How physiological and cultural conditions influence heterologous protein production in Kluyveromyces lactis

The optimization and scale-up of a specific protein production process have to take into account cultural conditions as well as cell physiology of growth and influence of foreign protein expression on host cell metabolism. Growth on cheap substrates, efficient secretion ability and a weaker tendency to hypermannosilate proteins than S. cerevisiae, make K. lactis an excellent and well-accepted host for heterologous protein production, even for human use. A fairly good heterologous glucoamylase yield and the setting of the optimal conditions to produce it were obtained expressing the Arxula adeninivorans glucoamylase in a strain of K. lactis and its isogenic mutant, which seems to have higher secretion ability. We performed batch cultures of both strains to analyze the influence of different physiological and environmental parameters on glucoamylase production/secretion. Interestingly, the maintenance of pH in the range of neutrality causes the consumption of a larger amount of carbon source, a longer time of production and a better stability of the active form of the enzyme, thus increasing biomass and glucoamylase production. Furthermore, the enrichment of the culture medium adds up to the action of pH control, forcing the mutant production/secretion to higher levels.     

Cardiovascular effects of growth hormone treatment: potential risks and benefits

Growth hormone (GH) and insulin-like growth factor-I are involved in heart development and in maintaining cardiac structure and performance. Cardiovascular disease has been reported to reduce life expectancy both in GH deficiency (GHD) and in GH excess. Patients with GHD suffer from abnormalities of left ventricular performance, i.e. reduced diastolic filling and impaired response to peak exercise. Patients with GHD also have increased intima-media thickness at the common carotid arteries, associated with a higher occurrence of atherosclerotic plaques, which may further aggravate the haemodynamic conditions. This may contribute to increased cardiovascular and cerebrovascular risk. These cardiovascular abnormalities can be reversed, at least partially, with GH replacement therapy. In recent years, GH therapy has been used to increase cardiac mass in ischaemic or dilated cardiomyopathy, but the results have produced contradictory data.     

Animal resources and subsistence strategies

Several faunal assemblages excavated in deposits of different antiquity (from Lower Paleolithic to Bronze Age), located in Northern, Central and Southern Italy, were studied from the archeozoological and taphonomic point of view. Data obtained by different Authors allow reconstruction of subsistence strategies adopted by prehistoric humans in these areas and through time, in particular as far as the exploitation of animal resources is concerned. The following assemblages were considered: Isernia La Pineta (Molise; Lower Paleolithic), Grotta Breuil (Latium; Middle Paleolithic), Grotta della Ghiacciaia (Verona; Middle Paleolithic), Riparo di Fumane and Riparo Tagliente (Verona; Middle and Upper Paleolithic), Riparo Mochi (Liguria; Upper Paleolithic), Grotta della Continenza (L'Aquila; Upper Paleolithic and Mesolithic), Grotta dell'Edera (Trieste; Mesolithic and Neolithic), Grotta della Cala at Marina di Camerota (Salerno; Eneolithic), Contraguda (Sassari; Neolithic), Castellaro Lagusello (Mantova; Bronze Age). Exploitation of the vegetal resources has been analyzed in the Neolithic sites of Colle Santo Stefano (Fucino), Settefonti (L'Aquila) and Catignano (Pescara).     

alpha and beta spectrin distribution during the differentiation of pyriform cells in follicles of lizard Podarcis sicula

Using alpha and beta spectrin mammalian antibodies on Western blotting, we demonstrated that lizard ovarian follicles contain two isoforms of alpha spectrin, Mr 94 and 134 kDa, and a 230 kDa beta spectrin, and that their pattern modifies in relation to pyriform cell differentiation. In fact, a positive immunoreaction is firstly evident within follicular epithelium of previtellogenic follicles when small cells differentiate into pyriform cells via intermediate cells. Later on, immunostain is present in pyriform cells and in the oocyte cortex that previously appears unstained. It is noteworthy that immunostain is also present on small cells located in contact with the oocyte membrane, but not on those located under the basal lamina and among pyriform cells, not engaged in pyriform cell differentiation. During the subsequent stages of previtellogenic phase, spectrin immunostain over the follicular epithelium and in the oocyte cortex does not change. By contrast, in vitellogenic follicles, when the follicular epithelium is constituted only by small cells, immunostain is evident at the level of the oocyte cortex and the cytoplasm of regressing pyriform cells. The present data strongly suggest that the alpha and beta spectrin pattern put in evidence during the different phases of lizard oocyte growth is related to the differentiation of small into pyriform cells, where such protein may guarantee a relationship between surface glycoproteins (Andreuccetti et al., 2001: Anat Rec 263:1-9), and the cytoskeleton distribution (Maurizii et al., 2000: Raf Mol Reprod Dev 57:159-166). Furthermore, the distribution of spectrin mRNA, similar to that observed for the protein, demonstrates that spectrin, once synthesized within pyriform cells, is transferred through intercellular bridges in the oocyte cortex, thus confirming that pyriform cells are nurse that significantly are involved in the oocyte growth. Finally, the present data demonstrate that alpha spectrin of lizard ovarian follicles has Mr quite different from those so far reported and may constitute a new group of isoforms. This important result will be the focus of future experiments. Mol. Reprod. Dev. 67: 101-107, 2004.     

Effects of simulated microgravity on the development and maturation of dissociated cortical neurons

Although a wealth of evidence supports the hypothesis that some functions of the nervous system may be altered during exposure to microgravity, the possible changes in basic neuronal physiology are not easy to assess. Indeed, few studies have examined whether microgravity affects the development of neurons in culture. In the present study, a suspension of dissociated cortical cells from rat embryos were exposed to 24 h of simulated microgravity before plating in a normal adherent culture system. Both preexposed and control cells were used after a period of 7-10 d in vitro. The vitality and the level of reactive oxygen species of cultures previously exposed did not differ from those of normal cultures. Cellular characterization by immunostaining with a specific antibody displayed normal neuronal phenotype in control cells, whereas pretreatment in simulated microgravity revealed an increase of glial fibrillary acidic protein fluorescence in the elongated stellate glial cells. Electrophysiological recording indicated that the electrical properties of neurons preexposed were comparable with those of controls. Overall, our results indicate that a short time of simulated microgravity preexposure does not affect dramatically the ability of dissociated neural cells to develop and differentiate in an adherent culture system.     

Limitations of Chromogranin A in clinical practice

Context: Usefulness of circulating Chromogranin A (CgA) for the diagnosis of neuroendocrine tumors (NEN) is controversial. The aim of the present study was to assess the actual role of this marker as diagnostic tool. Methods: Serum blood samples were obtained from 42 subjects affected with NEN, 120 subjects affected with non-endocrine neoplasias (non-NEN) and 100 non-neoplastic subjects affected with benign nodular goitre (NNG). Determination of CgA was performed by means of immunoradiometric assay. Results: The CgA levels among NEN-patients were not significantly different from NNG and non-NEN subjects. The Receiver operating characteristic (ROC) curves analysis failed to identify a feasible cut-off value for the differential diagnosis between NEN and the other conditions. Conclusion: Serum CgA is not helpful for the first-line diagnosis of NEN.     

Derivation and Validation of a Scoring System to Identify Patients with Bacteremia and Hematological Malignancies at Higher Risk for Mortality

Background

The aim of this study was to develop and validate a reliable clinical prediction rule that could be employed to identify patients at higher likelihood of mortality among those with hematological malignancies (HMs) and bacterial bloodstream infections (BBSIs).

Methods and Findings

We conducted a retrospective cohort study in nine Italian hematological units. The derivation cohort consisted of adult patients with BBSI and HMs admitted to the Catholic University Hospital (Rome) between January 2002 and December 2008. Survivors and nonsurvivors were compared to identify predictors of 30-day mortality. The validation cohort consisted of patients hospitalized with BBSI and HMs who were admitted in 8 other Italian hematological units between January 2009 and December 2010. The inclusion and exclusion criteria were identical for both cohorts, with type and stage of HMs used as matching criteria. In the derivation set (247 episodes), the multivariate analysis yielded the following significant mortality-related risk factors acute renal failure (Odds Ratio [OR] 6.44, Confidential Interval [CI], 2.36–17.57, P<0.001); severe neutropenia (absolute neutrophil count <100/mm³) (OR 4.38, CI, 2.04–9.43, P<0.001); nosocomial infection (OR, 3.73, CI, 1.36–10.22, P = 0.01); age ≥65 years (OR, 3.42, CI, 1.49–7.80, P = 0.003); and Charlson Comorbidity Index ≥4 (OR, 3.01, CI 1.36–6.65, P = 0.006). The variables unable to be evaluated at that time (for example, prolonged neutropenia) were not included in the final logistic model. The equal-weight risk score model, which assigned 1 point to each risk factor, yielded good-excellent discrimination in both cohorts, with areas under the receiver operating curve of 0.83 versus 0.93 (derivation versus validation) and good calibration (Hosmer-Lemshow P = 0.16 versus 0.75).

Conclusions

The risk index accurately identifies patients with HMs and BBSIs at high risk for mortality; a better initial predictive approach may yield better therapeutic decisions for these patients, with an eventual reduction in mortality.