Characterization of the DNA from the dinoflagellate Crypthecodinium cohnii and implications for nuclear organization.

Although dinoflagellates are eucaryotes, they possess many bacterial nuclear traits. For this reason they are thought by some to be evolutionary intermediates. Dinoflagellates also possess some unusual nuclear traits not seen in either bacteria or higher eucaryotes, such as a very large number of identical appearing, permanently condensed chromosomes suggesting polyteny or polyploidy. We have studied the DNA of the dinoflagellate Crypthecodinium cohnii with respect to DNA per cell, chromosome counts, and renaturation kinetics. The renaturation kinetic results tend to refute extreme polyteny and polyploidy as the mode of nuclear organization. This organism contains 55-60% repeated, interspersed DNA typical of higher eucaryotes. These results, along with the fact that dinoflagellate chromatin contains practically no basic protein, indicate that dinoflagellates may be organisms with a combination of both bacterial and eucaryotic traits.     

Mechanism of action of oritavancin and related glycopeptide antibiotics

Oritavancin (LY333328) is a semisynthetic glycopeptide antibiotic having excellent bactericidal activity against glycopeptide-susceptible and -resistant Gram-positive bacteria. Oritavancin is the N-alkyl-p-chlorophenylbenzyl derivative of chloroeremomycin (LY264826) and is currently in phase III clinical trials for use in Gram-positive infections. Studies show that oritavancin and related alkyl glycopeptides inhibit bacterial cell wall formation by blocking the transglycosylation step in peptidoglycan biosynthesis in a substrate-dependent manner. As with other glycopeptide antibiotics, including vancomycin, the effects of oritavancin on cell wall synthesis are attributable to interactions with dipeptidyl residues of peptidoglycan precursors. Unlike vancomycin, however, oritavancin is strongly dimerized and can anchor to the cytoplasmic membrane, the latter facilitated by its alkyl side chain. Cooperative interactions derived from dimerization and membrane anchoring in situ can be of sufficient strength to enable binding to either dipeptidyl or didepsipeptidyl peptidoglycan residues of vancomycin-susceptible and -resistant enterococci, respectively. This review describes the antibacterial activity of oritavancin, and examines the evidence supporting the proposed mechanism of action for this agent and related analogs.     

What is the role of arbuscular mycorrhizal fungi in plant-to-ecosystem responses to Elevated atmospheric CO2?

 We advocate the concept of an arbuscular mycorrhiza (AM) as a temporally and spatially complex symbiosis representing a suite of hosts and fungi, as against the more traditional "dual organism" view. We use the hierarchical framework presented in Fig. 1 as a basis for organizing many unanswered questions, and several questions that have not been asked, concerning the role of AM in responses to elevated atmospheric CO₂. We include the following levels: plant host, plant population, plant community, functional group and ecosystem. Measurements of the contributions of AM fungi at the various levels require the use of different response variables. For example, hyphal nutrient translocation rates or percent AM root infection may be important measures at the individual plant level, but hyphal biomass or glomalin production and turnover are more relevant at the ecosystem level. There is a discrepancy between our knowledge of the multifaceted role of AM fungi in plant and ecosystem ecology and most of the current research aimed at elucidating the importance of this symbiosis in global-change scenarios. Our framework for more integrated and multifactorial research on mycorrhizal involvement in regulating CO₂ responses may also serve to enhance communication between researchers working at different scales on large global-change ecosystem projects. Accepted: 12 February 1999     

Cloning and analysis of DNA sequences from Streptomyces hygroscopicus encoding geldanamycin biosynthesis

A gene library constructed from large (20 kb) fragments of total DNA from the geldananmycin-producing strain Streptomyces hygroscopicus 3602 cloned in the plasmid vector pIJ61 were used to transform S. lividans TK24. Three transformants of about 800 tested were found to have acquired the ability to produce an antibiotic lethal to a geldanamycin-sensitive strain of Bacillus subtilis. The plasmids isolated from these transformants, pIA101, pIA102 and pIA103, each contained an insert of 15 kb. A 4.5 kb DNA fragment from the insert in pIA102 hybridised to DNA from S. hygroscopicus 3602 and to DNA encoding part of the erythromycin polyketide synthase but not to S. lividans TK24 DNA. The integration-defective phage vector C31 KC515 containing this 4.5 kb fragment was able to lysogenise S. hygroscopicus 3602 to produce lysogens defective in geldanamycin production. Loss of the prophage restored the ability to produce geldanamycin. Extracts of fermentation broth cultures of S. lividans containing pIA101, pIA102 and pIA102 and pIA103 analysed by thin-layer chromatography (TLC) contained compounds identical or very similar to purified geldanamycin, which were not present in S. lividans. These compounds showed a mass spectrum indistinguishable from geldanamycin. The evidence suggests that the clones contain DNA sequences encoding functions required for geldanamycin biosynthesis including components of the polyketide synthase.     

Suspension of non-viable fungal spores as a standard for platelet counting.

In the search among biological particles for a standard for counting human platelets, a strain of Absidia corymbifera was found to have spores that resembled platelets. After fixing in formalin and autoclaving the spores had a similar mean cell volume to that of human platelets. A suspension of these killed spores was tested in 100 laboratories and gave consistent results as a standard for human platelet counting. The Absidia corymbifera standard can be used in electronic counting methods but not in sedimentation methods, as the spores will be removed by centrifugation.     

Comparative analysis of galactoside-binding lectins isolated from mammalian spleens

Galactoside-inhibitable lectins have been isolated from rabbit, rat, mouse, pig, lamb, calf, and human spleens. Native molecular mass, subunit structure, pI, and hemagglutinating activity have been compared for these lectins. The yields of lectin varied from 1.8 mg/kg for rabbit spleen to 79 mg/kg for lamb spleen. Pig, lamb, calf, and human spleen lectins yielded single protein peaks when subjected to Superose 12 fast-protein liquid chromatography. The apparent molecular mass for these lectins was 33-34 kDa. In contrast, rat and mouse spleen lectin preparations were separated into three components ranging from 8.4 to 34 kDa. Superose 12 chromatography of rabbit spleen lectin revealed the presence of at least six components. Gradient slab gel sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed the presence of single polypeptides for pig, calf, lamb, and human lectins corresponding to a molecular mass of 14-14.5 kDa. Multiple polypeptides were detected for the mouse, rat, and rabbit lectins. The molecular mass of the major polypeptides were 15, 15, and 17 kDa for rat, mouse, and rabbit, respectively. The presence of isolectins in all preparations was shown by isoelectric focusing. The major isolectins were acidic proteins with pI 4.38-4.80. Hemagglutination and hemagglutination inhibition assays demonstrated similarities as well as differences among the lectin preparations. Hemagglutinating activity could not be demonstrated in rabbit spleen extracts nor for isolated putative lectin. Human buffy coat cells were reversibly agglutinated by calf and human spleen lectins, demonstrating the presence of leucocyte cell surface lectin receptors.     

Dynamic viral populations in hypersaline systems as revealed by metagenomic assembly

Viruses of the Bacteria and Archaea play important roles in microbial evolution and ecology, and yet viral dynamics in natural systems remain poorly understood. Here, we created de novo assemblies from 6.4 Gbp of metagenomic sequence from eight community viral concentrate samples, collected from 12 h to 3 years apart from hypersaline Lake Tyrrell (LT), Victoria, Australia. Through extensive manual assembly curation, we reconstructed 7 complete and 28 partial novel genomes of viruses and virus-like entities (VLEs, which could be viruses or plasmids). We tracked these 35 populations across the eight samples and found that they are generally stable on the timescale of days and transient on the timescale of years, with some exceptions. Cross-detection of the 35 LT populations in three previously described haloviral metagenomes was limited to a few genes, and most previously sequenced haloviruses were not detected in our samples, though 3 were detected upon reducing our detection threshold from 90% to 75% nucleotide identity. Similar results were obtained when we applied our methods to haloviral metagenomic data previously reported from San Diego, CA: 10 contigs that we assembled from that system exhibited a variety of detection patterns on a timescale of weeks to 1 month but were generally not detected in LT. Our results suggest that most haloviral populations have a limited or, possibly, a temporally variable global distribution. This study provides high-resolution insight into viral biogeography and dynamics and it places "snapshot" viral metagenomes, collected at a single time and location, in context.     

Mesothelium of the murine allantois exhibits distinct regional properties

The rodent allantois is thought to be unique amongst mammals in not having an endodermal component. Here, we have investigated the mesothelium, or outer surface, of murine umbilical precursor tissue, the allantois (～7.25–8.5 days postcoitum, dpc) to discover whether it exhibits the properties of an epithelium. A combination of morphology, challenge with biotinylated dextran amines (BDAs), and immunohistochemistry revealed that the mesothelium of the mouse allantois exhibits distinct regional properties. By headfold stages (～7.75–8.0 dpc), distal mesothelium was generally squamous in shape, and highly permeable to BDA challenge, whereas ventral proximal mesothelium, referred to as “ventral cuboidal mesothelium” (VCM) for the characteristic cuboidal shape of its cells, was relatively impermeable. Although “dorsal cuboidal mesothelium” (DCM) resembled the VCM in cell shape, its permeability to BDA was intermediate between the other two regions. Results of immunostaining for Zonula Occludens‐1 (ZO‐1) and Epithelial‐cadherin (E‐cadherin), together with transmission electron microscopy (TEM), suggested that impermeability in the VCM may be due to greater cellular contact area between cells and close packing rather than to maturity of tight junctions, the latter of which, by comparison with the visceral yolk sac, appeared to be rare or absent from the allantoic surface. Both VCM and DCM exhibited an ultrastructure more favorable for protein synthesis than did the distal squamous mesothelium; however, at most stages, VCM exhibited robust afadin (AF‐6), whereas the DCM uniquely contained alpha‐4‐integrin. These observations demonstrate that the allantoic mesothelium is not a conventional epithelium but possesses regional ultrastructural, functional and molecular differences that may play important roles in the correct deployment of the umbilical cord and its associated vascular, hematopoietic, and other cell types. J. Morphol., 2011. © 2011 Wiley‐Liss, Inc.     

20(S)-Protopanaxadiol Inhibition of Progression and Growth of Castration-Resistant Prostate Cancer

Castration-resistant progression of prostate cancer after androgen deprivation therapies remains the most critical challenge in the clinical management of prostate cancer. Resurgent androgen receptor (AR) activity is an established driver of castration-resistant progression, and upregulation of the full-length AR (AR-FL) and constitutively-active AR splice variants (AR-Vs) has been implicated to contribute to the resurgent AR activity. We reported previously that ginsenoside 20(S)-protopanaxadiol-aglycone (PPD) can reduce the abundance of both AR-FL and AR-Vs. In the present study, we further showed that the effect of PPD on AR expression and target genes was independent of androgen. PPD treatment resulted in a suppression of ligand-independent AR transactivation. Moreover, PPD delayed castration-resistant regrowth of LNCaP xenograft tumors after androgen deprivation and inhibited the growth of castration-resistant 22Rv1 xenograft tumors with endogenous expression of AR-FL and AR-Vs. This was accompanied by a decline in serum prostate-specific antigen levels as well as a decrease in AR levels and mitoses in the tumors. Notably, the 22Rv1 xenograft tumors were resistant to growth inhibition by the next-generation anti-androgen enzalutamide. The present study represents the first to show the preclinical efficacy of PPD in inhibiting castration-resistant progression and growth of prostate cancer. The findings provide a rationale for further developing PPD or its analogues for prostate cancer therapy.     

Elevation of superior vena caval pressure increases extravascular lung water after endotoxemia

In many sheep Escherichia coli endotoxin results in pulmonary hypertension, increased microvascular permeability, pulmonary edema, and increased central venous pressure. Since lung lymph drains into the systemic veins, increases in venous pressure may impair lymph flow sufficiently to enhance the accumulation of extravascular fluid. We tested the hypothesis that, following endotoxin, elevating the venous pressure would increase extravascular fluid. Thirteen sheep were chronically instrumented with catheters to monitor left atrial pressure (LAP), pulmonary arterial pressure (PAP), and superior vena caval pressure (SVCP) as well as balloons to elevate LAP and SVCP. These sheep received 4 micrograms/kg endotoxin, and following the pulmonary hypertensive spike the left atrial balloon was inflated so that (PAP + LAP)/2 = colloid osmotic pressure. It was necessary to control PAP + LAP in this way to minimize the sheep-to-sheep differences in the pulmonary hypertension. We elevated the SVCP to 10 or 17 mmHg or allowed it to stay low (3.2 mmHg). After a 3-h period, we killed the sheep and removed the right lungs for determination of the extravascular fluid-to-blood-free dry weight ratio (EVF). Sheep with SVCP elevated to 10 or 17 mmHg had significant increases in EVF (5.2 +/- 0.1 and 5.6 +/- 1.2) compared with the sheep in which we did not elevate SVCP (EVF = 4.5 +/- 0.4). These results indicate that sustained elevation in central venous pressure in patients contributes to the amount of pulmonary edema associated with endotoxemia.