Enhanced Expression of Human Platelet-Derived Growth factor in \u3cem\u3ePichia Pastoris\u3c/em\u3e

Enhanced yields of the platelet-derived growth factor (PDGF) B-chain are obtained using the Pichia pastoris yeast system. Yields of both wild-type and mutant human proteins are enhanced when the yeast is transformed with a vector containing the yeast mating factor promoter fused to a sequence encoding the mature protein. The secreted wild-type protein is indistinguishable from mature 29-32 kDa protein isolated from human and Chinese hamster ovary (CHO) cells. An RKK→EEE mutant exhibited reduced association with the cell surface and accumulated in the culture medium as 29-32 kDa forms. Stable transfection of U87 astrocytoma cells with RKK→EEE mutants of either the A- or B-chain inhibited malignant growth in athymic nude mice. Despite altered receptor binding activities, each mutant retained full mitogenic activity when applied to cultured Swiss 3T3 cells. Circular dichroism spectrophotometric analysis of the RKK→EEE mutant revealed a secondary structure indistinguishable from the wild type. Yields of PDGF protein are increased many fold and in less time over previous methods

Targeted neutralization of calmodulin in the nucleus blocks DNA synthesis and cell cycle progression

AbstractCalmodulin (CaM) is a major intracellular calcium binding protein which has been implicated in the regulation of cell proliferation. Previous studies using chemically synthesized CaM antagonists and anti-sense RNA indicated that CaM is important for initiation of DNA synthesis and cell cycle progression. However, these methods reduce total intracellular CaM and globally interfering with all the CaM-dependent processes. In order to explore the function of nuclear CaM during the cell cycle, a CaM inhibitor peptide was targeted to the nucleus of intact mammalian cells. Cell progression through S-phase was assessed by incorporation of the thymidine analogue, BrdU. Cells were transfected for 48 h with either the CaM inhibitor peptide gene or the control plasmid prior to analysis. Approx. 70% of the control cells incorporated BrdU. In striking contrast, double immunofluorescent labeling demonstrated that none of the cells expressing the CaM inhibitor peptide entered S-phase. This result indicates that neutralization of nuclear CaM by targeted expression of a CaM inhibitor peptide blocks DNA synthesis and cell cycle progression

Evaluation of Exposure to Arsenic in Residential Soil

In response to concerns regarding arsenic in soil from a pesticide manufacturing plant, we conducted a biomonitoring study on children younger than 7 years of age, the age category of children most exposed to soil. Urine samples from 77 children (47% participation rate) were analyzed for total arsenic and arsenic species related to ingestion of inorganic arsenic. Older individuals also provided urine (n = 362) and toenail (n = 67) samples. Speciated urinary arsenic levels were similar between children (geometric mean, geometric SD, and range: 4.0, 2.2, and 0.89–17.7 μg/L, respectively) and older participants (3.8, 1.9, 0.91–19.9 μg/L) and consistent with unexposed populations. Toenail samples were < 1 mg/kg. Correlations between speciated urinary arsenic and arsenic in soil (r = 0.137, p = 0.39; n = 41) or house dust (r = 0.049, p = 0.73; n = 52) were not significant for children. Similarly, questionnaire responses indicating soil exposure were not associated with increased urinary arsenic levels. Relatively low soil arsenic exposure likely precluded quantification of arsenic exposure above background

Enhancement of Polymeric Immunoglobulin Receptor Transcytosis by Biparatopic VHH

The polymeric immunoglobulin receptor (pIgR) ensures the transport of dimeric immunoglobulin A (dIgA) and pentameric immunoglobulin M (pIgM) across epithelia to the mucosal layer of for example the intestines and the lungs via transcytosis. Per day the human pIgR mediates the excretion of 2 to 5 grams of dIgA into the mucosa of luminal organs. This system could prove useful for therapies aiming at excretion of compounds into the mucosa. Here we investigated the use of the variable domain of camelid derived heavy chain only antibodies, also known as VHHs or Nanobodies®, targeting the human pIgR, as a transport system across epithelial cells. We show that VHHs directed against the human pIgR are able to bind the receptor with high affinity (∼1 nM) and that they compete with the natural ligand, dIgA. In a transcytosis assay both native and phage-bound VHH were only able to get across polarized MDCK cells that express the human pIgR gene in a basolateral to apical fashion. Indicating that the VHHs are able to translocate across epithelia and to take along large particles of cargo. Furthermore, by making multivalent VHHs we were able to enhance the transport of the compounds both in a MDCK-hpIgR and Caco-2 cell system, probably by inducing receptor clustering. These results show that VHHs can be used as a carrier system to exploit the human pIgR transcytotic system and that multivalent compounds are able to significantly enhance the transport across epithelial monolayers

La proteína quinasa dependiente de Ca2+ y calmodulina (CaMKII): ¿es proarritmogénica en reperfusión?

La reperfusión del miocardio isquémico lo hace más propenso a la aparición de arritmias. Experimentos previos de nuestro laboratorio mostraron que al inicio de la reperfusión (R), momento en el que se detecta el mayor número de arritmias, aumenta la fosforilación dependiente de CaMKII del residuo PT17 de fosfolamban (PLN) (Vittone, 2002). El objetivo de este trabajo, fue evaluar la posibilidad de que la activación de CaMKII al inicio de la reperfusión sea un mecanismo proarritmogénico.Facultad de Ciencias Médica

Annexin-A5 assembled into two-dimensional arrays promotes cell membrane repair

Eukaryotic cells possess a universal repair machinery that ensures rapid resealing of plasma membrane disruptions. Before resealing, the torn membrane is submitted to considerable tension, which functions to expand the disruption. Here we show that annexin-A5 (AnxA5), a protein that self-assembles into two-dimensional (2D) arrays on membranes upon Ca2+ activation, promotes membrane repair. Compared with wild-type mouse perivascular cells, AnxA5-null cells exhibit a severe membrane repair defect. Membrane repair in AnxA5-null cells is rescued by addition of AnxA5, which binds exclusively to disrupted membrane areas. In contrast, an AnxA5 mutant that lacks the ability of forming 2D arrays is unable to promote membrane repair. We propose that AnxA5 participates in a previously unrecognized step of the membrane repair process: triggered by the local influx of Ca2+, AnxA5 proteins bind to torn membrane edges and form a 2D array, which prevents wound expansion and promotes membrane resealing

IgA in the horse: cloning of equine polymeric Ig receptor and J chain and characterization of recombinant forms of equine IgA

As in other mammals, immunoglobulin A (IgA) in the horse has a key role in immune defense. To better dissect equine IgA function, we isolated complementary DNA (cDNA) clones for equine J chain and polymeric Ig receptor (pIgR). When coexpressed with equine IgA, equine J chain promoted efficient IgA polymerization. A truncated version of equine pIgR, equivalent to secretory component, bound with nanomolar affinity to recombinant equine and human dimeric IgA but not with monomeric IgA from either species. Searches of the equine genome localized equine J chain and pIgR to chromosomes 3 and 5, respectively, with J chain and pIgR coding sequence distributed across 4 and 11 exons, respectively. Comparisons of transcriptional regulatory sequences suggest that horse and human pIgR expression is controlled through common regulatory mechanisms that are less conserved in rodents. These studies pave the way for full dissection of equine IgA function and open up possibilities for immune-based treatment of equine diseases

Regulation of the polymeric immunoglobulin receptor by the classical and alternative NF-κB pathways in intestinal epithelial cells

The polymeric immunoglobulin receptor (pIgR) transports IgA antibodies across intestinal epithelial cells (IECs). Expression of pIgR is upregulated by proinflammatory signaling pathways via activation of nuclear factor-κB (NF-κB). Here, we examined the contributions of the RelA-dependent classical and RelB-dependent alternative pathways of NF-κB to pIgR regulation in the HT-29 human IEC line following stimulation with tumor necrosis factor (TNF), lipopolysaccharide (LPS; Toll-like receptor 4 (TLR4) ligand), and polyinosinic: polycytidylic acid (pIC; TLR3 ligand). Whereas induction of proinflammatory genes such as interleukin-8 (IL-8) required only RelA, pIgR expression was regulated by complex mechanisms that involved both RelA and RelB. Upregulation of pIgR expression by ligation of the lymphotoxin-β receptor suggested a direct role for the alternative NF-κB pathway. Inhibition of mitogen-activated protein kinases reduced the induction of IL-8, but enhanced the induction of pIgR by TNF and TLR signaling. Regulation of pIgR through unique signaling pathways could allow IECs to sustain high levels of IgA transport while limiting the proinflammatory responses

Influence of ultra-low dose Aprotinin on thoracic surgical operations: a prospective randomized trial

<p>Abstract</p> <p>Background</p> <p>The blood saving effect of aprotinin has been well documented in cardiac surgery. In thoracic surgery, very few recent studies, using rather high doses of aprotinin, have shown a similar result. In a randomized prospective trial, we have tested the influence of aprotinin using an ultra-low dose drug regime.</p> <p>Methods</p> <p>Fifty-nine patients, mean age 58 ± 13.25 years (mean ± SD) undergoing general thoracic procedures were randomized into placebo (Group A) and treatment group (Group B). The group B (n = 29) received 500.000 IU of aprotinin after induction to anesthesia and a repeat dose immediately after chest closure. A detailed protocol with several laboratory parameters was recorded. Patients were transfused when perioperative Ht was less than 26%.</p> <p>Results</p> <p>The two groups were similar in terms of age, gender, diagnosis, pathology, co-morbidity and operations performed. The mean drainage of the first and second postoperative day in group B was significantly reduced (412.6 ± 199.2 vs. 764.3 ± 213.9 ml, p < 0.000, and 248.3 ± 178.5 vs. 455.0 ± 274.6, p < 0.001). Similarly, the need for fresh frozen plasma transfusion was lower in group B, p < 0.035. Both the operation time and the hospital stay were also less for group B but without reaching statistical significance (84.6 ± 35.2 vs 101.2 ± 52.45 min. and 5.8 ± 1.6 vs 7.2 ± 3.6 days respectively, p < 0.064). The overall transfusion rate did not differ significantly. No side effects of aprotinin were noted.</p> <p>Conclusion</p> <p>The perioperative ultra-low dose aprotinin administration was associated with a reduction of total blood losses and blood product requirements. We therefore consider the use of aprotinin safe and effective in major thoracic surgery.</p