Temporal shifts in physical habitat of the crayfish,Orconectes neglectus (Faxon)

A total of 300 samples was collected from February 1985 to August 1986 in a medium order Ozark Mountain stream. Physical habitat measurements of temperature, mean water column velocity, depth, and substrate character were recorded for each of the 25 monthly samples along with length and sex of all individuals of Orconectes neglectus (Faxon). Analysis of habitat utilization and suitability (or preference) was conducted using exponential polynomial models of hydraulic stress models. There appeared to be equal preference for depth over the range measured. Both substrate and velocity preference curves were bimodal with each mode designating certain crayfish size classes. Young-of-the-year were found primarily in cobbled, high velocity areas while adults were found in low velocity, macrophyte beds. Utilization curves for laminar sublayer thichness also reflected size-dependent phenomena where young-of-the-year were found in thin sublayer areas and adults were found primarily in thick sublayers. When separated by time and size, adults were found to occupy higher velocity, cobbled habitats during at least two months. This time period corresponded with the time of egg-bearing and further analysis yielded a time-dependent habitat suitability surface which accounted for this movement pattern. We suggest that the application of these suitability surfaces, which reflect habitat changes during the annual life cycle, will produce more accurate predictions of density and will allow better habitat management decisions under various regulated flow scenarios.     

Rapid Evaluation of Least-Squares and Minimum-Evolution Criteria on Phylogenetic Trees

We present fast new algorithms for evaluating trees with respectto least squares and minimum evolution (ME), the most commonlyused criteria for inferring phylogenetic trees from distancedata. The new algorithms include an optimal O(N2) time algorithmfor calculating the edge (branch or internode) lengths on atree according to ordinary or unweighted least squares (OLS);an O(N3) time algorithm for edge lengths under weighted leastsquares (WLS) including the Fitch-Margoliash method; and anoptimal O(N4) time algorithm for generalized least-squares (GLS)edge lengths (where N is the number of taxa in the tree). TheME criterion is based on the sum of edge lengths. Consequently,the edge lengths algorithms presented here lead directly toO(N2), O(N3), and O(N4) time algorithms for ME under OLS, WLS,and GLS, respectively. All of these algorithms are as fast asor faster than any of those previously published, and the algorithmsfor OLS and GLS are the fastest possible (with respect to orderof computational complexity). A major advantage of our new methodsis that they are as well adapted to multifurcating trees asthey are to binary trees. An optimal algorithm for determiningpath lengths from a tree with given edge lengths is also developed.This leads to an optimal O(N2) algorithm for OLS sums of squaresevaluation and corresponding O(N3) and O(N4) time algorithmsfor WLS and GLS sums of squares, respectively. The GLS algorithmis time-optimal if the covariance matrix is already inverted.The speed of each algorithm is assessed analytically—thespeed increases we calculate are confirmed by the dramatic speedincreases resulting from their implementation in PAUP* 4.0.The new algorithms enable far more extensive tree searches andstatistical evaluations (e.g., bootstrap, parametric bootstrap,or jackknife) in the same amount of time. Hopefully, the fastalgorithms for WLS and GLS will encourage the use of these criteriafor evaluating trees and their edge lengths (e.g., for approximatedivergence time estimates), since they should be more statisticallyefficient than OLS.     

Study of the in vitro bioactivation of albendazole in human liver microsomes and hepatoma cell lines

The metabolism of albendazole (ABZ), a benzimidazole anthelminthic, was studied in either microsomal preparations of human liver biopsies or cultured human hepatoma cell lines. Metabolites were analyzed by HPLC. Our data show that microsomes from human biopsies and two human cell lines, HepG2 and Hep3B, oxidize the drug to the sulfoxide very efficiently, whereas the third cell line tested, SK-HEP-1, does not. Both cytochrome P-450 dependent monooxygenases and favin-containing monooxygenases appear to be involved in human ABZ metabolism. Using the cell line displaying the highest ABZ-metabolizing activity, HepG2, the cytotoxic and the inducing effects of the parent drug ABZ and of two primary metabolites, the sulfoxide and the sulfone were studied. These three chemicals provoked a rise in mitotic index resulting from cell division blockage at the prophase or at the metaphase (ABZ metabolites) stage, and ABZ was more cytotoxic than its metabolites. With regard to enzyme-inducing effects, our data clearly demonstrate that the sulfoxide and, to a lesser degree, the sulfone are potent inducers of some drug metabolizing enzymes (i.e., cytochrome P-488 dependent monooxygenases and UDP glucuronyltransferase), whereas ABZ fails to increase and even slightly decreases these enzymatic activities. In conclusion, the HepG2 human hepatoma cell line appears to be suitable for the study of many parameters of metabolism and action of ABZ and other structurally related compounds in humans.Abbreviations ABZ albendazole - B[a]P benzo[a]pyrene - HPLC high-performance liquid chromatography - MC 3-methylcholanthrene - MFO mixed-function oxidase - UDPGT UDP-glucuronyltransferase     

SAR studies on dihydropyrimidinone antibiotics

There is an urgent need for the development of novel antimicrobial agents that offer effective treatment against MRSA. Using a new class of dipeptide antibiotic TAN-1057A/B as lead, we designed, synthesized and evaluated analogs of TAN-1057A/B. Several novel dihydropyrimidinone antibiotics demonstrating comparable antibiotic efficacy while possessing favorable selectivity were identified.     

In vitro characterization of a plastid terminal oxidase (PTOX).

The plastid terminal oxidase (PTOX) encoded by the Arabidopsis IMMUTANS gene was expressed in Escherichia coli cells and its quinone/oxygen oxidoreductase activity monitored in isolated bacterial membranes using NADH as an electron donor. Specificity for plastoquinone was observed. Neither ubiquinone, duroquinone, phylloquinone nor benzoquinone could substitute for plastoquinone in this assay. However, duroquinol (fully reduced chemically) was an accepted substrate. Iron is also required and cannot be substituted by Cu(2+), Zn(2+) or Mn(2+). This plastoquinol oxidase activity is independent of temperature over the 15-40 degrees C range but increases with pH (from 5.5 to 9.0). Unlike higher plant mitochondrial alternative oxidases, to which PTOX shows sequence similarity (but also differences, especially in a putative quinone binding site and in cysteine conservation), PTOX activity does not appear to be regulated by pyruvate or any other tested sugar, nor by AMP. Its activity decreases, however, with increasing salt (NaCl or KCl) concentration. Various quinone analogues were tested for their inhibitory activity on PTOX. Pyrogallol analogues were found to be inhibitors, especially octyl gallate (I50 = 0.4 microM ) that appears far more potent than propyl gallate or gallic acid. Thus, octyl gallate is a useful inhibitor for future in vivo or in organello studies aimed at studying the roles of PTOX in chlororespiration and as a cofactor for carotenoid biosynthesis.     

Growth and role of the corpus luteum throughout delayed gestation in the potoroo, Potorous tridactylus

The growth cycle of the corpus luteum (CL) of the potoroo is similar to that of other macropodids. During delayed gestation, the post-partum CL remains quiescent until it is reactivated by removal of the sucking pouch young. The CL then undergoes a period of growth, rapid from Day 6 until Day 12, followed by a gradual decline from Day 21 to Day 27. Excision of the CL before Day 6 of pregnancy either inhibited embryonic development or failed to support it. Excision of the CL between 6 and 21 days after removal of pouch young did not prevent embryos developing to full term but interfered with parturition. Excision on Day 25 after removal of pouch young allowed birth but impaired lactation, neonates dying within 2 days. By Day 27, the CL appeared to be no longer essential for embryonic development, birth or neonate survival. It is suggested that the CL of the potoroo is required for a slightly greater proportion of pregnancy than in most larger kangaroos because the birth canal must be prepared before each parturition.     

Characterization of a non-mitochondrial type I phosphatidylserine decarboxylase in Plasmodium falciparum

In search of key enzymes in Plasmodium phospholipid metabolism, we demonstrate the presence of a parasite-encoded phosphatidylserine decarboxylase (PSD) in the membrane fraction of Plasmodium falciparum-infected erythrocytes. PSD cDNA, encoding phosphatidylserine decarboxylase (PfPSD), was cloned by screening a directional cDNA library derived from the trophozoite erythrocytic stage. The corresponding PfPSD gene is located on chromosome 9 of P. falciparum, contains one intron of 938 nucleotides and is transcribed into a 3.7 kb mRNA. PfPSD cDNA encodes a putative protein of 362 amino acids, with a predicted molecular mass of 42.6 kDa, which clearly belongs to the type I PSD family. Only a 35 kDa polypeptide was detected in the parasite using a specific rabbit antiserum. PfPSD has a 314VGSS317 sequence near its carboxyl-terminus that is related to the Escherichia coli, yeast and human LGST motif, which is the site of proenzyme processing. PSD enzyme was expressed in E. coli with a KM of 63 +/- 19 microM and a VMAX of 680 +/- 49 nmol of phosphatidylethanolamine formed h-1 mg-1 protein. Site-directed mutagenesis of the VGSS active site demonstrated that the PfPSD proenzyme was processed into two non-identical subunits (alpha and beta) and revealed the crucial role played by each residue in enzyme processing and activity. Using indirect immunofluorescence, PfPSD labelling was co-localized with an endoplasmic reticulum marker, but not with a mitochondrial vital dye. This P. falciparum PSD is the first type I PSD identified in the endoplasmic reticulum compartment.     

Comparative population genetic structure in a plant-pollinator/seed predator system

Comparative analyses of spatial genetic structure of populations of plants and the insects they interact with provide an indication of how gene flow, natural selection and genetic drift may jointly influence the distribution of genetic variation and potential for local co‐adaptation for interacting species. Here, we analysed the spatial scale of genetic structure within and among nine populations of an interacting species pair, the white campion Silene latifolia and the moth Hadena bicruris, along a latitudinal gradient across Northern/Central Europe. This dioecious, short‐lived perennial plant inhabits patchy, often disturbed environments. The moth H. bicruris acts both as its pollinator and specialist seed predator that reproduces by laying eggs in S. latifolia flowers. We used nine microsatellite markers for S. latifolia and eight newly developed markers for H. bicruris. We found high levels of inbreeding in most populations of both plant and pollinator/seed predator. Among populations, significant genetic structure was observed for S. latifolia but not for its pollinator/seed predator, suggesting that despite migration among populations of H. bicruris, pollen is not, or only rarely, carried over between populations, thus maintaining genetic structure among plant populations. There was a weak positive correlation between genetic distances of S. latifolia and H. bicruris. These results indicate that while significant structure of S. latifolia populations creates the potential for differentiation at traits relevant for the interaction with the pollinator/seed predator, substantial gene flow in H. bicruris may counteract this process in at least some populations.     

Prerequisite for His4 in deltorphin A for highδ opioid receptor selectivity

Summary Analysis of deltorphin A position 4 analogues included: backbone constrained N MeHis, spinacine (Spi), N MePhe and the tetrahydroisoquinoline-3-carboxylic acid (Tic); spatially confined side-chain (Phg); and imidazole alkylation ofl- andd-His⁴ enantiomers. High selectivity was lost with the following replacements: N MeHis⁴, N MePhe⁴ and Phg⁴ reduced binding and the constrained residues also increasedµ binding; ring closure between the side-chain and amino group to yield Spi⁴ or Tic⁴ increasedµ affinity. Imidazole methylation of His⁴ marginally affected opioid binding and doubled selectivity; alkylatedd-His⁴-derivatives generally maintained selectivity in spite of decreased affinities. Thus, His⁴ imidazole preserves selectivity by facilitating high binding and by repulsion at theµ receptor. Several low energy conformers of deltorphin A indicated that the His⁴ imidazole preferred a spatial orientation parallel to the phenolic side-chain of Tyr¹ suggestive that this conformation might contribute to high affinity and selectivity.     

Adrm1, a putative cell adhesion regulating protein, is a novel proteasome-associated factor

We have identified Adrm1 as a novel component of the regulatory ATPase complex of the 26 S proteasome: Adrm1 was precipitated with an antibody to proteasomes and vice versa. Adrm1 co-migrated with proteasomes on gel-filtration chromatography and non-denaturing polyacrylamide gel electrophoresis. Adrm1 has been described as an interferon-gamma-inducible, heavily glycosylated membrane protein of 110 kDa. However, we found Adrm1 in mouse tissues only as a 42 kDa peptide, corresponding to the mass of the non-glycosylated peptide chain, and it could not be induced in HeLa cells with interferon. Adrm1 was present almost exclusively in soluble 26 S proteasomes, albeit a small fraction was membrane-associated, like proteasomes. Adrm1 was found in cells in amounts equimolar with S6a, a 26 S proteasome subunit. HeLa cells contain no pool of free Adrm1 but recombinant Adrm1 could bind to pre-existing 26 S proteasomes in cell extracts. Adrm1 may be distantly related to the yeast proteasome subunit Rpn13, mutants of which are reported to display no obvious phenotype. Accordingly, knock-down of Adrm1 in HeLa cells had no effect on the amount of proteasomes, or on degradation of bulk cell protein, or accumulation of polyubiquitinylated proteins. This indicates that Adrm1 has a specialised role in proteasome function.