Ectopic expression of β-lactoglobulin/human serum albumin fusion genes in transgenic mice: hormonal regulation andin situ localization

We produced transgenic mice carrying the native sheep -lactoglobulin (BLG) or fusion genes composed of the BLG promoter and human serum albumin (HSA) minigenes. BLG was expressed exclusively in the mammary glands of the virgin and lactating transgenic mice evaluated. In contrast, transgenic females carrying the BLG/HSA fusion constructs also expressed the HSA RNA ectopically in skeletal muscle, kidney, brain, spleen, salivary gland and skin. Ectopic expression of HSA RNA was detected only in strains that express the transgene in the mammary gland. There was no obvious correlation between the level of the HSA RNA expressed in the mammary gland and that found ectopically. In three transgenic strains analysed, the expression of HSA RNA in kidney and skeletal muscle increased during pregnancy and lactation, whereas in the brain HSA expression decreased during lactation in one of the strains. HSA protein was synthesized in skeletal muscle and skin of strain #23 and its level was higher in lactating mice compared with virgin mice. Expression of HSA was also analysed in males and was found to be more stringently controlled than in females of the same strains.In situ hybridization analyses localized the expressed transgene in the skin, kidney, brain and salivary glands of various transgenic strains. Distinct strain-specific and cell-type specific HSA expression patterns were observed in the skin. This is in contrast to the exclusive expression of the HSA transgene in epithelial cells surrounding the alveoli of the mammary gland. Taken together, these results suggest that the absence of sufficient mammary-specific regulatory elements in the BLG promoter sequences and/or the juxtaposition of the BLG promoter with the HSA coding sequences leads to novel tissue- and cell-specific expression in ectopic tissues of transgenic mice.     

Shear flow-induced formation of tubular cell protrusions in multiple myeloma cells

Exposure of live cells to shear flow induces major changes in cell shape, adhesion to the extracellular matrix, and migration. In the present study, we show that exposure of cultured multiple myeloma (MM) cells to shear flow of 4–36 dynes/cm² triggers the extension of long tubular protrusions (denoted flow‐induced protrusions, or FLIPs) in the direction of the flow. These FLIPs were found to be rich in actin, contain few or no microtubules and, apart from endoplasmic reticulum (ER)‐like membranal structures, are devoid of organelles. Studying the dynamics of this process revealed that FLIPs elongate at their tips in a shear force‐dependent manner, and retract at their bases. Examination of this force dependence revealed considerable heterogeneity in the mechanosensitivity of individual cells, most likely reflecting the diversity of the malignant B cell population. The mechanisms underlying FLIP formation following mechanical perturbation, and their relevance to the cellular trafficking of MM cells, are discussed. J. Cell. Physiol. 226: 3197–3207, 2011. © 2011 Wiley Periodicals, Inc.     

Combined static and dynamic analysis for determining the quality of time-series expression profiles

Expression profiling of time-series experiments is widely used to study biological systems. However, determining the quality of the resulting profiles remains a fundamental problem. Because of inadequate sampling rates, the effect of arrest-and-release methods and loss of synchronization, the measurements obtained from a series of time points may not accurately represent the underlying expression profiles. To solve this, we propose an approach that combines time-series and static (average) expression data analysis--for each gene, we determine whether its temporal expression profile can be reconciled with its static expression levels. We show that by combining synchronized and unsynchronized human cell cycle data, we can identify many cycling genes that are missed when using only time-series data. The algorithm also correctly distinguishes cycling genes from genes that specifically react to an environmental stimulus even if they share similar temporal expression profiles. Experimental validation of these results shows the utility of this analytical approach for determining the accuracy of gene expression patterns.     

Cultivable bacteria isolated from apple trees cultivated under different crop systems: Diversity and antagonistic activity against Colletotrichum gloeosporioides

This study evaluated the diversity of cultivable plant growth-promoting (PGP) bacteria associated with apple trees cultivated under different crop management systems and their antagonistic ability against Colletotrichum gloeosporioides. Samples of roots and rhizospheric soil from apple trees cultivated in organic and conventional orchards in southern Brazil were collected, together with soil samples from an area never used for agriculture (native field). Bacteria were identified at the genus level by PCR-RFLP and partial sequencing of the 16S rRNA, and were evaluated for some PGP abilities. The most abundant bacterial genera identified were Enterobacter (27.7%), Pseudomonas (18.7%), Burkholderia (13.7%), and Rahnella (12.3%). Sixty-nine isolates presented some antagonist activity against C. gloeosporioides. In a greenhouse experiment, five days after exposure to C. gloeosporioides, an average of 30% of the leaf area of plants inoculated with isolate 89 (identified as Burkholderia sp.) were infected, whereas 60 to 73% of the leaf area of untreated plants was affected by fungal attack. Our results allowed us to infer how anthropogenic activity is affecting the bacterial communities in soil associated with apple tree crop systems, and to obtain an isolate that was able to delay the emergence of an important disease for this culture.     

Endoplasmic Reticulum Stress Induces a Caspase-dependent N-terminal Cleavage of RBX1 Protein in B Cells

Endoplasmic reticulum (ER) stress develops when the ER is overloaded with too many proteins to fold. This elicits a signaling pathway called the unfolded protein response. The unfolded protein response is physiologically required for the terminal development of B cells into antibody-secreting plasma cells. Ring Box Protein 1 (RBX1) is a 14-kDa protein necessary for ubiquitin ligation activity of the multimeric cullin ring ubiquitin ligases (CRLs). As RBX1 is shared by a large number of CRLs, alterations in its activity may lead to global changes in protein stability. We discovered that RBX1 is cleaved in the course of LPS-induced plasma cell differentiation and in multiple myeloma cell lines upon induction of pharmacological ER stress. The cleavage is executed by several caspase proteases that cleave RBX1 eight amino acids from the N terminus. To address the possible implication of RBX1 cleavage for CRL activity, we replaced the endogenous RBX1 homolog of the yeast Saccharomyces cerevisiae, Roc1, with the wild type or the N-terminal Δ8 mutant human RBX1. We show that yeast expressing the cleaved RBX1 are hypersensitive to ER stress and are impaired in CRL-mediated ubiquitination and degradation. We propose a model by which N-terminal cleavage of RBX1 impairs its activity and promotes susceptibility to ER stress induction.     

Cellulolytic bacteria from soils in harsh environments

It is believed that the exposure of organisms to harsh climate conditions may select for differential enzymatic activities, making the surviving organisms a very promising source for bioprospecting. Soil bacteria play an important role in degradation of organic matter, which is mostly due to their ability to decompose cellulose-based materials. This work focuses on the isolation and identification of cellulolytic bacteria from soil found in two environments with stressful climate conditions (Antarctica and the Brazilian semi-arid caatinga). Cellulolytic bacteria were selected using enrichments at high and low temperatures (4 or 60°C) in liquid media (trypic soy broth—TSB and minimum salt medium—MM) supplemented with cellulose (1%). Many of the isolates (119 out of 254—46.9%) displayed the ability to degrade carboxymethyl-cellulose, indicating the presence of endoglucolytic activity, while only a minority of these isolates (23 out of 254—9.1%) showed exoglucolytic activity (degradation of avicel). The obtained isolates revealed a preferential endoglucolytic activity according to the temperature of enrichments. Also, the identification of some isolates by partial sequencing of the 16S rRNA gene indicated that the Bacteroidetes (e.g., Pedobacter, Chryseobacterium and Flavobacterium) were the main phylum of cellulolytic bacteria isolated from soil in Antarctica; the Firmicutes (e.g., Bacillus) were more commonly isolated from samples from the caatinga; and Actinobacteria were found in both types of soil (e.g., Microbacterium and Arthrobacter). In conclusion, this work reports the isolation of bacteria able to degrade cellulose-based material from soil at very low or very high temperatures, a finding that should be further explored in the search for cellulolytic enzymes to be used in the bioenergy industry.     

Persistence to anti-cancer treatments in the stationary to proliferating transition

The heterogeneous responses of clonal cancer cells to treatment is understood to be caused by several factors, including stochasticity, cell-cycle dynamics, and different micro-environments. In a tumor, cancer cells may encounter fluctuating conditions and transit from a stationary culture to a proliferating state, for example this may occur following treatment. Here, we undertake a quantitative evaluation of the response of single cancerous lymphoblasts (L1210 cells) to various treatments administered during this transition. Additionally, we developed an experimental system, a “Mammalian Mother Machine,” that tracks the fate of thousands of mammalian cells over several generations under transient exposure to chemotherapeutic drugs. Using our developed system, we were able to follow the same cell under repeated treatments and continuously track many generations. We found that the dynamics of the transition between stationary and proliferative states are highly variable and affect the response to drug treatment. Using cell-cycle markers, we were able to isolate a subpopulation of persister cells with distinctly higher than average survival probability. The higher survival rate encountered with cell-cycle phase specific drugs was associated with a significantly longer time-till-division, and was reduced by a non cell-cycle specific drug. Our results suggest that the variability of transition times from the stationary to the proliferating state may be an obstacle hampering the effectiveness of drugs and should be taken into account when designing treatment regimens.