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Biomedical subjects

L Brambilla

Publications and source records attributed to L Brambilla.

At least 19 recordsLinked to original sources

Alterations of the glucose metabolism in a triose phosphate isomerase-negative Saccharomyces cerevisiae mutant.

The absence of triose phosphate isomerase activity causes an accumulation of only one of the two trioses, dihydroxyacetone phosphate, and this produces a shift in the final product of glucose catabolism from ethanol to glycerol (Compagno et al., 1996). Alterations of glucose metabolism imposed by the deletion of the TPI1 gene in Saccharomyces cerevisiae were studied in batch and continuous cultures. The Deltatpi1 null mutant was unable to grow on glucose as the sole carbon source. The addition of ethanol or acetate in media containing glucose, but also raffinose or galactose, relieved this effect in batch cultivation, suggesting that the Crabtree effect is not the primary cause for the mutant's impaired growth on glucose. The addition of an energy source like formic acid restored glucose utilization, suggesting that a NADH/energy shortage in the Deltatpi1 mutant could be a cause of the impaired growth on glucose. The amount of glycerol production in the Deltatpi1 mutant could represent a good indicator of the fraction of carbon source channelled through glycolysis. Data obtained in continuous cultures on mixed substrates indicated that different contributions of glycolysis and gluconeogenesis, as well as of the HMP pathway, to glucose utilization by the Deltatpi1 mutant may occur in relation to the fraction of ethanol present in the media.

Bioreactors↗

CXCR4-activated astrocyte glutamate release via TNFalpha: amplification by microglia triggers neurotoxicity.

Astrocytes actively participate in synaptic integration by releasing transmitter (glutamate) via a calcium-regulated, exocytosis-like process. Here we show that this process follows activation of the receptor CXCR4 by the chemokine stromal cell-derived factor 1 (SDF-1). An extraordinary feature of the ensuing signaling cascade is the rapid extracellular release of tumor necrosis factor-alpha (TNFalpha). Autocrine/paracrine TNFalpha-dependent signaling leading to prostaglandin (PG) formation not only controls glutamate release and astrocyte communication, but also causes their derangement when activated microglia cooperate to dramatically enhance release of the cytokine in response to CXCR4 stimulation. We demonstrate that altered glial communication has direct neuropathological consequences and that agents interfering with CXCR4-dependent astrocyte-microglia signaling prevent neuronal apoptosis induced by the HIV-1 coat glycoprotein, gp120IIIB. Our results identify a new pathway for glia-glia and glia-neuron communication that is relevant to both normal brain function and neurodegenerative diseases.

Animals↗

Efficient homolactic fermentation by Kluyveromyces lactis strains defective in pyruvate utilization and transformed with the heterologous LDH gene.

A high yield of lactic acid per gram of glucose consumed and the absence of additional metabolites in the fermentation broth are two important goals of lactic acid production by microrganisms. Both purposes have been previously approached by using a Kluyveromyces lactis yeast strain lacking the single pyruvate decarboxylase gene (KlPDC1) and transformed with the heterologous lactate dehydrogenase gene (LDH). The LDH gene was placed under the control the KlPDC1 promoter, which has allowed very high levels of lactate dehydrogenase (LDH) activity, due to the absence of autoregulation by KlPdc1p. The maximal yield obtained was 0.58 g g(-1), suggesting that a large fraction of the glucose consumed was not converted into pyruvate. In a different attempt to redirect pyruvate flux toward homolactic fermentation, we used K. lactis LDH transformant strains deleted of the pyruvate dehydrogenase (PDH) E1alpha subunit gene. A great process improvement was obtained by the use of producing strains lacking both PDH and pyruvate decarboxylase activities, which showed yield levels of as high as 0.85 g g(-1) (maximum theoretical yield, 1 g g(-1)), and with high LDH activity.

Culture Media↗

Treatment of classical Kaposi's sarcoma with gemcitabine.

BACKGROUND: Several drugs are active in aggressive classical Kaposi's sarcoma (CKS); chemotherapeutic agents with fewer side-effects, more rapid response and able to overcome resistance to previous treatment are advisable when treating patients in a second line. Gemcitabine, an analogue of deoxycytidine with cytotoxic activity in the treatment of solid tumours, has been found to have no serious side-effects. OBJECTIVE: To evaluate the usefulness of treating patients affected by aggressive CKS with gemcytabine. METHODS: Twelve patients with a recurrent aggressive form of CKS previously treated with chemotherapy were treated with gemcitabine. The drug was administered intravenously at the dose of 1.2 g/week for 2 weeks, followed by a 1-week interval, until maximal response was reached. Objective responses and toxicity were evaluated according to WHO criteria. RESULTS: Eleven evaluable patients achieved an objective response: CR in 1/11 and PR in 10/11. Toxicity was limited. CONCLUSION: This study shows the usefulness of treating patients affected with aggressive CKS with gemcitabine, in order to obtain control of the disease and to reduce the related symptoms as well as to overcome a possible resistance to previous treatments.

Aged↗

Human herpesvirus 8 DNA in the skin and blood of patients with Mediterranean Kaposi's sarcoma: clinical correlations.

BACKGROUND: Kaposi's sarcoma is a multifocal lympho-angioproliferative disease that appears in elderly subjects of Mediterranean origin (classical form), young Africans and immunodepressed patients (as a result of organ transplantation or AIDS). In 1994, DNA sequences of a new human herpesvirus, called HHV-8, were detected in skin lesions and peripheral blood of patients with AIDS-related Kaposi's sarcoma by confirmational display analysis and polymerase chain reaction. OBJECTIVE: As HHV-8 in peripheral blood mononuclear cells is detected in about 50% of Mediterranean Kaposi's sarcoma patients and its presence fluctuates in time in the same patient, maybe its detection correlates with the clinical behaviour of the disease. METHODS: By using routine and nested polymerase chain reaction we evaluated the presence of HHV-8-specific DNA sequences in the skin lesions, perilesional healthy skin and peripheral blood mononuclear cells of a group of 40 HIV-negative patients with Mediterranean Kaposi's sarcoma. RESULTS: HHV-8 DNA sequences have been found in 40/40 (100%) lesional skin of Mediterranean Kaposi's sarcoma, in 35/40 (85%) perilesional apparently normal skin and in 24/40 (60%) peripheral blood monuclear cell samples. The results of polymerase chain reaction on peripheral blood monuclear cells were positive in 41% of the patients with slowly evolving disease as opposed to 74% of those with rapidly evolving disease, and in 47.6% of the patients with stage I-II disease as opposed to 73.6% of those with stage III-IV. CONCLUSION: The detection of HHV-8 in peripheral blood monuclear cells seems to correlate with the more aggressive stages and the rapid evolution behaviour of Mediterranean Kaposi's sarcoma.

Adult↗

Potassium depletion and salt sensitivity in essential hypertension.

To evaluate the actual role of potassium depletion on blood pressure, 11 hypertensive patients were placed on a 10-day isocaloric diet providing a daily potassium intake of either 18 or 80 mmol, with each subject serving as his or her own control; the intake of sodium (220 mmol/day) and other minerals was kept constant. On day 11 each patient was also subjected to central volume expansion by water immersion associated with either normal or low potassium intake. After a 10-day period of low potassium intake, systolic blood pressure increased (P < 0.02) by 5 mm Hg, whereas serum potassium decreased (P < 0.001) by 0.9 mmol/L; no significant changes in urinary sodium and a marked increase in urinary calcium excretion (P < 0.001) were found during the 10-day low potassium intake. PRA (P < 0.02) and plasma aldosterone (P < 0.04) concentrations also decreased during low potassium intake in hypertensive patients. Even though an identical natriuretic response was found during the water immersion experiments with either high or low potassium in the whole hypertensive group, the evaluation of hypertensive subjects in relation to salt sensitivity enabled us to disclose pronounced differences in the natriuretic and calciuretic response. In fact, although an impaired natriuretic ability and moderate calcium loss were particularly found during water immersion in those hypertensive subjects exhibiting a lower salt sensitivity index, a predominant calcium depletion appeared to be the most important consequence of potassium depletion in the hypertensive subjects with a higher salt sensitivity index. By confirming that potassium depletion may exacerbate essential hypertension, our data also suggest that not only sodium restriction, but also potassium and calcium supplementation, could be particularly advisable in salt-sensitive hypertensive patients.

Adult↗

Current awareness.

In order to keep subscribers up-to-date with the latest developments in their field, this current awareness service is provided by John Wiley & Sons and contains newly-published material on yeasts. Each bibliography is divided into 10 sections. 1 Books, Reviews & Symposia; 2 General; 3 Biochemistry; 4 Biotechnology; 5 Cell Biology; 6 Gene Expression; 7 Genetics; 8 Physiology; 9 Medical Mycology; 10 Recombinant DNA Technology. Within each section, articles are listed in alphabetical order with respect to author. If, in the preceding period, no publications are located relevant to any one of these headings, that section will be omitted. (4 weeks journals - search completed 7th Mar. 2001)

Yeasts↗

An interlaboratory comparison of physiological and genetic properties of four Saccharomyces cerevisiae strains.

To select a Saccharomyces cerevisiae reference strain amenable to experimental techniques used in (molecular) genetic, physiological and biochemical engineering research, a variety of properties were studied in four diploid, prototrophic laboratory strains. The following parameters were investigated: 1) maximum specific growth rate in shake-flask cultures; 2) biomass yields on glucose during growth on defined media in batch cultures and steady-state chemostat cultures under controlled conditions with respect to pH and dissolved oxygen concentration; 3) the critical specific growth rate above which aerobic fermentation becomes apparent in glucose-limited accelerostat cultures; 4) sporulation and mating efficiency; and 5) transformation efficiency via the lithium-acetate, bicine, and electroporation methods. On the basis of physiological as well as genetic properties, strains from the CEN.PK family were selected as a platform for cell-factory research on the stoichiometry and kinetics of growth and product formation.

Journal Article↗

Author reply

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Journal Article↗

Relating growth dynamics and glucoamylase excretion of individual Saccharomyces cerevisiae cells.

We have developed a novel flow cytometric procedure that allows determinations of properties of protein excretion in the growth medium on a cell-by-cell basis in Saccharomyces cerevisiae. The procedure is based on labelling of a periplasmically secreted protein with antibodies conjugated to a fluorescent marker such as fluorescein isothiocyanate (FITC). The staining conditions did not perturb cell growth after resuspension of stained cells in growth medium. Decrease in fluorescence was found to correlate with excretion of glucoamylase into the growth medium. The analysis of the staining pattern over time provides information on the behaviour of individual cells belonging to different cell-cycle phases and can be used to calculate the specific excretion rate of the overall population.

Culture Media↗

Real-time flow cytometric quantification of GFP expression and Gfp-fluorescence generation in Saccharomyces cerevisiae.

A genetic and analytical methodology was developed based on a green fluorescent mutant protein (Gfp(S65T)) that allows the real-time quantification of gene expression in Saccharomyces cerevisiae. Using the UAS(GAL)(1-10)/CYC1 promoter and plasmids that are maintained in different copy numbers per cell, wild-type GFP and mutant GFP(S65T) were expressed in low to high concentration. Flow cytometric analysis was then applied to directly quantify Gfp((S65T)) (both wild type and mutant protein) expression at the single-cell level, and to indirectly measure the concentrations of non-fluorescent apoGfp((S65T)) and fluorescent Gfp((S65T)), which is autocatalytically formed from the apoprotein. Kinetics of apoGfp((S65T))/Gfp((S65T)) conversion during aerobic growth showed that the time required for complete apoGfp((S65T)) conversion is limited only by the amount of apoprotein that is expressed. When GFP(S65T) was expressed in single copy, the apoprotein did not accumulate and was instantly converted into its fluorescent form. The data indicate that an instant quantification of gene expression in S. cerevisiae is achievable based on Gfp(S65T), even if the gene is transcribed from a very strong promoter.

Colony Count, Microbial↗

Human herpesvirus-8 infection among heterosexual partners of patients with classical Kaposi's sarcoma.

BACKGROUND: DNA sequences of human herpesvirus-8 (HHV8) are found in lesions of Kaposi's sarcoma (KS). OBJECTIVES: To verify the hypothesis of the sexual transmission of HHV8 infection. METHODS: We used the immunoperoxidase assay to study the prevalence of serum antibodies to HHV8-related antigens in 27 consorts of patients with classical KS, and in a control group of 25 healthy women. RESULTS: Forty-four per cent of the study group were positive, compared with 8% of the controls. CONCLUSIONS: The sexual route may be one of the possible ways of transmission of the HHV8 virus.

Aged↗

Improved secretion of native human insulin-like growth factor 1 from gas1 mutant Saccharomyces cerevisiae cells.

We studied the secretion of recombinant human insulin-like growth factor 1 (rhIGF-1) from transformed yeast cells. The hIGF-1 gene was fused to the mating factor alpha prepro- leader sequence under the control of the constitutive ACT1 promoter. We found that the inactivation of the GAS1 gene in the host strain led to a supersecretory phenotype yielding a considerable increase, from 8 to 55 mg/liter, in rhIGF-1 production.

Fungal Proteins↗

Development, reliability and acceptability of a new version of the DSM-IV Social and Occupational Functioning Assessment Scale (SOFAS) to assess routine social functioning.

OBJECTIVE: Development of a scale to assess patients' social functioning, the Personal and Social Performance scale (PSP). METHOD: PSP has been developed through focus groups and reliability studies on the basis of the social functioning component of the DSM-IV Social and Occupational Functioning Assessment Scale (SOFAS). The last reliability study was carried out by 39 workers with different professional roles on a sample of 61 psychiatric patients admitted to the rehabilitation unit. Each patient was rated independently on the scale by the two workers who knew them best. RESULTS: The PSP is a 100-point single-item rating scale, subdivided into 10 equal intervals. The ratings are based mainly on the assessment of patient's functioning in four main areas: 1) socially useful activities; 2) personal and social relationships; 3) self-care; and 4) disturbing and aggressive behaviours. Operational criteria to rate the levels of disabilities have been defined for the above-mentioned areas. Excellent inter-rater reliability was also obtained in less educated workers. CONCLUSION: Compared to SOFAS, PSP has better face validity and psychometric properties. It was found to be an acceptable, quick and valid measure of patients' personal and social functioning.

Adult↗

Rotenone and pyruvate prevent the tert-butylhydroperoxide-induced necrosis of U937 cells and allow them to proliferate.

Exposure of U937 cells to tert-butylhydroperoxide (tB-OOH) led to cyclosporin A-sensitive mitochondrial membrane permeability transition and necrosis. Pyruvate and rotenone, which increase mitochondrial NADH via different mechanisms, prevented these responses and the cells which received these treatments proliferated with kinetics similar to those observed in untreated cells. In contrast with these results, cells rescued by cyclosporin A were unable to proliferate. Thus, mitochondrial NADH plays a pivotal role in preventing upstream events which result in the onset of mitochondrial membrane permeability transition and death in cells exposed to tB-OOH. These events appear to be critical for recovery of the ability of the cells to proliferate.

Apoptosis↗

NADH-Linked substrate-mediated enhancement of mitochondrial calcium accumulation and DNA single-strand breakage elicited by tert-butylhydroperoxide: the source of the cation is a ryanodine-sensitive calcium store.

We previously found that the membrane-permeant NADH-linked substrates pyruvate and beta-hydroxybutyrate enhance the formation of DNA single-strand breaks induced by tert-butylhydroperoxide (tB-OOH) in intact U937 cells. This effect is mediated by a process involving enforced mitochondrial calcium accumulation in the absence of discernible elevation in the cytosolic concentration of free calcium ions. We now show that the intracellular source of the cation is a ryanodine-sensitive calcium store. A high concentration of ryanodine, which suppressed the caffeine-mediated mobilization of calcium ions, also abolished the effects of the NADH-linked substrates on the mitochondrial accumulation of the cation as well as on the tB-OOH-induced genotoxic response. These data constitute a novel demonstration of a physiological mechanism with important pathological implications.

Caffeine↗

Human immunodeficiency virus negative Kaposi sarcoma and lymphoproliferative disorders.

BACKGROUND: The concomitant occurrence of more than one primary neoplasm in the same individual has led researchers to seek possible common etiopathogenetic factors. Kaposi sarcoma (KS) is a multicentric neoplasm of vascular origin and perhaps viral etiology. Four forms of KS are known: classic or Mediterranean, endemic or African, posttransplant, and epidemic or acquired immunodeficiency syndrome-associated KS. In its classic form KS mainly affects elderly people and often has a long and indolent course that occasionally allows other malignancies to appear. Previous studies of the possible association between human immunodeficiency virus (HIV) negative KS and lymphoproliferative disorders (LDs) have produced discordant results. METHODS: To verify a possibly significant association between HIV negative KS and LDs, data relating to 250 evaluable Italian patients with HIV negative KS were evaluated retrospectively. RESULTS: Of the 250 KS patients, only 6 (2.4%) were found to have had an LD: 2 with Hodgkin lymphoma, 1 with non-Hodgkin lymphoma, 1 with cutaneous T-cell lymphoma, 1 with acute promyelocytic leukemia, and 1 with B-chronic lymphocytic leukemia. CONCLUSIONS: No significant association was found between HIV negative KS and LDs in the patient population in the current study. The authors believe that age, LD, or therapy-related immunodepression played a role in the cases in which KS appeared after the LD by determining the passing to the lytic phase of the herpes-virus HHV8 already present in anatomic sites of latency/persistence.

Adult↗

NADH reoxidation does not control glycolytic flux during exposure of respiring Saccharomyces cerevisiae cultures to glucose excess.

Introduction of the Lactobacillus casei lactate dehydrogenase (LDH) gene into Saccharomyces cerevisiae under the control of the TPI1 promoter yielded high LDH levels in batch and chemostat cultures. LDH expression did not affect the dilution rate above which respiro-fermentative metabolism occurred (Dc) in aerobic, glucose-limited chemostats. Above Dc, the LDH-expressing strain produced both ethanol and lactate, but its overall fermentation rate was the same as in wild-type cultures. Exposure of respiring, LDH-expressing cultures to glucose excess triggered simultaneous ethanol and lactate production. However, the specific glucose consumption rate was not affected, indicating that NADH reoxidation does not control glycolytic flux under these conditions.

Aerobiosis↗