Characterization and expression of two cDNA encoding 3-hydroxy-3-methylglutaryl coenzyme A reductase isoforms in coffee (Coffea arabica L.).

In higher plants there are two independent pathways for isoprenoid biosynthesis, located in the cytosol (mevalonic acid or MVA pathway) or in the plastids (methylerythritol phosphate ? MEP pathway). The 3-hydroxy-3-methyglutaryl-CoA reductase (HMGR) is the first committed step in the MVA pathway. Using the information available from the Brazilian Coffee Genome Project, we found 13 ESTs that originated two isoforms, CaHMGR1 and CaHMGR2, for the enzyme HMGR of Coffea arabica. A complementary DNA encoding the isoform CaHMGR1 was cloned, and its complete nucleotide sequence determined. The full-length cDNA of CaHMGR1 was 2242 bp containing a 1812-bp ORF encoding 604 amino acids. Bioinformatic analyses revealed that the deduced CaHMGR1 had extensive homology with other plant HMGRs and contained two transmembrane domains and two putative HMGR binding sites and two NADP(H)-binding sites. Under normal growth conditions, transcripts of isoform CaHMRG1 were detected in fruit tissues (pulp, perisperm and endosperm) only at the initial stages of development, flower buds and leaves. CaHMRG2 was expressed in all tissues and during all fruit development stages examined. These results suggest a constitutive expression of isoform CaHMGR2, while the isoform CaHMGR1 shows temporal and tissue-specific transcriptional activation

Análise in silico e in vivo da via de isoprenóides em café.

Os diterpenos cafestol e caveol, presentes na fração lipídica em grãos de café, originam-se da via de síntese de isoprenóides. Estes compostos são sintetizados em todos os organismos, sendo abundantes em plantas com cerca de 10 mil componentes relatados. Apesar da diversidade de funções e estruturas todos os isoprenóides derivam de cinco comuns átomos de carbono, o isopentenil difosfato (IPP) e do isômero dimetilalil difosfato (DMAPP). Em vegetais superiores, duas vias localizadas em compartimentos intracelulares separados estão envolvidas na biossíntese de IPP e DMAPP. No citosol, IPP é derivado da via do ácido mevalônico (MVA) e no plastídeo, IPP é formado pela via do metileritritol fosfato (MEP ou não mevalonato). Com a disponibilidade das seqüências dos genes expressos (ESTs) pelo Projeto Genoma Café tornou-se possível a identificação in silico e o estudo funcional dos genes que codificam para as enzimas 3-hidroxi-3metilglutaril-CoA reductoisomerase (HMGR) e mevalonato difosfato decarboxilase (MPDC) para a via MVA e 1-deoxi-D-xilulose 5-fosfato reductoisomerase (DXR) e isopentenil difosfato sintase (IDS) para a via MEP. Foram obtidas 13 ESTs de HMGR, que originaram três contigs incompletos, resultando em duas isoformas. Para o gene MPDC foram encontradas 7 ESTs que clusterizaram em somente uma isoforma, diferentemente de A. thaliana onde duas isoformas são encontradas. Para os genes da via MEP foram encontrados 22 ESTs para DXR e 47 ESTs para IDS que formaram apenas um contig para cada um destes genes. Southern blots dos genes HMGR e DXR também demonstraram a presença de duas isoformas para HMGR e uma para DXR em C. arabica. Análise da expressão por Northern blots detectou transcritos do gene DXR no começo de desenvolvimento do perisperma e nas fases finais de desenvolvimento de endosperma e polpa. Transcritos da isoforma HMGR2 foram detectados em polpa, perisperma e endosperma, em todas as fases de desenvolvimento do fruto. Entretanto, HMGR1 apresentou transcritos apenas em polpa e fase inicial do desenvolvimento de perisperma e endosperma

Phenotypic and genotypic characterization of Bradyrhizobia nodulating the leguminous tree Acacia albida

A

Prosodic modulation in the babble of cochlear implanted and normally hearing infants: a perceptual study using a visual analogue scale

This study investigates prosodic modulation in the spontaneous canonical babble of congenitally deaf infants with cochlear implants (CI) and normally hearing (NH) infants. Research has shown that the acoustic cues to prominence are less modulated in CI babble. However acoustic measurements of individual cues to prominence give incomplete information about prosodic modulation. In the present study, raters are asked to judge prominence since they simultaneously take into account all prosodic cues. Disyllabic utterances produced by CI and NH infants were presented to naive adult raters who had to indicate the degree and direction of prosodic modulation between syllables on a visual analogue scale. The results show that the babble of infants with CI is rated as having less prosodic modulation. Moreover, segmentally more variegated babble is rated as having more prosodic modulation. Raters do not perceive the babble to be predominantly trochaic, which indicates that the predominant stress pattern of Dutch is not yet apparent in the children’s productions

The effect of bee venom on tumor growth and metastasis formation of mammary carcinoma in CBA mice

Abstract The effect of honeybee venom on the tumor growth and metastasis formation in mice was studied. Bee venom was injected into mice either subcutaneously (s.c.) or intravenously (i.v.) at different doses. The tumor was a transplantable mammary carcinoma (MCA) weakly immunogenic to the syngeneic CBA mouse. The tumor was generated by injecting lOS MCA cells i.v. When the tumor cells were injected s.c, into the footpad immediately after bee venom, the growth of the tumor was suppressed regardless of the dose of the venom. The survival of the mice treated with 0.30 mg of bee venom was prolonged as compared to the controls. The number of lung metastases in the mice treated i.v. with 0.15 or 0.075 mg of bee venom was significantly lower (P < 0.001) than that in nontreated mice. However, both doses of bee venom given s.c. did not reduce the number oflung metastases, indicating that the antitumor effect of the venom could be highly dependent on the route of injection. Key words: murine mammary carcinoma, antitumor and antimetastatic activity, honeybee venom. Kljucne rijeci: karcinom mlijecne zlijezde misa, protutumorsko i protumetastatsko djelovanje, pCelinji otrov

Working in group living homes for older people with dementia: the effects on job satisfaction and burnout and the role of job characteristics

ABSTRACT Background: Group living homes are a fast-growing form of nursing home care for older people with dementia. This study seeks to determine the differences in job characteristics of nursing staff in group living homes and their influence on well-being. Methods: We examined the Job Demand Control Support (JDCS) model in relation to 183 professional caregivers in group living homes and 197 professional caregivers in traditional nursing homes. Multilevel linear regression analysis was used to study the mediator effect of the three job characteristics of the JDCS-model (demands, control and social support) on job satisfaction and three components of burnout (emotional exhaustion, depersonalization and decreased personal accomplishment). Results: Demands were lower in group living homes, while control and social support from co-workers were higher in this setting. Likewise, job satisfaction was higher and burnout was lower in group living homes. Analysis of the mediator effects showed that job satisfaction was fully mediated by all three psychosocial job characteristics, as was emotional exhaustion. Depersonalization was also fully mediated, but only by control and social support. Decreased personal accomplishment was partially mediated, again only by job characteristics, control and support. Conclusion: This study indicates that working in a group living home instead of a traditional nursing home has a beneficial effect on the well-being of nursing staff, largely because of a positive difference in psychosocial job characteristic

Fast analysis of antibody-derived therapeutics by automated multidimensional liquid chromatography - mass spectrometry

Characterization of post-translational modifications (PTMs) of therapeutic antibodies is commonly performed by bottom-up approaches, involving sample preparation and peptide analysis by liquid chromatography-mass spectrometry (LC-MS). Conventional sample preparation requires extensive hands-on time and can increase the risk of inducing artificial modifications as many off-line steps - denaturation, disulfide-reduction, alkylation and tryptic digestion - are performed. In this study, we developed an on-line multidimensional (mD)-LC-MS bottom-up approach for fast sample preparation and analysis of (formulated) monoclonal antibodies and antibody-derived therapeutics. This approach allows on-column reduction, tryptic digestion and subsequent peptide analysis by RP-MS. Optimization of the 1D -and 2D flow and temperature improved the trapping of small polar peptides during on-line peptide mapping analysis. These adaptations increased the sequence coverage (95-98% versus 86-94% for off-line approaches) and allowed identification of various PTMs (i.e. deamidation of asparagine, methionine oxidation and lysine glycation) within a single analysis. This workflow enables a fast (<2 h) characterization of antibody heterogeneities within a single run and a low amount of protein (10 mu g). Importantly, the new mD-LC-MS bottom-up method was able to detect the polar, fast-eluting peptides: Fc oxidation at Hc-Met-252 and the Fc N-glycosylation at Hc-Asn-297, which can be challenging using mD-LC-MS. Moreover, the method showed good comparability across the different measurements (RSD of retention time in the range of 0.2-1.8% for polar peptides). The LC system was controlled by only a standard commercial software package which makes implementation for fast characterization of quality attributes relatively easy. (C) 2021 The Author(s). Published by Elsevier B.V.Proteomic