Antagonism between Cry1Ac1 and Cyt1A1 toxins of bacillus thuringiensis

Most strains of the insecticidal bacterium Bacillus thuringiensis have a combination of different protoxins in their parasporal crystals. Some of the combinations clearly interact synergistically, like the toxins present in B. thuringiensis subsp. israelensis. In this paper we describe a novel joint activity of toxins from different strains of B. thuringiensis. In vitro bioassays in which we used pure, trypsin-activated Cry1Ac1 proteins from B. thuringiensis subsp. kurstaki, Cyt1A1 from B. thuringiensis subsp. israelensis, and Trichoplusia ni BTI-Tn5B1-4 cells revealed contrasting susceptibility characteristics. The 50% lethal concentrations (LC50s) were estimated to be 4,967 of Cry1Ac1 per ml of medium and 11.69 ng of Cyt1A1 per ml of medium. When mixtures of these toxins in different proportions were assayed, eight different LC50s were obtained. All of these LC50s were significantly higher than the expected LC50s of the mixtures. In addition, a series of bioassays were performed with late first-instar larvae of the cabbage looper and pure Cry1Ac1 and Cyt1A1 crystals, as well as two different combinations of the two toxins. The estimated mean LC50 of Cry1Ac1 was 2.46 ng/cm2 of diet, while Cyt1A1 crystals exhibited no toxicity, even at very high concentrations. The estimated mean LC50s of Cry1Ac1 crystals were 15.69 and 19.05 ng per cm2 of diet when these crystals were mixed with 100 and 1,000 ng of Cyt1A1 crystals per cm2 of diet, respectively. These results indicate that there is clear antagonism between the two toxins both in vitro and in vivo. Other joint-action analyses corroborated these results. Although this is the second report of antagonism between B. thuringiensis toxins, our evidence is the first evidence of antagonism between toxins from different subspecies of B. thuringiensis (B. thuringiensis subsp. kurstaki and B. thuringiensis subsp. israelensis) detected both in vivo and in vitro. Some possible explanations for this relationship are discussed.     

Tumor‐associated macrophages (TAMs) depend on ZEB1 for their cancer‐promoting roles

Accumulation of tumor‐associated macrophages (TAMs) associates with malignant progression in cancer. However, the mechanisms that drive the pro‐tumor functions of TAMs are not fully understood. ZEB1 is best known for driving an epithelial‐to‐mesenchymal transition (EMT) in cancer cells to promote tumor progression. However, a role for ZEB1 in macrophages and TAMs has not been studied. Here we describe that TAMs require ZEB1 for their tumor‐promoting and chemotherapy resistance functions in a mouse model of ovarian cancer. Only TAMs that expressed full levels of Zeb1 accelerated tumor growth. Mechanistically, ZEB1 expression in TAMs induced their polarization toward an F4/80^low pro‐tumor phenotype, including direct activation of Ccr2. In turn, expression of ZEB1 by TAMs induced Ccl2, Cd74, and a mesenchymal/stem‐like phenotype in cancer cells. In human ovarian carcinomas, TAM infiltration and CCR2 expression correlated with ZEB1 in tumor cells, where along with CCL2 and CD74 determined poorer prognosis. Importantly, ZEB1 in TAMs was a factor of poorer survival in human ovarian carcinomas. These data establish ZEB1 as a key factor in the tumor microenvironment and for maintaining TAMs’ tumor‐promoting functions.     

Amino acid sequence versus morphological data and the interordinal relationships of mammals

To a large extent, the mutual affinities of the mammalian orders continue to puzzle systematists, even though comparative anatomy and amino acid sequencing offer a massive data base from which these relationships could potentially be adduced. In the present paper the consistency index--the number of character states less the number of characters in a data set, divided by the total number of changes in the character states on a cladogram--was used to examine the relative resolving powers of recently published morphological and molecular- sequence data. Consistency indices were calculated for previously published alpha crystallin A chain and myoglobin amino acid-sequence cladograms and for four original amino acid-sequence cladograms (alpha crystallin A, myoglobin, and alpha and beta hemoglobin); these were found to be comparable to the consistency indices of morphologically based cladograms. Qualitative comparisons between the morphologically based and molecularly based trees were also made; only moderate congruence between the two was observed. Moreover, there was a general lack of congruence between the cladograms specified by each of the four proteins. Amino acid-sequence and morphological data agreed on the placement of edentates as an early eutherian offshoot and on the grouping of hyracoids, proboscideans, and sirenians. Otherwise there was only limited congruence: morphology strongly supported the grouping of lagomorphs and rodents and the alliance of pholidotes and edentates, but sequence analyses did not. The placement of tubulidentates differed widely among proteins. Morphology indicated the close association of sirenians with proboscideans; proteins suggested a pairing of sirenians with hyracoids. Sequence data did not identify many (morphologically well-diagnosed) orders as monophyletic (e.g., Lagomorpha).(ABSTRACT TRUNCATED AT 250 WORDS)      

Histological and histochemical study of the uropygial gland of chimango caracara (Milvago chimango vieillot, 1816)

The uropygial glands of birds are sebaceous organs that contribute to the water-repellent properties of the feather coat. We studied the histological and histochemical characteristics of the uropygial gland of chimango caracara using hematoxylin and eosin (H & E), Gomori´s trichrome, orcein, Gomori´s reticulin, periodic acid-Schiff (PAS), Alcian blue (AB) and a variety of lectins. The gland is composed of two lobes and a papilla with 20 downy feathers. It is surrounded by a capsule of dense connective tissue that contains elastic, reticular and smooth muscle fibers. The papilla is delicate and has two excretory ducts. The gland mass relative to body mass was 0.143%. Both adenomer cells and their secretions were stained with Sudan IV, PAS and AB, and were positive for numerous lectins that indicated the presence of lipids and carbohydrates. Immunohistochemical techniques to detect PCNA confirmed cell proliferation in the basal stratum of the adenomer cells. The lipids and glycoconjugates secreted by the uropygial gland serve numerous functions including protection against microorganisms.     

Rheumatoid synovial fluid interleukin-17-producing CD4 T cells have abundant tumor necrosis factor-alpha co-expression,but little interleukin-22 and interleukin-23R expression

Introduction  

Th17 cells have been implicated in the pathogenesis of rheumatoid arthritis (RA). The aim of this study was to systematically analyse the phenotype, cytokine profile and frequency of interleukin-17 (IL-17) producing CD4-positive T cells in mononuclear cells isolated from peripheral blood, synovial fluid and synovial tissue of RA patients with established disease, and to correlate cell frequencies with disease activity.     

Transition of healthy to diseased synovial tissue in rheumatoid arthritis is associated with gain of mesenchymal/fibrotic characteristics

The healthy synovial lining layer consists of a single cell layer that regulates the transport between the joint cavity and the surrounding tissue. It has been suggested that abnormalities such as somatic mutations in the p53 tumor-suppressor gene contribute to synovial hyperplasia and invasion in rheumatoid arthritis (RA). In this study, expression of epithelial markers on healthy and diseased synovial lining tissue was examined. In addition, we investigated whether a regulated process, resembling epithelial to mesenchymal transition (EMT)/fibrosis, could be responsible for the altered phenotype of the synovial lining layer in RA. Synovial tissue from healthy subjects and RA patients was obtained during arthroscopy. To detect signs of EMT, expression of E-cadherin (epithelial marker), collagen type IV (indicator of the presence of a basement membrane) and alpha-smooth muscle actin (alpha-sma; a myofibroblast marker) was investigated on frozen tissue sections using immunohistochemistry. Fibroblast-like synoviocytes (FLSs) from healthy subjects were isolated and subjected to stimulation with synovial fluid (SF) from two RA patients and to transforming growth factor (TGF)-beta. To detect whether EMT/fibrotic markers were increased, expression of collagen type I, alpha-sma and telopeptide lysylhydroxylase (TLH) was measured by real time PCR. Expression of E-cadherin and collagen type IV was found in healthy and arthritic synovial tissue. Expression of alpha-sma was only found in the synovial lining layer of RA patients. Stimulation of healthy FLSs with SF resulted in an upregulation of alpha-sma and TLH mRNA. Collagen type I and TLH mRNA were upregulated after stimulation with TGF-beta. Addition of bone morphogenetic protein (BMP)-7 to healthy FLS stimulated with SF inhibited the expression of alpha-sma mRNA. The finding that E-cadherin and collagen type IV are expressed in the lining layer of healthy and arthritic synovium indicates that these lining cells display an epithelial-like phenotype. In addition, the presence of alpha-sma in the synovial lining layer of RA patients and induction of fibrotic markers in healthy FLSs by SF from RA patients indicate that a regulated process comparable to EMT might cause the alteration in phenotype of RA FLSs. Therefore, BMP-7 may represent a promising agent to counteract the transition imposed on synoviocytes in the RA joint.     

Distinct transthyretin oxidation isoform profile in spinal fluid from patients with Alzheimer’s disease and mild cognitive impairment

Background

Transthyretin (TTR), an abundant protein in cerebrospinal fluid (CSF), contains a free, oxidation-prone cysteine residue that gives rise to TTR isoforms. These isoforms may reflect conditions in vivo. Since increased oxidative stress has been linked to neurodegenerative disorders such as Alzheimer’s disease (AD) it is of interest to characterize CSF-TTR isoform distribution in AD patients and controls. Here, TTR isoforms are profiled directly from CSF by an optimized immunoaffinity-mass spectrometry method in 76 samples from patients with AD (n = 37), mild cognitive impairment (MCI, n = 17)), and normal pressure hydrocephalus (NPH, n = 15), as well as healthy controls (HC, n = 7). Fractions of three specific oxidative modifications (S-cysteinylation, S-cysteinylglycinylation, and S-glutathionylation) were quantitated relative to the total TTR protein. Results were correlated with diagnostic information and with levels of CSF AD biomarkers tau, phosphorylated tau, and amyloid β_1-42 peptide.

Results

Preliminary data highlighted the high risk of artifactual TTR modification due to ex vivo oxidation and thus the samples for this study were all collected using strict and uniform guidelines. The results show that TTR is significantly more modified on Cys(10) in the AD and MCI groups than in controls (NPH and HC) (p ≤ 0.0012). Furthermore, the NPH group, while having normal TTR isoform distribution, had significantly decreased amyloid β peptide but normal tau values. No obvious correlations between levels of routine CSF biomarkers for AD and the degree of TTR modification were found.

Conclusions

AD and MCI patients display a significantly higher fraction of oxidatively modified TTR in CSF than the control groups of NPH patients and HC. Quantitation of CSF-TTR isoforms thus may provide diagnostic information in patients with dementia symptoms but this should be explored in larger studies including prospective studies of MCI patients. The development of methods for simple, robust, and reproducible inhibition of in vitro oxidation during CSF sampling and sample handling is highly warranted. In addition to the diagnostic information the possibility of using TTR as a CSF oxymeter is of potential value in studies monitoring disease activity and developing new drugs for neurodegenerative diseases.     

Identification and characterization of a prevacuolar compartment in stigmas of nicotiana alata

The stigmas of the ornamental tobacco plant Nicotiana alata accumulate large quantities of a series of 6-kD proteinase inhibitors (PIs) in the central vacuole that are derived from a 40-kD precursor protein, Na-PI. The sorting information that directs Na-PI to the vacuole is likely to reside in a C-terminal propeptide domain of 25 amino acids that forms an amphipathic alpha helix. Using cell fractionation techniques, we have examined transit of Na-PI through the endomembrane system and have identified a prevacuolar compartment that contains Na-PI with an intact targeting signal. In contrast, the targeting signal is not present on the predominant form of Na-PI in the vacuole. The prevacuolar compartment is marked by the presence of homologs of both the t-SNARE, PEP12p, and the putative vacuolar sorting receptor BP-80. Cross-linking and affinity precipitation studies revealed that Na-PI associates with BP-80 within this compartment, providing in vivo evidence for the function of BP-80 as a sorting receptor for a protein with a C-terminal vacuolar targeting signal.     

Hes1 Increases the Invasion Ability of Colorectal Cancer Cells via the STAT3-MMP14 Pathway

The Notch pathway contributes to self-renewal of tumor-initiating cell and inhibition of normal colonic epithelial cell differentiation. Deregulated expression of Notch1 and Jagged1 is observed in colorectal cancer. Hairy/enhancer of split (HES) family, the most characterized targets of Notch, involved in the development of many cancers. In this study, we explored the role of Hes1 in the tumorigenesis of colorectal cancer. Knocking down Hes1 induced CRC cell senescence and decreased the invasion ability, whereas over-expression of Hes1 increased STAT3 phosphorylation activity and up-regulated MMP14 protein level. We further explored the expression of Hes1 in human colorectal cancer and found high Hes1 mRNA expression is associated with poor prognosis in CRC patients. These findings suggest that Hes1 regulates the invasion ability through the STAT3-MMP14 pathway in CRC cells and high Hes1 expression is a predictor of poor prognosis of CRC.     

Selective gray matter staining of human brain slices: optimized use of cadaver materials

We report a novel staining technique for human brain slices that distinguishes clearly gray from white matter. Previously described techniques using either Prussian blue (Berlin blue) or phthalocyanine dyes usually have included a hot phenol pretreatment to prevent white matter staining. The technique we describe here does not require hot phenol pretreatment and allows the use of brains stored for postmortem periods of one to two years prior to staining. Our technique involves staining with copper(II) phthalocyanine-tetrasulfonic acid tetrasodium salt 1% in water for 2 h followed by acetic acid treatment; this produces excellent blue staining of gray matter with little white matter staining. The stained brain slices are excellent for teaching human brain anatomy and/or pathology, or for research purposes.