Type V collagen as the target for type-3 fimbriae, enterobacterial adherence organelles

Tissue-binding specificity of the type-3 fimbriae of pathogenic enteric bacteria was determined using frozen sections of human kidney. A wild-type Klebsiella sp. strain and the recombinant strain Escherichia coli HB101(pFK12), both expressing type-3 fimbriae, as well as the purified type-3 fimbriae effectively bound to sites at or adjacent to tubular basement membranes, Bowman's capsule, arterial walls, and the interstitial connective tissue. Bacterial adherence to kidney was decreased after collagenase treatment of the tissue sections. Recombinant strains expressing type-3 fimbriae specifically adhered to type V collagen immobilized on glass slides, whereas other collagens, fibronectin or laminin did not support bacterial adherence. In accordance with these findings, specific binding of purified type-3 fimbriae to immobilized type V collagen was demonstrated. Specific adhesion to type V collagen was also seen with the recombinant strain HB101(pFK52/pDC17), which expresses the mrkD gene of the type-3 fimbrial gene cluster in association with the pap-encoded fimbrial filament of E. coli, showing that the observed binding was mediated by the minor lectin (MrkD) protein of the type-3 fimbrial filament. The interaction is highly dependent on the conformation of type V collagen molecules since type V collagen in solution did not react with the fimbriae. Specific binding to type V collagen was also exhibited by type-3 fimbriate strains of Yersinia and Salmonella, showing that the ability to use type V collagen as tissue target is widespread among enteric bacteria.     

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.     

Brain-derived neurotrophic factor in the hypothalamic paraventricular nucleus reduces energy intake

Recent studies show that brain-derived neurotrophic factor (BDNF) decreases feeding and body weight after peripheral and ventricular administration. BDNF mRNA and protein, and its receptor tyrosine kinase B (TrkB) are widely distributed in the hypothalamus and other brain regions. However, there are few reports on specific brain sites of actions for BDNF. We evaluated the effect of BDNF in the hypothalamic paraventricular nucleus (PVN) on feeding. BDNF injected unilaterally or bilaterally into the PVN of food-deprived and nondeprived rats significantly decreased feeding and body weight gain within the 0- to 24-h and 24- to 48-h postinjection intervals. Effective doses producing inhibition of feeding behavior did not establish a conditioned taste aversion. PVN BDNF significantly decreased PVN neuropeptide Y (NPY)-induced feeding at 1, 2, and 4 h following injection. BDNF administration in the PVN abolished food-restriction-induced NPY gene expression in the hypothalamic arcuate nucleus. In conclusion, BDNF in the PVN significantly decreases food intake and body weight gain, suggesting that the PVN is an important site of action for BDNF in its effects on energy metabolism. Furthermore, BDNF appears to interact with NPY in its anorectic actions, although a direct effect on NPY remains to be established.