Tyrosine phosphorylation of mitochondrial pyruvate dehydrogenase kinase 1 is important for cancer metabolism

Many tumor cells rely on aerobic glycolysis instead of oxidative phosphorylation for their continued proliferation and survival. Myc and HIF-1 are believed to promote such a metabolic switch by, in part, upregulating gene expression of pyruvate dehydrogenase (PDH) kinase 1 (PDHK1), which phosphorylates and inactivates mitochondrial PDH and consequently pyruvate dehydrogenase complex (PDC). Here we report that tyrosine phosphorylation enhances PDHK1 kinase activity by promoting ATP and PDC binding. Functional PDC can form in mitochondria outside of the matrix in some cancer cells and PDHK1 is commonly tyrosine phosphorylated in human cancers by diverse oncogenic tyrosine kinases localized to different mitochondrial compartments. Expression of phosphorylation-deficient, catalytic hypomorph PDHK1 mutants in cancer cells leads to decreased cell proliferation under hypoxia and increased oxidative phosphorylation with enhanced mitochondrial utilization of pyruvate and reduced tumor growth in xenograft nude mice. Together, tyrosine phosphorylation activates PDHK1 to promote the Warburg effect and tumor growth.     

Glycerol-induced formation of the molten globule from acid-denatured cytochrome c: implication for hierarchical folding

At high concentration (98% or higher, v/v), glycerol induces collapse of acid-denatured cytochrome c into a compact state, the G_U state, showing a molten globule character. The G_U state possesses a nativelike -helix structure but a tertiary conformation less packed with respect to the native state. The spectroscopic properties of the G_U state closely resemble those of the molten globule stabilized by the organic solvent from the native protein (called the G_N state), indicating that glycerol can stabilize the molten globule of cytochrome c either from the native or the acid-denatured protein. The G_U and the G_N states show spectroscopic (and, thus, structural) properties and stabilities comparable to those of molten globules stabilized by different effectors, despite the fact that the mechanisms involved in the molten globule formation may significantly differ. This implies in cytochrome c a hierarchy for the rupture (native-to-molten globule) or the formation (unfolded-to-molten globule) of intramolecular interactions leading to the stabilization of the molten globule state of the protein, independently from the effector responsible for the structural transition, in accord with the sequential model proposed by Englander and collaborators.     

Copper catechol-driven Fenton reactions and their potential role in wood degradation

Metals can potentially play a role in the non-enzymatic processes involved in wood biodegradation. Dihydroxybenzenes reduce Cu(II)–Cu(I), which then react with H₂O₂ driving a Fenton reaction. In this work the degradation of veratryl alcohol (VA), the simplest non-phenolic lignin model compound, via a cuprous Fenton reaction mediated by 1,2-dihydroxybenzene (catechol, CAT) was studied. A factorial experimental design was performed to assess the impact of several experimental variables including, pH, and CAT, CuCl₂ and H₂O₂ concentrations on VA degradation. Optimized conditions were determined using a response surface modeling methodology (RSM). The greatest amount of VA degradation occurred at a CAT:CuCl₂:H₂O₂ ratio of 0.287:0.313:4.062, a pH of 3.6. A time-course measurement for VA degradation was performed under these experimental conditions and after an 8 h reaction period, 31% of the VA was degraded. Under the same experimental conditions, VA degradation by an iron CAT-driven Fenton reaction was more effective than the copper CAT-driven Fenton reaction. In a similar experiment, carboxymethyl cellulose (CMC) depolymerization was also determined. Only the iron CAT-driven Fenton reaction was found to depolymerize CMC. We suggest that the greater redox potential of the Fe(III)CAT complex compared to the Cu(II)CAT complex would dictate that under most environmental conditions, degradation of VA would occur by the iron complex only. This research has important implications for the mechanisms of brown rot fungal degradation in wood because it eliminates a pathway that had previously been proposed as a mechanism explaining free radical generation in the oxidative depolymerization of cellulose in the cell wall.     

Endogenous abscisic acid during the germination of chick-pea seeds

Over the past twenty years many studies have been undertaken to elucidate the regulation of seed germination. Abscisic acid (ABA) and the gibberellins (GAs) are the hormones proposed to control this process, the first by inhibiting and the second by inducing germination. It has been proposed that a high water potential increases the growth potential of the embryo, presumably permitting the production or activation by GA of the cell wall hydrolases and thus decreasing the yield threshold of the endosperm close to the radicle tip. A low water potential, e.g., imbibition in an osmoticum. imposes a stress on cell metabolism, by reducing the turgor of the radicle cells, and there is a decrease in growth potential. Exogenous ABA also causes a decline in growth potential of the radicle: however, the actions of low water potential in preventing germination are not mediated through an increase in ABA in the seeds. In the present paper an attempt is made to asses the role of ABA and polyethylene glycol (PEG) in the germination of chick-pea (Cicer arietinum L.) seeds. The endogenous ABA of chick-pea seeds was purified by reversed-phase HPLC and quantified by GC-ECD. The variations in the ABA levels in the embryonic axes and the cotyledons were studied during 120 h. of imbibition. The highest ABA level in the embryome axes was found at 18 h. coinciding with an increase in fresh weight and a high germination percentage. ABA was not detected in the cotyledons during incubation which probably indicates that the hormone is more involved in the active growth of the embryonic axes itself than in the mobilization process of the reserves. When seeds were treated with different PEG-cycles. PEG delayed germination, reduced the fresh weight of embryonic axes, and retarded the onset of ABA synthesis. It is concluded that endogenous ABA is related to the onset of germination and the growth of the embryonic axis. In addition, there is no correlation among the different PEG-cycles and the level of ABA and germination. Germination was related more to the water conditions inside the embryo's cells than to ABA levels.     

Chemical Profile and Antioxidant Properties of Extracts and Essential Oils from Citrus × limon (L.) Burm. cv. Femminello Comune

Citrus × limon cv. Femminello Comune (Rutaceae) from Rocca Imperiale (Italy), one of the six Protected Geographical Indication (PGI) Italian lemon crops, has been recently received renewed interest. In this work, fresh and dried peels and leaves were extracted by hydrodistillation, supercritical fluid extraction (SFE), and Soxhlet apparatus. Chemical profile was assessed by gas chromatography (GC) and gas chromatography/mass spectrometry (GC/MS). Except for leaves extracts obtained by Soxhlet apparatus, the monoterpene hydrocarbons fraction dominated. Limonene, γ‐terpinene, and β‐pinene were the main identified compounds. The antioxidant activity was investigated using different in vitro assays namely 2,2‐diphenyl‐1‐picrylhydrazyl (DPPH), ABTS, ferric reducing ability power (FRAP), and β‐carotene bleaching test. In DPPH test, the essential oil obtained by hydrodistillation of fresh peel exhibited the highest activity (IC₅₀ of 1.17 mg/ml). Leaves extracted by SFE showed a good activity in both DPPH and β‐carotene bleaching test with IC₅₀ values of 2.20 and 6.66 mg/ml, respectively. Monoterpene hydrocarbons fraction exhibited a positive Pearson's correlation coefficient with all antioxidant assays. Leaves, often considered waste material, should be considered from a different point because they represent a matrix of indisputable interest.     

HLA class I antigen and HLA-A, -B, and -C haplotype frequencies in Uruguayans

HLA class I antigens were determined for 959 unrelated Uruguayans. The predominant HLA alleles were A2, Cw4, and B35, and the most frequently observed two-loci haplotypes were A2-B44 and B35-Cw4. The most frequent three-loci HLA haplotype was A2-Cw5-B44. We compared the Uruguayan sample with similar data from other populations.     

The Conformational State of Actin Filaments Regulates Branching by Actin-related Protein 2/3 (Arp2/3) Complex

Actin is a highly ubiquitous protein in eukaryotic cells that plays a crucial role in cell mechanics and motility. Cell motility is driven by assembling actin as polymerizing actin drives cell protrusions in a process closely involving a host of other actin-binding proteins, notably the actin-related protein 2/3 (Arp2/3) complex, which nucleates actin and forms branched filamentous structures. The Arp2/3 complex preferentially binds specific actin networks at the cell leading edge and forms branched filamentous structures, which drive cell protrusions, but the exact regulatory mechanism behind this process is not well understood. Here we show using in vitro imaging and binding assays that a fragment of the actin-binding protein caldesmon added to polymerizing actin increases the Arp2/3-mediated branching activity, whereas it has no effect on branch formation when binding to aged actin filaments. Because this caldesmon effect is shown to be independent of nucleotide hydrolysis and phosphate release from actin, our results suggest a mechanism by which caldesmon maintains newly polymerized actin in a distinct state that has a higher affinity for the Arp2/3 complex. Our data show that this new state does not affect the level of cooperativity of binding by Arp2/3 complex or its distribution on actin. This presents a novel regulatory mechanism by which caldesmon, and potentially other actin-binding proteins, regulates the interactions of actin with its binding partners.     

Grossly Defective nef Gene Sequences in a Human Immunodeficiency Virus Type 1-Seropositive Long-Term Nonprogressor

We have been investigating a long-term nonprogressor who was found to be human immunodeficiency virus type 1 (HIV-1) seropositive in 1985 and has survived with stable CD4⁺ T-cell counts (>1,000 CD4 cells/μl) without any AIDS-related illness. We have previously reported that repeated attempts to measure HIV-1 RNA in the peripheral mononuclear cells obtained from this subject have invariably failed. In the present study, we have analyzed the molecular nature of the HIV-1 quasispecies infecting this patient by PCR amplification of two proviral regions, the 5′ long terminal repeat (5′LTR)/gag leader and the nef gene, directly from fresh uncultured peripheral mononuclear cells, followed by length polymorphism analysis (with 1994, 1995, and 1996 samples) and sequencing (with a 1996 sample). Only proviral forms with nef deletions were revealed by length polymorphism analysis in samples from all three time points. Sequence analysis of the nef gene from the 1996 sample confirmed the presence of similar proviral quasispecies characterized by the presence of several deletions located in the nef-alone and the nef/U3 overlapping regions. Length polymorphism analysis of the 5′LTR/gag leader region suggested the existence of two major quasispecies populations, one characterized by the presence of forms carrying deletions in the U3 region and the other showing a completely intact, full-length 5′LTR. Evidence of the role of nef gene defects in long-term survival of HIV-1-infected patients has been provided so far in two independent investigations involving patients infected with HIV through blood transfusion. Here we show the existence of a similar condition in a subject who acquired HIV-1 seropositivity through the sexual route.     

Structural characterization of the N-glycans of gpMuc from Mucuna pruriens seeds

Mucuna pruriens seeds are used in some countries as a human prophylactic oral anti-snake remedy. Aqueous extracts of M. pruriens seeds possess in vivo activity against cobra and viper venoms, and protect mice against Echis carinatus venom. It was recently demonstrated that the seed immunogen generating the antibody that cross-reacts with the venom proteins is a multiform glycoprotein (gpMuc), and the immunogenic properties of gpMuc seemed to mainly reside in its glycan chains. In the present study, gpMuc was found to contain only N-glycans. Part of the N-glycans could be released with peptide-(N ⁴-(N-acetyl-β -glucosaminyl)asparagine amidase F (PNGase F-sensitive N-glycans); the PNGase F-resistant N-glycans were PNGase A-sensitive. The oligosaccharides released were analyzed by a combination of MALDI-TOF mass spectrometry, HPLC profiling of 2-aminobenzamide-labelled derivatives and ¹H NMR spectroscopy. The PNGase F-sensitive N-glycans comprised a mixture of oligomannose-type structures ranging from Man₅GlcNAc₂ to Man₉GlcNAc₂, and two xylosylated structures, Xyl₁Man₃GlcNAc₂ and Xyl₁Man₄GlcNAc₂. The PNGase A-sensitive N-glycans, containing (α 1-3)-linked fucose, were identified as Fuc₁Xyl₁Man₂GlcNAc₂ and Fuc₁Xyl₁Man₃GlcNAc₂. In view of the determined N-glycan ensemble, the immunoreactivity of gpMuc was ascribed to the presence of core (β 1-2)-linked xylose- and core α (1-3)-linked fucose-modified N-glycan chains.