D. John Faulkner

Gβγ-Mediated Regulation of Golgi Organization Is through the Direct Activation of Protein Kinase D

Abstract We have shown previously that the βγ subunits of the heterotrimeric G proteins regulate the organization of the pericentriolarly localized Golgi stacks. In this report, evidence is presented that the downstream target of Gβγ is protein kinase D (PKD), an isoform of protein kinase C. PKD, unlike other members of this class of serine/threonine kinases, contains a pleckstrin homology (PH) domain. Our results demonstrate that Gβγ directly activates PKD by interacting with its PH domain. Inhibition of PKD activity through the use of pharmacological agents, synthetic peptide substrates, and, more specifically, the PH domain of PKD prevents Gβγ-mediated Golgi breakdown. Our findings suggest a possible mechanism by which the direct interaction of Gβγ with PKD regulates the dynamics of Golgi membranes and protein secretion.

Complete vesiculation of Golgi membranes and inhibition of protein transport by a novel sea sponge metabolite, ilimaquinone

We have identified a novel natural metabolite, ilimaquinone (IQ), from sea sponges that causes Golgi membranes to break down completely in vivo into small vesicular structures (called vesiculated Golgi membranes [VGMs]). Under these conditions, transport of newly synthesized proteins from endoplasmic reticulum (ER) to the cis-Golgi-derived VGMs is unaffected; however, further transport along the secretory pathway is blocked. Upon removal of the drug, VGMs reassemble rapidly into a Golgi complex, and protein transport is restored. By employing a cell-free system that reconstitutes vesicular transport between successive Golgi cisternae, we provide evidence that the inhibition of protein transport by IQ is specifically due to an inhibition of transport vesicle formation. In addition, like brefeldin A (BFA), IQ treatment prevents the association of beta-COP and ADP-ribosylation factor to the Golgi membranes; however, unlike BFA treatment, there is no retrograde transport of Golgi enzymes into ER.

Lithistid Sponges: Star Performers or Hosts to the Stars

Extremely careful separation of the symbiotic bacteria from the cells of lithistid sponges has provided evidence that in the case of Theonella swinhoei the chemically diverse and biologically active metabolites, swinholide A and theopalauamide (1), are produced by symbionts. Marine sponges of the order Lithistida contain a diverse range of exotic natural products with valuable biological and pharmacological activities.

Vertical Transmission of Diverse Microbes in the Tropical Sponge Corticium sp.

ABSTRACT Sponges are host to extremely diverse bacterial communities, some of which appear to be spatiotemporally stable, though how these consistent associations are assembled and maintained from one sponge generation to the next is not well understood. Here we report that a diverse group of microbes, including both bacteria and archaea, is consistently present in aggregates within embryos of the tropical sponge Corticium sp. The major taxonomic groups represented in bacterial 16S rRNA sequences amplified from the embryos are similar to those previously described in a variety of marine sponges. Three selected bacterial taxa, representing proteobacteria, actinobacteria, and a clade including recently described sponge-associated bacteria, were tested and found to be present in all adult samples tested over a 3-year period and in the embryos throughout development. Specific probes were used in fluorescence in situ hybridization to localize cells of the three types in the embryos and mesohyl. This study confirms the vertical transmission of multiple, phylogenetically diverse microorganisms in a marine sponge, and our findings lay the foundation for future work on exploring vertical transmission of specific, yet diverse, microbial assemblages in marine sponges.

Regulation of Golgi Structure through Heterotrimeric G Proteins

We have previously shown that ilimaquinone (IQ), a marine sponge metabolite, causes complete vesiculation of the Golgi stacks. By reconstituting the IQ-mediated vesiculation of the Golgi apparatus in permeabilized cells, we now demonstrate that this process does not require ARF and coatomers, which are necessary for the formation of Golgi-derived COPI vesicles. We find that IQ-mediated Golgi vesiculation is inhibited by G alpha(s)-GDP and G alpha(i3)-GDP. Interestingly, adding betagamma subunits in the absence of IQ is sufficient to vesiculate Golgi stacks. Our findings reveal that IQ-mediated Golgi vesiculation occurs through activation of heterotrimeric G proteins and that it is the free betagamma, and not the activated alpha subunit, that triggers Golgi vesiculation.

Defensive chemicals of the Spanisch dancer nudibranch Hexabranchus sanguineus and its egg ribbons: macrolides derived from a sponge diet

Abstract The Spanish dancer nudibranch Hexabranchus sanguineus (Ruppell et Leuckart), a large brightly colored shell-less sea slug (Gastropoda : Opisthobranchia) common to Indo-Pacific coral reefs, derives a potent chemical defense from a sponge that it eats ( Halichondria sp.). In turn, the nudibranch passes defensive compounds to its egg ribbons, which are similarly conspicuous and physically defenseless. Slices of the dorsal mantle tissue of the nudibranch were rejected in laboratory feeding assays employing two common sympatric predators: an Indo-Pacific reef fish, Thalassoma lunare (Linnaeus), and a reef hermit crab, Dardanus megistos (Herbst). The defensive metabolites, a suite of unusual oxazole-containing macrolides, were isolated from the sponge, the nudibranch, and the nudibranch egg masses at 0.14–0.38, 0.14–0.62, and 2.65% of dry weight, respectively, and were effective inhibitors of feeding by T. lunare at minimum concentrations of 0.01–0.02% dry weight of food pellet. The macrolides were concentrated in the dorsal mantle of the nudibranch, which is most vulnerable to predatory attack, and in the combined digestive gland/gonad, site of both sponge digestion and egg production. The most abundant macrolide in the sponge tissue was not present in the nudibranch or its egg masses, suggesting that chemical modification of this compound takes place upon digestion. In a reef environment dominated by visually oriented predators, the striking color pattern and behavioral responses of Hexabranchus may have arisen with a concomitant elaboration of dietarily derived chemical defenses.

A chemical defense mechanism for the nudibranch cadlina luteomarginata

Abstract The nudibranch Cadlina luteomarginata from San Diego, California, concentrates selected metabolites from the sponges that constitute its diet. Gut analyses revealed that C. leutomarginata consumes at least ten sponges although Axinella sp. and Myxilla incrustans are most frequently eaten. Field observations and analysis of metabolites suggest that keratose sponges Leiosella idia (= Spongia idia ) and Dysidea amblia are also consumed. Three novel compounds, the furan 20 , the isonitrile 23 and the isothiocyanate 24 were identified by analysis of spectral data. The secondary metabolites of C. leuteomarginata were found only in the dorsum, which is exposed to potential predators. Five metabolites of C. luteomarginata were screened for antifeedant activity against fish and all showed some activity at 10–100 μ/mg in food pellets.

A new method for bromination of carbazoles, β-carbolines and iminodibenzyls by use of N-bromosuccinimide and silica gel

Carbazole, N-ethylcarbazole, iminodibenzyl (10,11-dihydro-5H-dibenz[b,f]azepine), N-ethyliminodibenzyl and imipramine are readily mono-, di- or polybrominated in high yields at ambient temperature in dichloromethane by use of the appropriate quantity of N-bromosuccinimide in the presence of silica gel. By contrast, the brominations of the β-carbolines, harmane and norharman, are less selective and give mixtures of products, some of which have unusual substitution patterns.

Alkaloids from the antarctic sponge Kirkpatrickia varialosa. Part 2: Variolin A and N(3′)-methyl tetrahydrovariolin B

Abstract Two pyridopyrrolopyrimidine alkaloids, variolin A (2) and N(3′)-methyl tetrahydrovariolin B (3), have been isolated from the Antarctic sponge Kirkpatrickia varialosa, and their structures determined by X-ray crystallography and interpretation of spectral data respectively. N(3′)-Methyl tetrahydrovariolin B (3) is moderately cytotoxic and showed antifungal activity, while variolin A (2) is weakly cytotoxic.

Antibiotic Metabolites from a Marine Pseudomonad

An antibiotic-producing pseudomonad was isolated from a seawater sample from a La Jolla, Calif., tidepool. The pseudomonad produces two novel antibacterial compounds, 2-n-pentyl-4-quinolinol and 2-n-heptyl-4-quinolinol. It also synthesizes indole-3-carboxaldehyde, 6-bromoindole-3-carboxaldehyde, and the known antibiotic p-hydroxybenzaldehyde. Each of these compounds was identified by analysis of spectral data, and the structures were confirmed by synthesis or comparison with authentic samples.