotherapy, including intensified doses of idarubicin. Venetoclax should also be investigated in combination with direct inhibitors of MCL1 as a chemotherapy-free approach in the future.

A comparative study of metal contamination in soil using the borehole method.

The present study deals with possible contamination of the soil by metal ions which have been affecting the environment. The concentrations of metal ions in 14 borehole samples were studied using the ICP-OES standard method. The degree of contamination was determined on the basis of single element pollution index (SEPI), combined pollution index (CPI), soil enrichment factor (SEF), and geo-accumulation index (Igeo). Geo-accumulation indices and contamination factors indicated moderate to strong contaminations for eight boreholes (BL-1, BL-2, BL-6, BL-8, BL-9, BL-10, BL-12, and BL-13) while the rest were extremely contaminated. Among all the boreholes, BL-3 and BL-11 demonstrated the highest level of Cd(II) and Pb(II) which were found the most polluted sites. The level of metal contamination was also compared with other countries. The development, variation, and limitations regarding the regulations of soil and groundwater contamination can be provided as a helpful guidance for the risk assessment of metal ions in developing countries.

How do consumer attitudes influence acceptance of a novel wild blueberry-soy product?

Acceptance of healthful foods by consumers is not yet well understood. In this study, 3 formulations of frozen dessert bars were prepared containing both soy and wild blueberries. Soy content was controlled to provide an amount of soy protein that qualified for the health claim for soy and reduced risks for cardiovascular disease. Consumers were asked to complete the Health and Taste Attitude Scales (HTAS) and then evaluate the acceptability of the 3 frozen bar types using a 9-point hedonic scale. One week after the 1st session, the participants returned. Approximately half were given information to read regarding the health benefits of soy protein, the other participants were given no information. The samples were then presented a 2nd time and labeled with their soy protein content. Changes in hedonic scores between sessions were compared and correlated with HTAS ratings. Nutrition information generally did not affect acceptability scores.

Biophysical characterization of interactions involving importin-alpha during nuclear import.

Proteins containing the classical nuclear localization sequences (NLSs) are imported into the nucleus by the importin-alpha/beta heterodimer. Importin-alpha contains the NLS binding site, whereas importin-beta mediates the translocation through the nuclear pore. We characterized the interactions involving importin-alpha during nuclear import using a combination of biophysical techniques (biosensor, crystallography, sedimentation equilibrium, electrophoresis, and circular dichroism). Importin-alpha is shown to exist in a monomeric autoinhibited state (association with NLSs undetectable by biosensor). Association with importin-beta (stoichiometry, 1:1; K(D) = 1.1 x 10(-8) m) increases the affinity for NLSs; the importin-alpha/beta complex binds representative monopartite NLS (simian virus 40 large T-antigen) and bipartite NLS (nucleoplasmin) with affinities (K(D) = 3.5 x 10(-8) m and 4.8 x 10(-8) m, respectively) comparable with those of a truncated importin-alpha lacking the autoinhibitory domain (T-antigen NLS, K(D) = 1.7 x 10(-8) m; nucleoplasmin NLS, K(D) = 1.4 x 10(-8) m). The autoinhibitory domain (as a separate peptide) binds the truncated importin-alpha, and the crystal structure of the complex resembles the structure of full-length importin-alpha. Our results support the model of regulation of nuclear import mediated by the intrasteric autoregulatory sequence of importin-alpha and provide a quantitative description of the binding and regulatory steps during nuclear import.

Essential role of the N-terminal autoregulatory sequence in the regulation of phenylalanine hydroxylase.

Phenylalanine hydroxylase (PAH) is activated by its substrate phenylalanine and inhibited by its cofactor tetrahydrobiopterin (BH(4)). The crystal structure of PAH revealed that the N-terminal sequence of the enzyme (residues 19-29) partially covered the enzyme active site, and suggested its involvement in regulation. We show that the protein lacking this N-terminal sequence does not require activation by phenylalanine, shows an altered structural response to phenylalanine, and is not inhibited by BH(4). Our data support the model where the N-terminal sequence of PAH acts as an intrasteric autoregulatory sequence, responsible for transmitting the effect of phenylalanine activation to the active site.

Sevoflurane induction in the elderly.

The effects of 2% and 4% sevoflurane in oxygen and nitrous oxide for induction of anaesthesia in 60 unpremedicated elderly patients was compared to those obtained during an intravenous Thiopentone induction. Intravenous induction induced anaesthesia in 27 +/- 5 seconds, significantly faster than a 2% or 4% sevoflurane induction (109 +/- 36 and 71 +/- 24 seconds respectively). One patient in both the thiopentone and 2% sevoflurane groups, and 2 patients in the 4% sevoflurane group coughed during induction. The postinduction reduction in mean arterial pressure was greatest in the thiopentone group followed by the 4% and the 2% sevoflurane groups. Heart rate changes were minimal in all groups. We conclude that 2% or 4% sevoflurane offered suitable conditions for induction of anaesthesia in the elderly with minimal cardiovascular derangement.

Protective and nonprotective epitopes from amino termini of M proteins from Australian aboriginal isolates and reference strains of group A streptococci.

The M protein is the primary vaccine candidate to prevent group A streptococcal (GAS) infection and the subsequent development of rheumatic fever (RF). However, the large number of serotypes have made it difficult to design a vaccine against all strains. We have taken an approach of identifying amino-terminal M protein epitopes from GAS isolates that are highly prevalent in GAS-endemic populations within the Northern Territory (NT) of Australia. Australian Aboriginals in the NT experience the highest incidence of RF worldwide. To develop a vaccine for this population, 39 peptides were synthesized, representing the amino-terminal region of the M protein from endemic GAS. Mice immunized with these peptides covalently linked to tetanus toxoid and emulsified in complete Freund's adjuvant raised high-titer antibodies. Over half of these sera reduced bacterial colony counts by >80% against the homologous isolate of GAS. Seven of the peptide antisera also cross-reacted with at least three other heterologous peptides by enzyme-linked immunosorbent assay. Antiserum to one peptide, BSA10(1-28), could recognize six other peptides, and five of these peptides could inhibit opsonization mediated by BSA10(1-28) antiserum. Cross-opsonization studies showed that six of these sera could opsonize at least one heterologous isolate of GAS. These data reveal vaccine candidates specific to a GAS-endemic area and show the potential of some to cross-opsonize multiple isolates of GAS. This information will be critical when considering which epitopes may be useful in a multiepitope vaccine to prevent GAS infection.

FHA domain boundaries of the dun1p and rad53p cell cycle checkpoint kinases.

Dun1p and Rad53p of the budding yeast Saccharomyces cerevisiae are members of a conserved family of cell cycle checkpoint protein kinases that contain forkhead-associated (FHA) domains. Here, we demonstrate that these FHA domains contain 130-140 residues, and are thus considerably larger than previously predicted by sequence comparisons (55-75 residues). In vivo, expression of the proteolytically defined Dun1p FHA domain, but not a fragment containing only the predicted domain boundaries, inhibited the transcriptional induction of repair genes following replication blocks. This indicates that the non-catalytic FHA domain plays an important role in the transcriptional function of the Dun1p protein kinase.

Structural basis of recognition of monopartite and bipartite nuclear localization sequences by mammalian importin-alpha.

Importin-alpha is the nuclear import receptor that recognizes cargo proteins which contain classical monopartite and bipartite nuclear localization sequences (NLSs), and facilitates their transport into the nucleus. To determine the structural basis of the recognition of the two classes of NLSs by mammalian importin-alpha, we co-crystallized an N-terminally truncated mouse receptor protein with peptides corresponding to the monopartite NLS from the simian virus 40 (SV40) large T-antigen, and the bipartite NLS from nucleoplasmin. We show that the monopartite SV40 large T-antigen NLS binds to two binding sites on the receptor, similar to what was observed in yeast importin-alpha. The nucleoplasmin NLS-importin-alpha complex shows, for the first time, the mode of binding of bipartite NLSs to the receptor. The two basic clusters in the NLS occupy the two binding sites used by the monopartite NLS, while the sequence linking the two basic clusters is poorly ordered, consistent with its tolerance to mutations. The structures explain the structural basis for binding of diverse NLSs to the sole receptor protein.

The effect of polishing systems on microleakage of tooth coloured restoratives: Part 1. Conventional and resin-modified glass-ionomer cements.

The purpose of this in vitro study was to investigate the effect of polishing systems on the microleakage of conventional and resin-modified glass-ionomer cements. Class V cavities were prepared at the cemento-enamel junction of 80 freshly extracted posterior teeth. The prepared teeth were randomly divided into two groups and restored with conventional or resin-modified glass-ionomer cements. The restored teeth were stored in distilled water at 37 degrees C for 1 week after removal of excess restorative with diamond finishing burs. The restored teeth were then divided into four groups of 10 and finished and polished using the following systems: Two Striper MFS; Sof-Lex XT; Enhance Composite Finishing and Polishing System; Shofu Composite Finishing Kit. The finished restorations were subjected to dye penetration testing. Results showed that the microleakage at dentin margins of conventional glass-ionomer cements and enamel margins of resin-modified glass-ionomer cements are significantly affected by the different polishing systems.

Turn up the HEAT.

The recently determined crystal structure of the PR65/A subunit of protein phosphatase 2A reveals the architecture of proteins containing HEAT repeats. The structural properties of this solenoid protein explain many functional characteristics and account for the involvement of solenoids as scaffold, anchoring and adaptor proteins.

Crystallization of importin alpha, the nuclear-import receptor.

Crystals of recombinant importin alpha, the nuclear-import receptor, have been obtained at two different pH conditions by vapour diffusion using sodium citrate as precipitant and dithiothreitol as an additive. At pH 4-5, the crystals have the symmetry of the trigonal space group P3121 or P3221 (a = b = 78.0, c = 255.8 A, gamma = 120 degrees ); at pH 6-7, the crystals have the symmetry of the orthorhombic space group P212121 (a = 78.5, b = 89.7, c = 100.5 A). In both cases, there is probably one molecule of importin alpha in the asymmetric unit. At least one of the crystal forms diffracts to a resolution higher than 3 A using the laboratory X-ray source; the crystals are suitable for crystal structure determination.

Crystallization of a trimeric human T cell leukemia virus type 1 gp21 ectodomain fragment as a chimera with maltose-binding protein.

We present a novel protein crystallization strategy, applied to the crystallization of human T cell leukemia virus type 1 (HTLV-1) transmembrane protein gp21 lacking the fusion peptide and the transmembrane domain, as a chimera with the Escherichia coli maltose binding protein (MBP). Crystals could not be obtained with a MBP/gp21 fusion protein in which fusion partners were separated by a flexible linker, but were obtained after connecting the MBP C-terminal alpha-helix to the predicted N-terminal alpha-helical sequence of gp21 via three alanine residues. The gp21 sequences conferred a trimeric structure to the soluble fusion proteins as assessed by sedimentation equilibrium and X-ray diffraction, consistent with the trimeric structures of other retroviral transmembrane proteins. The envelope protein precursor, gp62, is likewise trimeric when expressed in mammalian cells. Our results suggest that MBP may have a general application for the crystallization of proteins containing N-terminal alpha-helical sequences.

Mammalian AMP-activated protein kinase subfamily.

The mammalian 5'-AMP-activated protein kinase (AMPK) is related to a growing family of protein kinases in yeast and plants that are regulated by nutritional stress. We find the most prominent expressed form of the hepatic AMPK catalytic subunit (alpha 1) is distinct from the previously cloned kinase subunit (alpha 2). The alpha 1 (548 residues) and alpha 2 (552 residues) isoforms have 90% amino acid sequence identity within the catalytic core but only 61% identity elsewhere. The tissue distribution of the AMPK activity most closely parallels the low abundance 6-kilobase alpha 1 mRNA distribution and the alpha 1 immunoreactivity rather than alpha 2, with substantial amounts in kidney, liver, lung, heart, and brain. Both alpha 1 and alpha 2 isoforms are stimulated by AMP and contain noncatalytic beta and gamma subunits. The liver alpha 1 isoform accounts for approximately 94% of the enzyme activity measured using the SAMS peptide substrate. The tissue distribution of the alpha 2 immunoreactivity parallels the alpha 2 8.5-kilobase mRNA and is most prominent in skeletal muscle, heart, and liver. Isoforms of the beta and gamma subunits present in the human genome sequence reveal that the AMPK consists of a family of isoenzymes.

Insert regions in domain X of the casein kinase II catalytic subunit.

Casein kinase II, cyclin-dependent kinases, and glycogen synthase kinase-3 are members of the protein kinase subfamily with a prominent insert in domain X of their catalytic subunit sequence. The function of the insert sequence in casein kinase II was investigated utilising synthetic peptides corresponding to the insert, cross-linking experiments, and the generation of casein kinase II insert region mutants. The mutation of basic residues (R276-->A, R278-->A, R281-->A, K277-->A) within the major insert sequence (PRFHDILQRHSRKRWERFVHSDNQHL, positions 265-290) did not affect alpha/beta subunit association, enzyme tetramerisation, thermal stability, and peptide (RRRDDDSDDD-NH2) phosphorylation. Similarly, replacement of residues 276-290 within the major insert with the corresponding residues from the cell-cycle kinase cyclin-dependent kinase 2 (CDK2) (FPKWKPGSLASHVKN) had no significant effect. The mutation of charged residues (H232-->A, H234-->A, D235-->A) within a nearby minor insert sequence (HGHDNY, positions 232-237), or replacement of residues 234-237 with the corresponding residues from CDK2 (DSEI) also did not affect alpha/beta subunit association and tetramerisation, but reduced enzyme thermal stability to more closely resemble the stability of the isolated alpha-subunit. In addition, mutations within the minor insert caused approximately a threefold increase in the apparent Km for peptide substrate. The results indicate that the major and minor inserts are not essential for alpha/beta subunit association, but the minor insert region influences substrate binding and thermal stability.

Catalytic subunits of the porcine and rat 5'-AMP-activated protein kinase are members of the SNF1 protein kinase family.

The 5'-AMP-activated protein kinase (AMPK) regulates the fatty acid and sterol synthesizing pathways via phosphorylation of acetyl-CoA carboxylase and HMG-CoA reductase, respectively. Highly purified kinase from porcine liver contains three apparent subunits of molecular mass 63 kDa, 40 kDa and 38 kDa. Peptide sequencing of the 63 kDa protein (AMPK63cat) revealed that this polypeptide is the catalytic subunit of the kinase. Porcine peptide sequences were used to clone by RT-PCR partial length cDNAs for the catalytic domains of the porcine AMPK63cat, and its rat homolog, which were virtually identical in deduced amino acid sequence. Screening of a rat liver cDNA library with these partial length cDNAs and with degenerate oligonucleotides yielded several unique clones, some of which had a 142 bp deletion in the catalytic domain of the kinase. A consensus full-length sequence with a 1.7 kb open reading frame has been constructed from overlapping library and PCR-derived clones. A large mRNA for rat AMPK63cat (8.5 kb) is expressed in nearly all rat tissues, with highest levels detectable in heart and skeletal muscle. Using PCR, the presence of two mRNA species with or without the 142 bp deletion in the catalytic domain was noted in all rat tissues examined. Comparison of the deduced protein sequence of AMPK63cat reveals highly conserved homologies in both the catalytic and non-catalytic domains to several members of the SNF1 kinase family, including kinases from Arabidopsis, barley, rye, and S. cerevesiae, as well as to other mammalian kinases and to a C. elegans kinase. The high evolutionary conservation of both kinase structure and function (metabolite sensing) coupled with their pattern of tissue/organism expression suggest that the mammalian members of this kinase family likely play wider roles than the regulation of cellular lipid metabolism.

Influence of prepartum protein and energy concentrations for dairy goats during pregnancy and early lactation.

Sixty-three multiparous Alpine does were blocked by pregnancy type (single vs. multiple) on d 90 of pregnancy and assigned to one of nine diets to evaluate the interaction of prepartum protein and energy intake on BW change, kidding, and subsequent production and composition of milk. Treatments were factorial with three percentages of CP (8.5, 11.5, and 14.5% of DM) and three concentrations of metabolizable energy (1.80, 2.16, and 2.53 Mcal/kg of DM). Does were fed for ad libitum intake during pregnancy and switched to a lactation diet (16% CP and 2.35 Mcal of metabolizable energy/kg of DM) after parturition. Milk production and composition were recorded for the first 15 wk of lactation. Prepartum BW gain increased quadratically as protein amount increased but was unaffected by energy. Kidding rate, litter weight, and gestation length were unaffected by protein or energy amounts. Milk production in the subsequent lactation increased quadratically in response to prepartum CP (2.59, 3.26, and 3.07 kg/d for 8.5, 11.5, and 14.5% CP, respectively). Milk production increased linearly in response to prepartum metabolizable energy concentration (2.63, 3.05, and 3.26 kg/d for 1.80, 2.16, and 2.53 Mcal/kg of DM, respectively). Milk fat percentage increased linearly in response to increased prepartum energy. Production of milk fat, protein, SNF, FCM, and SCM were affected quadratically by increased prepartum CP and linearly by prepartum energy, following the pattern for milk production. The present recommendations for prepartum CP and energy appear to be adequate for gestation and subsequent lactation performance of dairy goats.

Mammalian 5'-AMP-activated protein kinase non-catalytic subunits are homologs of proteins that interact with yeast Snf1 protein kinase.

The 5'-AMP-activated protein kinase is responsible for the regulation of fatty acid synthesis by phosphorylation and inactivation of acetyl-CoA carboxylase. The porcine liver 5'-AMP-activated protein kinase 63-kDa catalytic subunit co-purifies 14,000-fold with a 38- and 40-kDa protein (Mitchelhill, K.I. et al. (1994) J. Biol. Chem. 269, 2361-2364). The 63-kDa subunit is homologous to the Saccharomyces cerevisiae Snf1 protein kinase, which regulates gene expression during glucose derepression. Peptide amino acid and polymerase chain reaction-derived partial cDNA sequences of both the pig and rat liver enzymes show that the 38-kDa protein is homologous to Snf4p (CAT3) and that the 40-kDa protein is homologous to the Sip1p/Spm/GAL83 family of Snf1p interacting proteins. Sucrose density gradient and cross-linking experiments with purified 5'-AMP-activated protein kinase suggest that both the 38- and 40-kDa proteins associate tightly with the 63-kDa catalytic polypeptide in either a heterotrimeric complex or in dimeric complexes. The 40-kDa subunit is autophosphorylated within the 63-kDa subunit complex. The sequence relationships between the mammalian 5'-AMP-activated protein kinase and yeast Snf1p extend to the subunit proteins consistent with conservation of the functional roles of these polypeptides in cellular regulation by this family of metabolite-sensing protein kinases.

Varying amounts of rumen-inert fat for high producing goats in early lactation.

This study determined the responses of early lactating goats fed varying amounts of rumen-inert fat. Forty multiparous high producing Alpine does in the first 2 wk of lactation were assigned randomly to four isonitrogenous dietary treatments containing 0, 3, 6, and 9% added fat. The study consisted of a 2-wk preliminary and a 10-wk experimental period. Feed intake, blood glucose, and rumen pH were not affected by dietary treatments. Body weight gain and milk production decreased linearly as dietary fat increased. Peak production was higher with 3% added fat than with 6 and 9%. The SCC, total solids, and lactose contents of milk were unaffected by fat percentage. Milk fat percentage increased linearly as fat percentage increased; milk protein content was highest at 6% dietary fat addition. The rumen-inert fat tended to increase long-chain fatty acids and to reduce short-chain fatty acids of milk. These findings suggest that fat supplementation at 3% of the total diet can increase fat percentage in milk from high producing dairy goats in early lactation.

Effect of source and amount of protein on milk production in dairy cows.

Forty multiparous Alpine does (mean BW of 61.5 kg) were utilized in a 13-wk trial to investigate the effects of a TMR differing in CP amount (13 or 17%) and source (solvent-extracted soybean meal or heat-treated soybean meal with or without urea) on lactational performance. Protein supplements contributed 30% of the N in 13% CP diets and 50% of the N in 17% CP diets. All diets were isoenergetic (2.5 Mcal of metabolizable energy/kg of DM) and were fed for ad libitum intake for the entire trial. Mean DMI (2.88 kg/d), milk production (2.65 kg/d), milk fat (4.05%), milk protein (2.68%), milk lactose (4.54%), and milk SNF (7.81%) did not differ among dietary treatments. Plasma urea N was greater (23.2 vs. 10.9 mg/dl) in does receiving the 17% CP diets; however, blood hematocrit (27.4%), beta-hydroxybutyrate (843 microM), plasma glucose (68.8 mg/dl), NEFA (600 mu eq/dl), and plasma total protein (74.% g/L) were not significantly affected by treatment. The apparent absence of a dietary effect on lactational performance may be due to the high DMI of the does (4.7% when expressed as DMI per kilogram of BW) and high CP intake providing a surfeit of protein relative to requirements.