Isolation and characterization of a subset of erythropoietin glycoforms with cytoprotective but minimal erythropoietic activity

Although historically used for the treatment of anemia, erythropoietin (EPO) has emerged as a neurotrophic and neuroprotective agent in different conditions of neuronal damage (traumatic brain injury, ischemia, spinal cord compression, peripheral neuropathy, retinal damage, epilepsy, Parkinson's Disease, among others). Nonetheless, EPO's therapeutic application is limited due to its hematological side‐effects. With the aim of obtaining EPO derivatives resembling the hormone isolated from cells and tissues of neural origin, a novel combination of less acidic EPO glycoforms ‐designated as neuroepoetin (rhNEPO)‐ was purified to homogeneity from the supernatant of a CHO‐producing cell line by a four‐step chromatographic procedure. This simple and single process allowed us to prepare two EPO derivatives with distinct therapeutic expectations: the hematopoietic version and a minimally hematopoietic, but mainly in vitro cytoprotective, alternative. Further biological characterization showed that the in vivo erythropoietic activity of rhNEPO was 25‐times lower than that of rhEPO. Interestingly, using different in vitro cytoprotective assays we found that this molecule exerts cytoprotection equivalent to, or better than, that of rhEPO in cells of neural phenotype. Furthermore, despite its shorter plasma half‐life, rhNEPO was rapidly absorbed and promptly detected in the cerebrospinal fluid after intravenous administration in rats (5 min postinjection, in comparison with 30 min for rhEPO). Therefore, our results support the study of neuroepoetin as a potential drug for the treatment of neurological diseases, combining high cytoprotective activity with reduced hematological side‐effects. © 2011 American Institute of Chemical Engineers Biotechnol. Prog., 2011     

Prevalence of potentially thermophilic microorganisms in biofilms from greenhouse-enclosed drip irrigation systems

Drip irrigation systems using reclaimed water often present clogging events of biological origin. Microbial communities in biofilms from microirrigation systems of an experimental greenhouse in Almería, SE Spain, which used two different qualities of water (treated wastewater and reclaimed water), were analyzed by denaturing gradient gel electrophoresis and subsequent sequencing of amplified 16S rRNA gene bands. The most remarkable feature of all biofilms was that regardless of water origin, sequences belonging to Firmicutes were prevalent (53.5 % of total mean band intensity) and that almost all sequences recovered had some similarity (between 80.2 and 97 %) to thermophilic microorganisms. Mainly, sequences were closely related to potentially spore-forming organisms, suggesting that microbial communities able to grow at high temperatures were selected from the microbiota present in the incoming water. These pioneer results may contribute to improve management strategies to minimize the problems associated to biofouling in irrigation systems.     

Regulatory domain of human protein kinase Cα dominantly inhibits protein kinase Cβ-I regulated growth and morphology in Saccharomyces cerevisiae

This study demonstrates that the isolated regulatory (R) domain (amino acids 1–270) of human protein kinase Cα (PKCα) is a potent inhibitor of PKCβ-I activity in a yeast expression system. The PKCα R domain fused to glutathione-S-transferase competitively inhibited the activity of yeast-expressed rat PKCβ-I in vitro (Ki = 0.2 μM) and was 400-fold more potent than a synthetic pseudosubstrate peptide corresponding to amino acids 19–36 from PKCα. In contrast, the fusion protein did not affect the activity of the purified catalytic subunit of cAMP-dependent protein kinase. The PKCα R domain (without glutathione-S-transferase [GST]) also was tested for its ability to inhibit PKCβ-I activity in vivo, in a yeast strain expressing rat PKCβ-I. Upon treatment with a PKC-activating phorbol ester, yeast cells expressing rat PKCβ-I were growth-inhibited and a fraction of the cells appeared as long chains. Coexpression of the R domain with rat PKCβ-I blocked the phorbol ester-induced inhibition of yeast cell growth and the phorbol ester-dependent alterations in yeast cell morphology. These results indicate that the R domain of PKCα acts as a dominant inhibitor of PKC activity in vivo and thus provides a useful genetic tool to assess the roles of PKC in various signal transduction processes. © 1996 Wiley-Liss, Inc.     

The ESCRT-deubiquitinating enzyme USP8 in the cervical spinal cord of wild-type and Vps54-recessive (wobbler) mutant mice

Usp8 is a deubiquitinating enzyme that works as regulator of endosomal trafficking and is involved in cell proliferation. “In vivo” USP8 is predominantly expressed in the central nervous system and testis, two organs with highly polarized cells. Considering that neuronal cell functionality is strictly dependent on vesicular traffic and ubiquitin-mediated sorting of the endocytosed cargo, it could be of relevance to investigate about USP8 in neuronal cells, in particular motor neurons. In this study, we found that USP8 is expressed in the gray and white matter of the spinal cord, labeling neuronal cell bodies, axonal microtubules and synaptic terminals. The glia component is essentially USP8-immunonegative. The partial colocalization of USP8 with EEA1 in motor neurons indicates that USP8 is involved in early endosomal trafficking while that with Vps54 suggests an involvement in the retrograde traffic. The variant Vps54(L967Q) is responsible for the wobbler phenotype, a disorder characterized by motor neuron degeneration. We searched for USP8/Vps54 in wobbler spinal cord. The most worth-mention result was that wobbler oligodendrocytes, in contrast to the wild-type, are heavily USP8-immunoreactive; no significant modification was appreciated about the cellular expression of mutated Vps54. On the other hand, as to the neuronal intracellular localization, both USP8 and Vps54(L967Q) did not show the typical spot-like distribution, but seemed to accumulate in proteinaceous aggregates. Collectively, our study suggests that in neuronal cells USP8 could be involved in endosomal trafficking, retrograde transport and synaptic plasticity. In disorders leading to neurodegeneration USP8 is upregulated and could influence the neuron–oligodendrocyte interactions.     

Spleen metastasis from thyroid carcinoma. Report of a case with diagnosis by fine needle aspiration cytology

BACKGROUND: Lymphoreticular malignancies are the most common neoplasms involving the spleen. Metastasis can be caused by direct invasion from surrounding tumors or from hematogenous spread. Spleen metastases from thyroid carcinoma are unusual, and only 1 case has been reported; none have been diagnosed by fine needle aspiration cytology (FNAC). CASE: A 75-year-old female was diagnosed 6 months earlier with a poorly differentiated thyroid carcinoma with wide lymphatic and vascular invasion. Abdominal computed tomography (CT) and magnetic resonance imaging showed several spleen nodules. FNAC was performed under CT guidance. Cytologic examination showed atypical epithelial cells with thyroidal characteristics. CONCLUSION: This case had the cytologic findings of a poorly differentiated carcinoma of the thyroid metastatic to spleen. We confirmed the rarity of this pathology and the efficacy of splenic FNAC in the diagnosis.     

The use of the normal tissue non-complication probability (NTCP0) methodology as a new alternative of assessing side-effects in brachytherapy treatments

BackgroundThe NTCP methodology evaluating side-effects (S-Es) was initially used in radiotherapy (RT), and later was extended to brachytherapy (BT). The NTCP0 methodology has been recently introduced in RT. Given the advantages, this methodology could replace NTCP.Materials and methodsRevisions of studies related to use of NTCP in the evaluations of S-Es in BT. Development of the first versions of two Matlab applications of the NTCP0 methodology. These applications have three options. Two of them employ the well-known aspects of a phenomenological model, or the probabilistic relationship between NTCP0 and total NTCP (TNTCP) that is the sum(NTCP(x_i)) i: i^th complication i:1..nc: Number of complications; where NTCP0 = 100% — TNTCP; and the third option assumes a NTCP(x_i) discrete probabilistic distribution generated by the binomial distribution, where one of its parameters is automatically obtained from a databased of the Disease locations Vs. Late complications.ResultsThe NTCP0cal and NTCP0calDr Matlab applications have been developed, and respectively used for fractional continuous low dose-rate BT.ConclusionsNTCP0 is defined as the ratio of the number of patients without acute/late complications and total of them, and also can be obtained using our Matlab applications. NTCP0 works do not disregard the last 10–15 years of NTCP research; but NTCP0 was not considered during these years. A generic example was used for showing the variations of the late complications and NTCP0 for a BT treatment of a constant number of fractions and six different dose per fraction values.     

The respiratory syncytial virus G protein conserved domain induces a persistent and protective antibody response in rodents

Respiratory syncytial virus (RSV) is an important cause of severe upper and lower respiratory disease in infants and in the elderly. There are 2 main RSV subtypes A and B. A recombinant vaccine was designed based on the central domain of the RSV-A attachment G protein which we had previously named G2Na (aa130-230). Here we evaluated immunogenicity, persistence of antibody (Ab) response and protective efficacy induced in rodents by: (i) G2Na fused to DT (Diphtheria toxin) fragments in cotton rats. DT fusion did not potentiate neutralizing Ab responses against RSV-A or cross-reactivity to RSV-B. (ii) G2Nb (aa130-230 of the RSV-B G protein) either fused to, or admixed with G2Na. G2Nb did not induce RSV-B-reactive Ab responses. (iii) G2Na at low doses. Two injections of 3 μg G2Na in Alum were sufficient to induce protective immune responses in mouse lungs, preventing RSV-A and greatly reducing RSV-B infections. In cotton rats, G2Na-induced RSV-reactive Ab and protective immunity against RSV-A challenge that persisted for at least 24 weeks. (iv) injecting RSV primed mice with a single dose of G2Na/Alum or G2Na/PLGA [poly(D,L-lactide-co-glycolide]. Despite the presence of pre-existing RSV-specific Abs, these formulations effectively boosted anti-RSV Ab titres and increased Ab titres persisted for at least 21 weeks. Affinity maturation of these Abs increased from day 28 to day 148. These data indicate that G2Na has potential as a component of an RSV vaccine formulation.     

Estrogen receptors mediate estradiol's effect on sensitization and CPP to cocaine in female rats: Role of contextual cues

Preclinical studies show that estradiol enhances sensitization to cocaine in females by mechanisms not fully understood. These studies consistently show that ovariectomized (OVX) rats exhibit little or no sensitization to cocaine compared to OVX rats administered estradiol. In this study we varied the dose of cocaine (10, 15, and 30 mg/kg), the length of cocaine treatment (from 5 to 10 days) and the context of cocaine injections to determine if these factors play a role on estradiol's effects on cocaine sensitization. Because OVX rats are hormonally compromised, they are not representative of the natural state of the animal, and thus the physiological context of these studies remains unclear. To address this issue, we blocked ERs in gonadally intact females by icv administration of the antiestrogen ICI-182,780.