The Sulfonylurea-Inhibited NADH Oxidase Activity of HeLa Cell Plasma Membranes has Properties of a Protein Disulfide–Thiol Oxidoreductase with Protein Disulfide–Thiol Interchange Activity

Plasma membrane vesicles of HeLa cells are characterized by a drug-responsive oxidation of NADH. The NADH oxidation takes place in an argon or nitrogen atmosphere and in samples purged of oxygen. Direct assay of protein thiols by reaction with 5,5-dithiobis-(2-nitrobenzoic acid) (DTNB; Ellman's reagent), suggests that protein disulfides may be the natural electron acceptors for NADH oxidation by the plasma membrane vesicles. In the presence of NADH, protein disulfides of the membranes were reduced with a concomitant stoichiometric increase in protein thiols. The increase in protein thiols was inhibited in parallel to the inhibition of NADH oxidation by the antitumor sulfonylurea LY181984 with an EC₅₀ of ca. 30 nM. LY181984, with an EC₅₀ of 30 nM, also inhibited a protein disulfide–thiol interchange activity based on the restoration of activity to inactive (scrambled) RNase and thiol oxidation. The findings suggest that thiol oxidation, NADH-dependent disulfide reduction (NADH oxidation), and protein disulfide–thiol interchange in the absence of NADH all may be manifestations of the same sulfonylurea binding protein of the HeLa plasma membrane. A surface location of the thiols involved was demonstrated using detergents and the impermeant thiol reagent p-chloromercuriphenylsulfonic acid (PCMPS). The surface location precludes a physiological role of the protein in NADH oxidation. Rather, it may carry out some other role more closely related to a function in growth, such as protein disulfide–thiol interchange coupled to cell enlargement.     

Use of dipyridyl-dithio substrates to measure directly the protein disulfide-thiol interchange activity of the auxin stimulated NADH: Protein disulfide reductase (NADH oxidase) of soybean plasma membranes

Dipyridyl-dithio substrates were cleaved by isolated vesicles of plasma membranes prepared from etiolated hypocotyls of soybean. The cleavage was stimulated by auxins at physiological concentrations. The substrates utilized were principally 2,2-dithiodippyrine (DTP) and 6,6-dithiodinicotinic acid (DTNA). The DTP generated 2 moles of 2-pyridinethione whereas the 6,6-dithiodinicotinic acid generated 2 moles of 6-nicotinylthionine. Both products absorbed at 340 nm. The auxin herbicide, 2,4-dichlorophenoxyacetic acid (2,4-D) stimulated the activity approximately 2-fold to a maximum at about 10 M. Concentrations of 2,4-D greater than 100 M inhibited the activity. Indole-3-acetic acid stimulated the activity as well. The growth-inactive auxin, 2,3-dichlorophenoxyacetic acid (2,3-D), was without effect. DTNA cleavage correlated with oxidation of NADH and reduction of protein disulfide bonds reported earlier in terms of location at the external plasma membrane surface, absolute specific activity, pH dependence and auxin specificity. The dipyridyl-dithio substrates provide, for the first time, a direct measure of the disulfide-thiol interchange activity of the protein previously measured only indirectly as an auxin-dependent ability of isolated plasma membrane vesicles to restore activity to scrambled and inactive RNase.     

Compositional similarity among urban floras within and across continents: biogeographical consequences of human-mediated biotic interchange

Anthropogenic activities have weakened biogeographical barriers to dispersal resulting in the global spread and establishment of an increasing number of non‐native species. We examine the broad‐scale consequences of this phenomenon based on an analysis of compositional similarity across urban floras in the northeastern United States and Europe. We test the prediction that homogenization of species composition is uniquely defined within vs. between continents based on the time and place of origin of non‐native species. In this case, for archaeophytes and neophytes in Europe (introduced before and after ad 1500, respectively) and non‐native species originating from within and outside the United States. More species in urban floras were shared within than between continents. Within Europe, archaeophytes shared more species across urban floras compared with neophytes; strong associations were not observed for non‐native species across US urban floras. Between the two continents, non‐native species in the United States that originated from outside the United States shared species primarily with archaeophytes but also with European natives and neophytes. These results suggest that the direction of biotic interchange was unidirectional with species moving primarily from Europe to the United States with archaeophytes playing a primary and non‐native species originating from outside the two continents a secondary role as a homogenizing source. Archaeophytes, based on combination of biogeographical, evolutionary, and ecological factors in association with a long history of anthropogenic influence, appear to have played a prominent role in the continental and intercontinental homogenization of species composition. This suggests that the uniform homogenization of the Earth's biota is not imminent and is presently directed by a combination of biogeographically defined anthropogenic and historical factors.     

Correlation between NQR parameters and residue size of aliphatic amino acids and their dimers

Nuclear quadrupole coupling constants (NQCC), chi, and asymmetry parameters, eta, of 2D, 14N and 17O nuclei have been calculated for aliphatic amino acids and their dimers using MP2/6-311++G** method to shed some light on the differences between the structural parameters in the aliphatic amino acids and their dimers. For this purpose, electric field gradient (EFG) at the sites of quadrupolar nuclei have been calculated and evaluated for each compound. A correlation is observed between the calculated NQCC parameters and the conformational structures of the compounds, showing that extraction of structural data from the NQR spectra might be promising. Our results showed that 17O NQCC of terminal carboxylic acid and 14N NQCC of the terminal amino groups are, respectively, the least and the most sensitive parameters to the variation of the size of the residue. It is found also that conformation of R (i.e. values of the dihedral angles) plays a very effective role in the determination of the values of the calculated NQCC parameters. Sensitivity of the NQR parameters to the changes in the conformational structure is significantly greater (nearly 20-fold) than that to the changes in the other structural parameters such as bond lengths.     

Laccase-mediated dimerization of the flavonolignan silybin

Flavonolignan silybin (1) present in the seeds of the milk thistle (Silybum marianum) is widely used in human therapy of liver dysfunctions and as a hepatoprotectant thanks to its dual function: it acts as a highly effective radical scavenger (antilipoperoxidant) and also as an antioxidant. Molecular mechanisms of antiradical action of 1 and even functional groups responsible for this activity are not well known so far. Silybin forms during in vitro reaction with stable radicals (e.g., DPPH) or with enzyme laccase (Trametes pubescens) complex mixtures of oligomeric and polymeric products whose structural analysis is virtually impossible. Methylation of 7-OH in 1 yields under laccase-mediated oxidation C–O and C–C dimers in the ratio ca. 1:2.5. Using this approach, the hydroxyl groups responsible for antiradical activity of silybin (20-OH group) were determined and the molecular mechanism of the E-ring antiradical activity was explained.     

Studies on the porcine liver esterase-catalyzed hydrolysis of pentaacetyl catechin and epicatechin: application to the synthesis of novel dimers and trimers

Porcine liver esterase-catalyzed hydrolysis of 3,5,7,3′,4′-pentaacetylated catechin was studied. The selectivity of the enzyme in hydrolyzing the acetate moiety is time dependent. Careful control of the duration of hydrolysis makes it possible to isolate the differentially protected catechins. Similar result was also obtained in the epicatechin series. These results are important for elaboration of epicatechin or catechin into different derivatives with defined regiochemistry. These include novel dimeric and trimeric architectures.     

Characterization of creatine kinase isoforms in herring (Clupea harengus) skeletal muscle

It is known that mitochondrial creatine kinase (MtCK) in mammals is always expressed in conjunction with one of the cytosolic forms of creatine kinase (CK), either muscle-type (MM-CK) or brain-type (BB-CK) in tissues of high, sudden energy demand. The two creatine kinase (CK) isoforms were detected in herring (Clupea harengus) skeletal muscle: cytosolic CK and mitochondrial CK (MtCK) that displayed the different electrophoretic mobility. These isoforms differ in molecular weight and some biochemical properties. Isolation and purification procedures allowed to obtain purified enzymes with specific activity of the 206 μmol/min/mg for cytosolic CK and 240 μmol/min/mg for MtCK. Native M_rs of the cytosolic CK and MtCK determined by gel permeation chromatography were 86.000 and 345.000, respectively. The results indicate that one of isoforms found in herring skeletal muscle is a cytosolic dimer and the other one, is a mitochondrial octamer. Octamerization of MtCK is not an advanced feature and also exists in fish. These values correspond well with published values for MtCKs and cytosolic CK isoforms from higher vertebrate classes and even from lower invertebrates.     

TIME OF ORIGIN AND BIOGEOGRAPHICAL HISTORY OF SPECIALIZED RELATIONSHIPS BETWEEN NORTHERN MARINE PLANTS AND HERBIVOROUS MOLLUSCS

A survey of cool-temperate North Pacific and North Atlantic marine plants and herbivorous molluscs and their descendants was undertaken to determine the time of origin and the biogeographical history of specialized host-guest relationships between plants and herbivores. Of 12 such associations, 6 (50%) were established no earlier than the Pliocene. These six associations resulted from the invasion of either the plant (three cases) or the herbivore (three cases) from geographical regions other than the one in which the association was forged. These results are contrary to the widely held view that specialized (“coevolved”) relationships tend to be ancient, and highlight the importance of biotic interchange as a biogeographical process influencing the opportunity for trophic specialization.     

Stalk region of kinesin-related protein Unc104 has moderate ability to form coiled-coil dimer

Unc104/KIF1A, a kinesin family member, is reported to be monomeric in solution, though its polypeptide has regions that potentially form coiled coils. For a better understanding of the mechanism underlying Unc104/KIF1A's motility, it is important to evaluate the dimerization ability of this protein. The CD measurement of relevant segments of Caenorhabditis elegans Unc104 indicated that peptides having a common region (N358-K379) showed spectra characteristic to an alpha-helix. Dimerization by coiled-coil formation was confirmed by analytical ultracentrifugation. By analyzing the concentration dependence of the CD spectra, the monomer-dimer dissociation constant, Kd, of (N354-E388) was estimated to be about 5 microM, which is considerably larger than that of the corresponding segment of human kinesin (62 nM). Though its dimerization ability is rather moderate, Unc104/KIF1A could nonetheless dimerize and therefore could move by the same mechanism as human kinesin when the concentration of Unc104 is high due to, e.g., local crowding. This suggests that the motility could be controlled by the concentration of the motor protein.     

Sex differences in scent‐marking in sifaka: Mating conflict or male services?

Male and female interests can either be in conflict or serve as a basis for exchange. Communication is thus an important aspect of intersexual relationships. Verreaux's sifaka (Propithecus verreauxi verreauxi), like many prosimians, uses chemical signals as one form of communication. The goals of this study were to determine 1) if males and females exhibit sex differences in their scent-marking behavior, and 2) if scent-marking is an example of mating conflict or cooperation. All occurrences of scent-marks, scent-mark context, and scent-mark style were collected on 23 sifaka in the Kirindy Forest of western Madagascar for 7 months (September 2001-March 2002). Scent-mark rates were collected using continuous focal animal sampling from November 2000-March 2002. Home-range data were collected using monthly censuses and instantaneous focal sampling throughout those 17 months. The pressures of behavioral ecology seem to have shaped scent-marking in sifaka: the sexes exhibited significantly different scent-marking behavior. Results from this study are consistent with the hypotheses that 1) females scent-mark to advertise their presence and mark their resources, 2) clean-chested males use scent-marks as between-group communication to advertise their presence, and 3) stained-chested males use scent-marks as a form of olfactory mate-guarding. Scent-marking does not appear to be a "service" that males provide to females, because overmarking limits female communication rather than adding to the overall number of scent-marks. Scent-marking behavior is a crucial aspect of the mating conflict and for understanding intersexual relationships in sifaka.