Development of In Vivo Sponge Cultures: Particle Feeding by the Tropical Sponge Pseudosuberites aff. andrewsi

The rate of food particle uptake of the tropical sponge Pseudosuberites aff. andrewsi was studied in relation to particle concentrations and particle size. A range of different concentrations of either the marine microalga Dunaliella tertiolecta (∼5–8 μm) or the marine cyanobacterium Synechococcus sp. (∼1 μm) was supplied to the sponges. D. tertiolecta had a pronounced effect on the filtration activity of the sponges: at concentrations higher than approximately 4 × 10⁵ cells/cm³, the filtration rates dropped dramatically. Such a clear effect was not found for Synechococcus sp. The results further showed that the maximal amount of food (when expressed in organic carbon) that can be taken up per cubic centimeter of sponge volume per unit of time should in principle be sufficient to enable growth (irrespective of the food particle type). At the maximal food particle concentration that did not affect the filtration rates, the uptake of organic carbon is already highly in excess of the amount of organic carbon that the sponges need to cope with their respiratory demand. Based on these findings, a series of growth experiments was carried out in which the sponges were subjected to a constant concentration of different types of food particles (Synechococcus sp. and the microalgae Chlorella sorokiniana and Nannochloropsis sp). Although initial growth was sometimes observed, continuous growth at a constant rate could not be obtained. It is concluded that qualitative aspects of feeding rather than quantitative aspects are the key to successful in vivo sponge culture. Received December 20, 2000; accepted March 26, 2001     

PCR template preparation for capillary DNA sequencing

Fluorescence-based capillary DNA sequencing has facilitated the early completion of several complex sequencing projects. While capillary systems offer great benefits in terms of ease of use and automation, we find that they are sufficiently different from slab gel separation methodologies, demanding re-examination of the protocols used to generate and use DNA sequencing templates. We have recently initiated a large-scale Human Open Reading Frame EST project involving 30 laboratories feeding 11 MegaBace 1000 capillary sequencers. The group has already produced more than 300,000 valid sequences. The most successful template preparation protocol we have found is described here. We have found that a crucial step is the standardization of the quantity and quality of the templates, which have been achieved by overnight bacterial culture followed by PCR using limiting amounts of primers. Using this protocol, there is no need for post-PCR purification, and the final preparation cost is US $0.09/template. After sequencing 10,848 templates using this protocol, 78% of the reads were accepted (after discarding vectors without inserts and inserts smaller than 100 nucleotides), and 85% of the total number of bases had Phred scores of 15 or above.     

Regulation of secretory granule pH in insulin-secreting cells

Luminal acidification is important for the maturation of secretory granules, yet little is known regarding the regulation of pH within them. A pH-sensitive green fluorescent protein (EGFP) was targeted to secretory granules in RIN1046-38 insulinoma cells by using a construct in which the EGFP gene was preceded by the nucleotide sequence for human growth hormone. Stimulatory levels of glucose doubled EGFP secretion from cell cultures, and potentiators of glucose-induced insulin secretion enhanced EGFP release. Thus this targeted EGFP is useful for population measurements of secretion. However, less than ~4% of total cell EGFP was released after 1.5 h of stimulation. Consequently, when analyzed in single cells, fluorescence of the targeted EGFP acts as an indicator of pH within secretory granules. Glucose elicited a decrease in granule pH, whereas inhibitors of the V-type H(+)-ATPase increased pH and blocked the glucose effect. Granule pH also was modified by effectors of the protein kinase A pathway, with activation eliciting granule alkalinization, suggesting that potentiation of peptide release by cAMP may involve regulated changes in secretory granule pH.     

The evolutionary history of kinetoplastids and their kinetoplasts

Despite extensive phylogenetic analysis of small subunit ribosomal RNA (SSUrRNA) genes, the deep-level relationships among kinetoplastids remain poorly understood, limiting our grasp of their evolutionary history, especially the origins of their bizarre mitochondrial genome organizations. In this study we examine the SSUrRNA data in the light of a new marker--cytoplasmic heat shock protein 90 (hsp90) sequences. Our phylogenetic analyses divide kinetoplastids into four main clades. Clades 1-3 include the various bodonid kinetoplastids. Trypanosomatids comprise the fourth clade. SSUrRNA analyses give vastly different and poorly supported positions for the root of the kinetoplastid tree, depending on the out-group and analysis method. This is probably due to the extraordinary length of the branch between kinetoplastids and any out-group. In contrast, almost all hsp90 analyses place the root between clade 1 (including Dimastigella, Rhynchomonas, several Bodo spp., and probably Rhynchobodo) and all other kinetoplastids. Maximum likelihood and maximum likelihood distance analyses of hsp90 protein and second codon-position nucleotides place trypanosomatids adjacent to Bodo saltans and Bodo cf. uncinatus (clade 3), as (weakly) do SSUrRNA analyses. Hsp90 first codon- plus second codon-position nucleotide analyses return a slightly different topology. We show that this may be an artifact caused, in part, by the different evolutionary behavior of first- and second-codon positions. This study provides the most robust evidence to date that trypanosomatids are descended from within bodonids and that B. saltans is a close relative of trypanosomatids. A total reevaluation of the high-level systematics within kinetoplastids is needed. We confirm that the interlocking network organization of kinetoplast DNA seen in trypanosomatids is a derived condition within kinetoplastids but suggest that open-conformation minicircles may have arisen early in kinetoplastid evolution. Further understanding of the evolution of kinetoplast structure and RNA editing is hampered by a paucity of data from basal (i.e., clade 1) bodonids.     

Evolutionary history of "early-diverging" eukaryotes: the excavate taxon Carpediemonas is a close relative of Giardia

Diplomonads, such as Giardia, and their close relatives retortamonads have been proposed as early-branching eukaryotes that diverged before the acquisition-retention of mitochondria, and they have become key organisms in attempts to understand the evolution of eukaryotic cells. In this phylogenetic study we focus on a series of eukaryotes suggested to be relatives of diplomonads on morphological grounds, the "excavate taxa". Phylogenies of small subunit ribosomal RNA (SSU rRNA) genes, alpha-tubulin, beta-tubulin, and combined alpha- + beta-tubulin all scatter the various excavate taxa across the diversity of eukaryotes. But all phylogenies place the excavate taxon Carpediemonas as the closest relative of diplomonads (and, where data are available, retortamonads). This novel relationship is recovered across phylogenetic methods and across various taxon-deletion experiments. Statistical support is strongest under maximum-likelihood (ML) (when among-site rate variation is modeled) and when the most divergent diplomonad sequences are excluded, suggesting a true relationship rather than an artifact of long-branch attraction. When all diplomonads are excluded, our ML SSU rRNA tree actually places retortamonads and Carpediemonas away from the base of the eukaryotes. The branches separating excavate taxa are mostly not well supported (especially in analyses of SSU rRNA data). Statistical tests of the SSU rRNA data, including an "expected likelihood weights" approach, do not reject trees where excavate taxa are constrained to be a clade (with or without parabasalids and Euglenozoa). Although diplomonads and retortamonads lack any mitochondria-like organelle, Carpediemonas contains double membrane-bounded structures physically resembling hydrogenosomes. The phylogenetic position of Carpediemonas suggests that it will be valuable in interpreting the evolutionary significance of many molecular and cellular peculiarities of diplomonads.     

Long range molecular wire behaviour in a metal complex of DNA

M-DNA is a complex of metal ions such as Zn(2+) with duplex DNA. Previous results showed that the fluorescence of a donor fluorophore was quenched when an acceptor fluorophore was placed at the opposite end of a short M-DNA duplex. In order to investigate further the molecular wire behaviour of M-DNA, 30-mer duplexes were constructed with fluorescein as donor and rhodamine, pyrene and the cyanine dyes, Cy5 and Cy5.5 as acceptors. Good quenching was observed in all cases even though the efficiency of resonance energy transfer was calculated to be < 5%. The distance dependence of quenching was investigated by preparing doubly-labelled duplexes ranging in length from 20 to 1,000 base pairs. Upon formation of M-DNA significant quenching of the fluorescence of the donor fluorophore was observed in duplexes up to 500 base pairs in length. The amount of quenching decreased with increasing length of the duplexes with a shallow distance dependence. The results are consistent with an electron transfer mechanism in which the electron hops between metal centers. This process can occur efficiently over long distances.     

Developing a branch-mimicking trap for adult plum curculios

We conducted field and semi-field experiments to determine the most visually stimulating and acceptable version of a branch-mimicking trap (PVC cylinder topped with an inverted screen funnel) to capture adult plum curculios, Conotrachelus nenuphar (Herbst) (Coleoptera: Curculionidae) foraging within canopies of host trees. Cylinder traps (25 cm tall × 6 cm diam) coated with different colors of gloss or flat latex paint captured similar numbers of feral adults in field tests. Tall cylinder traps (50 cm tall × 6 cm diam) captured significantly more feral adults in field tests in 1999 than cylinder traps of lesser height and/or different diameter, and significantly more released adults arrived at traps with these dimensions compared to cylinder traps of lesser height in semi-field tests. In field and semi-field tests, respectively, significantly more adults were captured by and numerically more adults arrived at tall cylinder traps (50 cm tall × 6 cm diam) attached in an upright, standard position to a horizontal limb than attached at angles of 45°, 90°, 135°, or 180° to horizontal limbs.     

Use of fine needle aspiration biopsy in radiofrequency ablation

OBJECTIVE: To demonstrate the utility of fine needle aspiration biopsy (FNAB) in radiofrequency ablation (RFA) of suspected metastatic tumors at various sites. STUDY DESIGN: Eighteen patients referred for RFA underwent 21 aspirations prior to the procedure. A radiologist performed the FNAB and RFA with radiographic guidance. On-site preliminary evaluation of Diff-Quik-stained smears were followed with Papanicolaou staining. A final diagnosis was rendered and compared to the preliminary diagnosis. RESULTS: Liver was aspirated in 17 cases, lung in 3 cases and pubic bone in 1. Fifteen aspirates were deemed on site as positive or suspicious for malignancy. A preliminary, on-site diagnosis of benign was given in one case and adequate with deferment for review of all slides in four others. One FNAB was unsatisfactory. All but one (patient with benign diagnosis) then immediately proceeded to RFA of the lesion. After review of additional slides, the final diagnosis confirmed metastatic adenocarcinoma in 16, hepatocellular carcinoma in 2 and metastatic squamous cell carcinoma in 1. One FNAB yielded benign hepatocytes, and one was unsatisfactory. CONCLUSION: FNAB is an accurate, safe and rapid method of confirming disease in patients just prior to undergoing RFA.