Identification, sequence and developmental expression of invertebrate flotillins from Drosophila melanogaster

Caveolae are vesicular organelles that represent a sub-compartment of the plasma membrane. Caveolins (Cav-1, -2 and -3) and flotillins {FLO-1 and FLO-2 [also known as epidermal surface antigens (ESAs)]} are two families of mammalian caveolae-associated integral membrane proteins. Although a caveolin gene family has recently been described in the invertebrate Caenorhabditis elegans, it remains unknown as to whether flotillin homologues exist in invertebrates.

Here, we report the identification, cDNA sequence and embryonic expression pattern of the first invertebrate flotillin, i.e. flotillin from Drosophila melanogaster (FLO^Dm). FLO^Dm is most closely related to mammalian flotillin-1. Remarkably, the invertebrate FLO^Dm protein behaves like mammalian flotillins and is targeted to the caveolae-enriched membrane fraction after transient expression in mammalian cells. Localization of the FLO^Dm message in D. melanogaster embryos reveals that expression of FLO^Dm is confined primarily to the developing nervous system. This is consistent with our previous observation that mammalian flotillin-1 mRNA and protein is expressed abundantly in brain tissue. Interestingly, the FLO^Dm gene is localized to chromosomal region 52 B1–B2. In addition, we find that at least two flotillin-related genes are expressed in D. melanogaster. Our current results provide a starting point and systematic basis for dissecting the role of flotillin in caveolae and neuronal development using Drosophila as a genetic system.     

Unique features in the mitochondrial D-loop region of the European seabass Dicentrarchus labrax

We have cloned and sequenced the displacement-loop (D-loop) region of the mitochondrial DNA (mtDNA) from the European seabass Dicentrarchus labrax (Dl). This sequencing revealed the presence of four tandemly repeated elements (R1, R2, R3 and R4); the individual variation in mtDNA total length is entirely accounted for by their variable number. The individuals examined also possessed an imperfect copy of one of the tandem repeats (ΨR2). At least one termination-associated sequence (TAS) is present in each of the repeats and in two copies 5′ upstream from the tandem array as well. The alignment of the Dl D-loop region with D-loop sequences from four other Teleosts and one Chondrosteus showed the Dl sequence to be larger than that of other fish. The extraordinary length of the D1 D-loop sequence is also due to the 5′ and 3′ regions that are flanking the tandem array, the largest ones to date analyzed in fish. In this study, we also report the unique organization and localization of putative TAS and conserved-sequence block (CSB) elements, and the presence of a conserved 218-bp sequence in the D1 D-loop region.     

Microsatellite markers from sugarcane (Saccharum spp.) ESTs cross transferable to erianthus and sorghum

Analysis of a sugarcane (Saccharum spp.) EST (expressed sequence tag) library of 8678 sequences revealed approximately 250 microsatellite or simple sequence repeats (SSRs) sequences. A diversity of dinucleotide and trinucleotide SSR repeat motifs were present although most were of the (CGG)_n trinucleotide motif. Primer sets were designed for 35 sequences and tested on five sugarcane genotypes. Twenty-one primer pairs produced a PCR product and 17 pairs were polymorphic. Primer pairs that produced polymorphisms were mainly located in the coding sequence with only a single pair located within the 5′ untranslated region. No primer pairs producing a polymorphic product were found in the 3′ untranslated region. The level of polymorphism (PIC value) in cultivars detected by these SSRs was low in sugarcane (0.23). However, a subset of these markers showed a significantly higher level of polymorphism when applied to progenitor and related genera (Erianthus sp. and Sorghum sp.). By contrast, SSRs isolated from sugarcane genomic libraries amplify more readily, show high levels of polymorphism within sugarcane with a higher PIC value (0.72) but do not transfer to related species or genera well.     

Association of Drosophila cysteine string proteins with membranes

Cysteine string proteins are putative synaptic vesicle proteins that lack a transmembrane domain. Our analysis shows that Drosophila cysteine string proteins are extensively modified by hydroxylamine-sensitive fatty acylation. This modification could be responsible for association of csp's with membranes. Extensive deacylation of Dcsp's by a 20 h incubation in 1 M hydroxylamine, pH 7.0, or methanolic KOH produces a protein of 6–7 kDa lower mass than untreated Dcsp's. Surprisingly, the hydroxylamine treatment does not cause release of Dcsp's from membranes. On the other hand, alkaline stripping of membranes isolated from Drosophila brain by 0.1 M sodium carbonate, pH 11.5, causes a significant release of Dcsp's from membranes into the cytosol. These results indicate that fatty acylation may not form the main anchor of Dcsp's in membranes. Taking advantage of the endocytotic block in the Drosophila mutant shibire^ts1, we analyzed the acylation states of Dcsp's in two stages during synaptic vesicle recycling and found no evidence for an acylation/ deacylation cycle of Dcsp's in the brain nerve terminals.     

On the ecological energetics of 0-group Carcinus maenas (L.) from a shallow sandy bottom in gullmar fjord, Sweden

0-group Carcinus maenas (L.) was investigated from June 1975 to September 1976 on a shallow sandy bottom at Kvarnbukten Bay, Gullmar Fjord (58° 15′N: 11°28′E), Sweden, at an average salinity of 25% and a range of monthly mean temperatures of −0.3 to 197. °C.

The new year-class settles from August to early September at a carapace breath of 2 to 3 mm and a calorific content of 32 cal. The distribution is restricted to clusters of the mussel Mytilus edulis L. Depth, type of substratum, and patches of the eel-grass Zostera marina L. are of no importance for their spatial distribution. There is no migration to deeper water in the autumn. The carapace breadth is ≈ 9.5 mm after one year of benthic life. Sexual maturity is reached after two years. Growth occurs at temperatures above 10 °C, i.e., from August to October and from May to July. During the first year of benthic life the animals moult 7 times. The 0-group seems to be micro-carnivores feeding on the sediment meiofauna.

The individual energy budget for the first year of benthic life is: consumption (C_c) 905 cal., production (P_1c) 236 cal., cast carapaces (P_2c) 153 cal., respiration (R_c) 404 cal., and rejectiction (F_c) 112 cal. The assimilation efficiency (U_c⁻¹) is 88%, the gross growth efficiency (K_1c) 43%, and the net growth efficiency (K_2c) 49%.

At Kvarnbukten Bay there are large variations in size between the separate year-classes. The energy content of the food consumed by the 1975/76 cohort was used as follows: 4% was stored in living biomass after one year, 36% was released to other trophic levels as dead animals and cast carapaces, 13% rejected as faeces, and 47% was lost through respiration.     

Neolignans from Ocotea catharinensis

Bark, wood and leaves of Ocotea catharinensis contain respectively 10 (average yield 0.7%.), 15 (average yield 0.004%.) and one (yield 0.4%.) neolignans of the bicyclo[3.2.1]octanoid and the hydrobenzofuranoid structural types, including the new rel-(7S,8R,1′R,4′S,5′R,6′R)-Δ^8′-4′,6′-dihydroxy-5′-methoxy-3,4-methylenedioxy-3′-oxo-8.1′,7.5′-neolignan, (7S,8S)-Δ^{1′,3′,5′,8′}-5,3′,5′-trimethoxy-3,4-methylenedioxy-8.1′,7.O.6′,4.O.7′-neolignan, (7R,8S,1′R,3′R)-Δ^5′,8′-3,4,3′,5′-tetramethoxy-4′-oxo-8.1′,7.O.6′-neolignan and rel-(7R,8S,1′R,2′S)-Δ^4′,8′-2′-hydroxy-3,4-dimethoxy-3′-oxo-8.1′,7.O.2′-neolignan.     

Further farnesyl-homogentisic acid derivatives from Otoba parvifolia

The seeds of Otoba parvifolia contain three novel compounds apparently derived from homogentisic acid, rel-(1′R,5′R)-2-(1′-farnesyl-5′-hydroxy-2′-oxocyclohex-3′-en-1′-yl)-acetic acid and its acetate as well as rel-(1′R,4′S,5′R)-2-(1′-farnesyl-4′,5′-dihydroxy-2′-oxocyclohexan-1′-yl)-acetic acid δ-lactone. The structure of an additional isolate, previously described as 2-(1′-farnesyl-2′-hydroxy-5′-oxocyclohex-3′-en-1′-yl)-acetic acid γ-lactone was revised to rel-(1′R,5′R)-2-(1′-farnesyl-5′-hydroxy-2′-oxocyclohex-3′-en-1′-yl)-acetic acid δ-lactone.     

Esterified, optical pure (3S, 3′S)-astaxanthin from flowers of Adonis annua

The quantitative carotenoid composition of the red flower petals of Adonis annua is reported. Optically pure (3S, 3′S)-astaxanthin occurs both as a diester (64% of total carotenoid) and as a monoester (11%). The optical purity was determined by hydrolysis of the natural esters in the absence of oxygen and subsequent HPLC analysis of the paren -ketol esterified with (−)-camphanic acid. All non-animal sources hitherto examined synthesize pure 3S,3′S- or 3R,3′R-isomers of astaxanthin, whereas marine animal sources contain mixtures of all three optical isomers, including the meso form.     

Tissue distribution, hormone regulation and evidence for a human homologue of the estrogen-inducible Xenopus laevis vitellogenin mRNA binding protein

17β-estradiol induces the synthesis of massive amounts of the hepatic mRNA encoding the Xenopus laevis egg yolk precursor protein, vitellogenin. Vitellogenin mRNA exhibits a half life of approx. 500 h when 17β-estradiol is present, and 16 h after removal of 17β-estradiol from the culture medium. We recently reported that Xenopus liver contains a protein, which is induced by 17β-estradiol and binds with a high degree of specificity to a binding site in a segment of the 3′-untranslated region (3′-UTR) of vitellogenin mRNA implicated in 17β-estradiol stabilization of vitellogenin mRNA. To determine if this mRNA binding protein was specific to this system, or if it was present elsewhere, and regulated by other steroids, we examined the tissue distribution and androgen regulation of this protein. Substantial amounts of the vitellogenin 3′-UTR binding protein were found in several Xenopus tissues including testis, ovary and muscle. In the absence of hormone treatment, lung and intestine contained minimal levels of the mRNA binding protein. Testosterone administration induced the vitellogenin 3′-UTR RNA binding protein in several tissues. Additionally, we found a homologous mRNA binding protein in MCF-7, human breast cancer cells. Although the MCF-7 cell protein was not induced by 17β-estradiol, the MCF-7 cell mRNA binding protein appears to be closely related to the Xenopus protein since: (i) the human and Xenopus proteins elicit gel shifted bands with the same electrophoretic mobility using the vitellogenin mRNA 3′-UTR binding site; (ii) The human and Xenopus proteins exhibit similar binding specificity for the vitellogenin 3′-UTR RNA binding site; and (iii) RNA from MCF-7 cells is at least as effective as RNA from control male Xenopus liver in blocking the binding of the Xenopus and human proteins to the vitellogenin mRNA 3′-UTR binding site. Its broad tissue distribution and regulation by both 17β-estradiol and testosterone suggests that this mRNA binding protein may play a significant role in steroid hormone regulation of mRNA metabolism in many vertebrate cells.     

Novel antioxidants isolated from plants of the genera Ferula, Inula, Prangos and Rheum collected in Uzbekistan

We examined the effects of 48 compounds isolated from Ferula pallida, F. penninervis, Inula macrophylla, Prangos pabularia, P. tschimganica and Rheum maximowiczii collected in Uzbekistan on ADP/Fe²⁺-induced lipid peroxidation of egg yolk phosphatidylcholine liposomes. Of those compounds, 23 inhibited ADP/Fe²⁺-induced lipid peroxidation and nine showed especially strong inhibition of lipid peroxidation. Most compounds that inhibited peroxidation scavenged the 1,1′-diphenyl-2-picrylhydrazyl (DPPH) radical, indicating that the inhibition was due to radical scavenging. However, some compounds did not scavenge DPPH but inhibited lipid peroxidation significantly, suggesting that their inhibitory effect was not due to radical scavenging but to some other mechanism, such as prevention of Fe²⁺ function. Thus, we found various new antioxidants, some of which had a unique mechanism of action, in Ferula, Inula, Prangos and Rheum plants collected in Uzbekistan as seeds used in medicine.     

Developmental profiles of glial enzymes in the chick embryo: In vivo and in culture

The activities of two glial cell enzymes, glutamine synthetase (a marker for astrocytes) and 2′,3′-cyclic nucleotide 3′-phosphohydrolase (a marker for oligodendrocytes and myelination) were studied in the developing chick embryo brain in vivo and in cultures derived from chick embryos. The in vivo findings showed that the activities of both enzymes parallel the patterns of gliogenesis and myelination. Glutamine synthetase follows similar patterns in culture and in vivo, whereas the developmental profile of 2′,3′-cyclic nucleotide 3′-phosphohydrolase appears to be affected by the culture conditions.     

A Drosophila minisatellite contains multiple Chi sequences

We demonstrate the existence of polymorphic DNA minisatellites in Drosophila mauritiana, a close relative of D. melanogaster. One of these sequences (minisatellite mD4.2) consists of 13 tandemly repeated monomers, 10 of which are 33 base pairs long. Each of the repeat monomers contains sequences identical or very similar to the Chi sequence (GCTGGTGG), a signal for recBCD-dependent recombination in Escherichia coli. Sequences hybridizing to the mD4.2 minisatellite are present in at least 20–25 genomic locations and exhibit substantial variability among different populations of three Drosophila species and two populations of the house fly, Musca domestica. Interpopulational variation is a result of length differences rather than restriction site polymorphisms and genetic crosses establish that the hybridizing restriction fragment patterns have an underlying genetic basis. The presence of these sequences in the genetically well known Drosophila species allows critical examination of processes that produce and maintain the remarkable variability associated with these genomic regions.     

Effects of electronic structure on chiral induction in outer-sphere electron transfer reactions of complexes with the C3 symmetric ligand, 1,4,7-triazacyclononane-1,4,7-tris[2′(R)-2′-propionate](-3)

The ligand 1,4,7-triazacyclononane-1,4,7-tris[2′(R)-2′-propionate](-3)((R)-tacntp³⁻), binds stereospecifically to transition metal ions. The structures of the complexes [Cr((R)-tacntp)]·NaBr and [Fe((R)-tacntp)]·H₂O have been determined by X-ray crystallography. Both complexes have the Λ-configuration but the conformation of the chelate rings in Λ-[Cr((R)-tacntp)] is (λ,λ,λ) with a geometry close to octahedral while in Λ-[Fe((R)-tacntp)] it is (δ,δ,δ) and the geometry is closer to that of a trigonal prism. Chiral induction in the electron transfer reactions of Λ-[Co((R)-tacntp)], Λ-[Fe((R)-tacntp)] and Λ-[Mn((R)-tacntp)] with [Co((RR,SS)-chxn)₃]²⁺ has been investigated. All three reactions are outer-sphere and four isomeric [Co((RR,SS)-chxn)₃]³⁺ products are identified in each case. The oxidants Λ-[Fe((R)-tacntp)] and Λ-[Mn((R)-tacntp)] show very similar selectivities, quite different from those of Λ-[Co((R)-tacntp)]. Reasons for this behavior are discussed.     

Mutations at the W locus affect survival of neural crest-derived melanocytes in the mouse

The development of melanoblasts in normally pigmented and dominant spotting (W) embryos was followed by in situ hybridisation to TRP-2/DT mRNA, which labels migratory melanoblasts from 10 days post coitum. Numerous melanoblasts migrate to the inner ear around 11 days. In contrast, few migratory melanoblasts are associated with the eye or skin at this stage and melanoblast distribution within the trunk and tail is patchy. The distribution of melanoblasts in 10.5–11-day-old W^v/W^v, W^sh/W^sh and W⁴¹/W⁴¹ mutants was similar to that in controls but melanoblast density was lower and by 12 days was severely reduced. These results suggest that mutations of the c-kit receptor tyrosine kinase encoded at the W locus do not alter early migration or differentiation of melanoblasts but severely affect melanoblast survival.     

(−)-di-de-o-methylgrandisin, a lignan from Virola pavonis leaves

Leaves of Virola pavonis yielded a (7,7′β,8β,8′)-4,4′-dihydroxy-3,3′5,5′-tetramethoxy-7,7′-epoxyligna (−)-di-de-O-methylgrandisin.     

Dihydrochalcones, coumarins and alkaloids from Metrodorea nigra

Two novel dihydrochalcones, 2′,3,4′,6′-tetrahydroxy-4-methoxy-3′,5-di-(3,3-dimethylallyl)-dihydrochalcone and 2′,.3,6′-trihydroxy-4-methoxy-5-(3,3-dimethylallyl)-3′,4′-(2″,2″-dimethyldihydropyran)-dihydrochalcone, have been isolated from fresh fruits of Metrodorea nigra. Stems and leaves showed a similar composition and we have isolated common steroids, simple coumarins, several furocoumarins, furoquinoline alkaloids and a furofuran lignan. From stems, we have also isolated the pentacyclic 6-C-monoterpenyl-5,7-dioxycoumarin, deoxybruceol. Structures of the isolated compounds were elucidated on the basis of spectral data.