Integrated approach to produce a recombinant, His-tagged human α-galactosidase A in mammalian cells

Successful production of recombinant proteins (r-proteins) by transient gene expression (TGE) depends on several parameters (including producer cells, culture conditions, transfection procedure, or expression vector) that should be optimized when producing any recombinant product. In this work, TGE-based production of human α-galactosidase A (GLA) is described. Producer cells, expression vectors, and parameters influencing cell metabolism after transfection have been tested. The enzyme is secreted, has the right molecular weight, and is enzymatically active. Productivities of up to 30-40 mg/L have been achieved, with a simple, fast procedure. A 6 × His tag allows enzyme purification in a single step, rendering a highly pure product. We propose a TGE-based protocol able to produce up to several milligrams per liter of highly pure, active GLA in a time as short as a few days. By this, enough amounts of engineered versions of the enzyme can be easily produced to be tested in vitro or in preclinical trials.     

F-box proteins everywhere

The ubiquitin proteasome system is a key regulator of many biological processes in all eukaryotes. This mechanism employs several types of enzymes, the most important of which are the ubiquitin E3 ligases that catalyse the attachment of polyubiquitin chains to target proteins for their subsequent degradation by the 26S proteasome. Among the E3 families, the SCF is the best understood; it consists of a multi-protein complex in which the F-box protein plays a crucial role by recruiting the target substrate. Strikingly, nearly 700 F-box proteins have been predicted in Arabidopsis, suggesting that plants have the capacity to assemble a multitude of SCF complexes, possibly controlling the stability of hundreds of substrates involved in a plethora of biological processes. Interestingly, viruses and even pathogenic bacteria have also found ways to hijack the plant SCF and to reprogram it for their own purposes.     

Modulatory Effects of the Piccolo Genotype on Emotional Memory in Health and Depression

Major depressive disorder (MDD) has been associated with biased memory formation for mood-congruent information, which may be related to altered monoamine levels. The piccolo (PCLO) gene, involved in monoaminergic neurotransmission, has previously been linked to depression in a genome-wide association study. Here, we investigated the role of the PCLO risk allele on functional magnetic resonance imaging (MRI) correlates of emotional memory in a sample of 89 MDD patients (64 PCLO risk allele carriers) and 29 healthy controls (18 PCLO risk allele carriers). During negative word encoding, risk allele carriers showed significant lower activity relative to non-risk allele carriers in the insula, and trend-wise in the anterior cingulate cortex and inferior frontal gyrus. Moreover, depressed risk allele carriers showed significant lower activity relative to non-risk allele carriers in the striatum, an effect which was absent in healthy controls. Finally, amygdalar response during processing new positive words vs. known words was blunted in healthy PCLO+ carriers and in MDD patients irrespective of genotype, which may indicate that signalling of salient novel information does not occur to the same extent in PCLO+ carriers and MDD patients. The PCLO risk allele may increase vulnerability for MDD by modulating local brain function with regard to responsiveness to salient stimuli (i.e. insula) and processing novel negative information. Also, depression-specific effects of PCLO on dorsal striatal activation during negative word encoding and the absence of amygdalar salience signalling for novel positive information further suggest a role of PCLO in symptom maintenance in MDD.     

The Tissue Bank at the National Nuclear Research Institute in Mexico

The Instituto Nacional de Investigaciones Nucleares (ININ, The National Nuclear Research Institute) received during 1997-1998 strong support of the International Atomic Energy Agency (IAEA), to establish the first and only one tissue bank (BTR ININ tissue bank) in Mexico that uses ionising radiation as sterilising agent. In that time, the BTR staff was trained in different tissue banks in several countries. Basic equipment for tissue processing donated by the IAEA was received in 1998. In July, 1999 the Mexican Health Secretariat gave the Sanitary License No. 1062000001 to the BTR to operate as an official organ and tissue bank. In August, 2001 the ININ and the Hospital Materno Infantil (HMI-ISSEMYM) signed an agreement to collaborate in amnion processing. The hospital is responsible for donor selection, serology tests, tissue procurement and washing, since this hospital is the BTR amnion supplier. The tissues are collected by ININ weekly with complete documentation. The BTR is responsible for processing: cleaning, air drying, packaging, labelling, microbiological control and sterilisation by gamma irradiation. The sterilised tissue is kept under quarantine for 6 months to obtain the results of the donor second serology test. From March to June, 2002 the BTR has processed 347.86 units (50 cm(2) each), is say, 17,393 cm(2). In addition, the pig skin xenograft process has been implemented and a protocol for clinical applications of it is running at the Hospital Central Sur de Alta Especialidad (PEMEX). Also the ININ tissue bank present status and perspectives are described.     

GPCR-induced migration of breast carcinoma cells depends on both EGFR signal transactivation and EGFR-independent pathways

The epidermal growth factor receptor (EGFR) plays a key role in the regulation of important cellular processes under normal and pathophysiological conditions such as cancer. In human mammary carcinomas the EGFR is involved in regulating cell growth, survival, migration and metastasis and its activation correlates with the lack of response in hormone therapy. Here, we demonstrate in oestrogen receptor-positive and -negative human breast cancer cells and primary mammary epithelial cells a cross-communication between G protein-coupled receptors (GPCRs) and the EGFR. We present evidence that specific inhibition of ADAM15 or TACE blocks GPCR-induced and proHB-EGF-mediated EGFR tyrosine phosphorylation, downstream mitogenic signalling and cell migration. Notably, activation of the PI3K downstream mediator PKB/Akt by GPCR ligands involves the activity of sphingosine kinase (SPHK) and is independent of EGFR signal transactivation. We conclude that GPCR-induced chemotaxis of breast cancer cells is mediated by EGFR-dependent and -independent signalling pathways, with both parallel pathways having to act in concert to achieve a complete migratory response.     

Discovery,characterization and comparison of inhibitors of Bacillus anthracis and Staphylococcus aureus replicative DNA helicases

Antibacterial compounds with new mechanisms of action are needed for effective therapy against drug-resistant pathogens in the clinic and in biodefense. Screens for inhibitors of the essential replicative helicases of Bacillus anthracis and Staphylococcus aureus yielded 18 confirmed hits (IC₅₀ ? 25 μM). Several (5 of 18) of the inhibitors were also shown to inhibit DNA replication in permeabilized polA-deficient B. anthracis cells. One of the most potent inhibitors also displayed antibacterial activity (MIC ～5 μg/ml against a range of Gram-positive species including bacilli and staphylococci) together with good selectivity for bacterial versus mammalian cells (CC₅₀/MIC > 16) suitable for further optimization. This compound shares the bicyclic ring of the clinically proven aminocoumarin scaffold, but is not a gyrase inhibitor. It exhibits a mixed mode of helicase inhibition including a component of competitive inhibition with the DNA substrate (K_i = 8 μM) and is rapidly bactericidal at 4 × MIC.     

CD8 T cells expressing NK associated receptors are increased in melanoma patients and display an effector phenotype

CD8⁺ T cells can express NK-associated receptors (NKRs) that may regulate their cytolytic function. We have characterized the expression of several NKRs on peripheral blood CD8⁺ T cells from melanoma patients and compared them to age-matched healthy donors. The analysis performed includes HLA class I specific receptors (KIRs, LILRB1 and CD94/NKG2) and other NK receptors like CD57, CD56 and CD16. Melanoma patients showed a higher variability in the expression of NKRs on circulating CD8⁺ T cells than age-matched healthy donors. NKR expression on CD8⁺ T cells from melanoma patients showed a significant increase of KIR2DL2/L3/S2 (mAb gl183), CD244, CD57, CD56 and CD16. We have also found an increase of CD8⁺ CD28^– CD27^– T cells in melanoma patients. This subset represents terminally differentiated effector cells expressing CD244 and high levels of perforin. The expression of NKRs was also mainly restricted to this T cell subset. Altogether, circulating CD8⁺ T cells from melanoma patients display a distinct phenotype characterized by downregulation of costimulatory molecules and higher expression of NKRs. We suggest that the increased expression of NKRs on T cells may contribute to the final outcome of the immune response against melanoma both stimulating or inhibiting activation and differentiation to effector cells. Blocking inhibitory receptor function and enhancing activating receptors may represent new strategies with therapeutic potential against melanoma.     

Microtubule and microfilament organization in immature, in vitro matured and in vitro fertilized prepubertal goat oocytes

The aim of our study was to analyse the cytoskeletal organization of prepubertal goat oocytes. Microtubule and microfilament organization during in vitro maturation of prepubertal and adult goat oocytes and presumptive zygotes of in vitro matured-in vitro fertilized (IVM-IVF) prepubertal goat oocytes were analysed. Oocytes were matured in M-199 with hormones and serum and inseminated with frozen-thawed sermatozoa. Oocytes and presumptive zygotes were treated with anti-alpha-tubulin antibody and fluorescein isothiocyanate (FITC)-labelled goat anti-mouse antibody to stain the microtubules. Microfilaments were localized by means of phalloidin 5 microg/ml conjugated with fluorescein isothiocyanate (FITC-phalloidin). DNA was stained with propidium iodide. Stained oocytes were observed under a confocal laser scanning microscope. At the germinal vesicle nuclear stage, microfilaments were distributed at the cortex of the oocytes. After in vitro maturation, 91.7% of metaphase II (MII) oocytes from adult goats displayed microfilaments in the cortex and within the polar body and were characterized by the presence of a microfilament thickening at the cortical region over the meiotic spindle. In prepubertal goat MII oocytes only 5.7% of oocytes displayed microfilaments at the cortex and within the polar body. After insemination, most of the zygotes displayed microfilaments distributed at the cortex. An undefined microtubular network was observed in adult and prepubertal goat oocytes at the germinal vesicle stage. After in vitro maturation, 100% of MII oocytes from adult goats displayed microtubules on the meiotic spindle and within the polar body. This pattern of distribution was observed in 71.6% of prepubertal goat oocytes. Undefined microtubule networks were present in most of the zygotes analysed. In conclusion, cytoskeletal differences were found between prepubertal and adult goat MII oocytes. Furthermore, most of the zygotes from IVM-IVF prepubertal goat oocytes displayed cytoskeletal anomalies.