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Biomedical subjects

A F Borghetti

Publications and source records attributed to A F Borghetti.

At least 37 records · Page 2Linked to original sources
PubMed1993

Effect of betaine on HSP70 expression and cell survival during adaptation to osmotic stress.

Induced expression of the HSP70 gene in 3T3 and SV-3T3 cells was monitored by measurements of the synthesis of HSP70 and of the cellular contents of both HSP70 and its mRNA. The presence of betaine (N-trimethylglycine) at concentrations of 2.5-25 mM decreased the induction of HSP70 gene expression caused by incubation of 3T3 and SV-3T3 cells in hypertonic (0.5 osM) medium. This effect was accompanied by an enhancement of SV-3T3 cell adaptation, assayed by colony formation, to the hyperosmotic conditions. In contrast, the presence of betaine did not affect HSP70 gene expression induced in these cells by heat shock. After 6 h incubation with 25 mM betaine under hypertonic (0.5 osM) conditions the intracellular concentration of betaine in SV-3T3 cells was about 195 mM, compared with about 70 mM under isotonic (0.3 osM) conditions. Hence, with this concentration of extracellular betaine, the marked increase in the accumulation of betaine within the cells presumably counteracts the imposed osmotic pressure and eliminates the signal that otherwise initiates increased expression of the HSP70 gene.

3T3 Cells↗
PubMed1993

Different HSP70 expression and cell survival during adaptive responses of 3T3 and transformed 3T3 cells to osmotic stress.

Responses both to hyperosmotic stress and to heat shock were compared in 3T3 cells, spontaneously transformed cells (ST3T3) and simian virus 40-transformed cells (SV3T3). Cell adaptation to these stresses was measured in terms of surviving cell viability and plating efficiency, while their induced synthesis of stress proteins was monitored in terms of the presence of mRNA for HSP70, the pattern of polypeptides synthesised and the accumulation of HSP70 detectable by monoclonal antibodies. All three types of cells responded similarly to heat shock in their expression of HSP70 and showed no clear differences in ability to recover. In contrast, both ST3T3 and SV3T3 cells adapted more poorly and much more slowly to hyperosmotic stress (0.5 osM incubation) than did normal 3T3 cells. This different pattern of adaptation to hyperosmotic stress was parallelled by the cells' different expression of a stress protein that could not be distinguished from the heat-induced HSP70 by any of the methods listed above. In view of these findings it seems possible that hyperosmotic treatment might be useful in selectively affecting the survival of tumour cells.

3T3 Cells↗
PubMed1992

Modulation by betaine of cellular responses to osmotic stress.

Various solutes were tested to see if they could modify the responses of SV-3T3 cells to hyperosmotic (0.5 osM) conditions, which cause an inhibition of general cell protein synthesis and of the rate of cell proliferation, coupled with an induction of amino acid transport activity. The added solutes were glycerol, proline, taurine, betaine, dimethylglycine and sarcosine. Of these, betaine produced the most dramatic and consistent effects. Addition of 10-25 mM-betaine to the hyperosmotic medium largely prevented the 90% inhibition of cell proliferation that occurred in its absence. Whether it was added initially or after the cells were exposed to hyperosmotic medium, 25 mM-betaine also converted a 50% recovery of the rate of protein synthesis into 100%. Similarly, the same concentrations of betaine prevented a 30% decrease in cell volume and decreased the induction of amino acid transport via system A by 73%. Lower concentrations of betaine produced smaller but still significant changes in these functional responses. With chick-embryo fibroblasts, under identical hyperosmotic conditions, 25 mM-betaine completely counteracted a 75% inhibition of the rate of protein synthesis. At present it is not clear how betaine modulates these effects of hyperosmolarity on cell functions.

3T3 Cells↗
PubMed1992

Modulation of cell growth and host protein synthesis during HIV infection in vitro.

During HIV infection of CEM cells cultured in vitro, significant differences in growth rate and protein turnover were observed with different viral preparations. There was a significant inhibition of proliferation after infection with crude HIV supernatants. On the other hand, infection with purified HIV particles obtained by filtration, differential centrifugation, and isopycnic sedimentation led to a progressively increasing stimulation of cell growth. This early stimulation was prevented by neutralizing the virus with soluble CD4 molecules. Study of cell growth in the presence of a purified membrane preparation indicated that membrane fragments contaminating the crude HIV supernatant were responsible for the observed growth inhibition. Interestingly, the stimulation of proliferation was also observed with heat-inactivated virus or after inhibition of viral replication with ZDV. In the presence of purified HIV virions, the rate of general protein synthesis was not inhibited, as is usually observed with crude viral supernatants. However, a marked reduction in protein content and increased protein degradation was found in cultures infected with either crude or purified HIV preparations.

CD4-Positive T-Lymphocytes↗
PubMed1992

New developments in synthetic bone replacement materials.

Synthetic bone replacement materials continue to be much discussed in the current periodontal literature. Numerous reports have shown their clinical use in the treatment of intraosseous defects. Periodontal treatment aims also include regeneration of a new functional attachment. Although histologic studies have shown that most of the synthetic bone substitutes can enhance bone formation, they are not able to promote new attachment of periodontal tissues to the root surface previously exposed. Future studies are needed to assess whether these materials could be of use together with growth factors in composite grafts or in conjunction with guided tissue regeneration techniques.

Alveolar Bone Loss↗
PubMed1991

Differential adaptive response to hyperosmolarity of 3T3 and transformed SV3T3 cells.

Both 3T3 and simian virus 40-transformed 3T3 (SV3T3) cells were used to investigate differences in population kinetics, protein synthesis, monovalent ion levels, and amino acid accumulations between normal and transformed cells exposed to hyperosmolarity at 0.5 Osm. Under similar culture conditions, SV3T3 cells were found to be more sensitive in their proliferative response than normal cells to the hyperosmolar treatment. In the normal 3T3 cells, the increase in transport of amino acids was less sustained and was associated with higher levels of accumulated amino acids. The equilibrium distribution of intracellular monovalent cations and the rate of protein synthesis also returned faster to baseline values in the normal cells than in the transformed cells. Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) analysis revealed the induction of a 69-kDa polypeptide in the 3T3 cells but not in the SV3T3 cells after exposure to hyperosmolarity. On electrofocusing and relative mass analysis, this polypeptide closely migrated with the 70-kDa heat shock protein (hsp) family, although it was unrelated immunologically to the inducible 72-kDa hsp.

Amino Acids↗
PubMed1990

Induction of amino acid transport activity in chick embryo fibroblasts by replacement of extracellular sodium chloride with disaccharide.

The activity of amino acid transport System A in avian fibroblasts was increased following incubation of the cells in a medium in which most of the NaCl normally present had been isoosmotically replaced by sucrose. This increase was detectable after 2 h of incubation, reached a maximum at about 4 h, and remained constant thereafter. Transfer of treated cells back to a normal medium resulted in decay of the induced transport activity, with a half-life of less than 2 h. Kinetic analysis revealed that the increase in transport activity arose from an increase in Vmax, with little change in Km. This induction of System A activity did not occur if an inhibitor of either RNA or protein synthesis was present in the modified medium. The use of various different solutes as replacements for NaCl in the incubation medium showed that, although each replacement caused a decrease in both cellular Na+ content and protein synthesis, only disaccharides produced the increase in amino acid transport activity. In addition, estimates of cell volume indicated that, even under iso-osmotic conditions, incubation in the sucrose-containing medium caused initial cell shrinkage, followed by swelling. It is concluded that this induction of System A activity is associated with a volume regulatory process and that this process probably accounts for the parallel responses previously observed when cells were incubated in hyperosmolar media. Induction of amino acid transport activity by this process is distinct from adaptive regulation, caused by amino acid starvation; but the two processes are not strictly additive, and so appear to converge at some step.

Animals↗
PubMed1990

Stimulatory effect of serum albumin on the proliferation of serum-free SV40-transformed Balb/c 3T3 cells.

Commercial serum albumins have been found to be able to stimulate the proliferation of Balb/c 3T3 cells transformed by SV40, but not that of the normal counterpart. The effect is most pronounced with crystalline samples of albumin depleted of both globulin and fatty acid components, and depends on conditions used for the attachment and on seeding density. Physical and chemical treatments aimed to remove tightly bound impurities do not abolish the activity of fatty acid free serum albumin, thus supporting the idea that albumin per se is mitogenic towards these cells.

Animals↗
PubMed1990

Protease activation during HIV infection in a CD4-positive cell line.

The mechanism of cytopathic effects associated with HIV infection in a continuous line of CD4-positive lymphocytes (CEM cells, clone 13) has been studied. Here we report the following observations: (1) HIV infection killed a variable but always significant number of cells without a strict relationship with the syncytia formation; (2) an important decrease in the proliferation rate occurred soon after infection; (3) a marked inhibition of protein synthesis took place within the first few hours of infection and clearly before the beginning of viral protein expression. In addition, when three-day-old cultures were incubated in serum-free medium, a larger degradation of proteins was observed in infected cells in comparison to controls. An increase in protein degradation activity was observed also in vitro with extracts obtained from HIV-infected cells and incubated in the presence of endogenous- or exogenous-labeled substrates. Extracts from cells infected with heat-inactivated HIV did not show a similar degradative activity. The possible induction or activation of latent proteases during the development of the HIV infection is discussed.

CD4 Antigens↗
PubMed1989

Density-dependent regulation of amino acid transport in a Burkitt lymphoma cell line.

Rate of proliferation and amino acid transport were assessed in the Burkitt's lymphoma-derived Namalwa cells by measurements of growth rate and proline and serine uptake. Cell density of the cultures was varied by modifying the number of cells initially seeded and growing for different periods of time. Under these experimental conditions the growth rate was not correlated with cell density. In contrast, the activity of amino acid transport through Systems A and ASC, as assessed by the uptake of proline and serine, respectively, decreased as a function of cell density. This marked decrease of transport activity cannot be explained by large alterations of cell morphology since it was observed at a cell density range where minimal change of cell volume and surface area occurred. When a constant number of cells suspended in an identical volume of medium sedimented on different settling areas, a marked effect on amino acid transport activity occurred. These results indicate that cell to cell contacts may be involved in the density-dependent regulation of transport.

Amino Acids↗
PubMed1989

Control of protein synthesis by extracellular Na+ in cultured fibroblasts.

In chick embryo fibroblasts (CEFs), a partial substitution of extracellular Na+ with other cations or carbohydrates decreased the intracellular Na+ content without altering the K+ level. Concomitantly, a significant decrease in the serum-dependent rate of protein synthesis occurred. This phenomenon appeared to be quickly reversible upon reconstitution of the correct extracellular Na+ concentration in the culture medium. The presence of a transcriptional inhibitor such as actinomycin D during the treatment did not inhibit the reversibility of the phenomenon. The presence in the culture medium of K+ in such excess as to dissipate the membrane potential did not alter the observed relationship between the protein synthesis rate and the internal Na+ content. Analysis of the amino acid pool indicated that the observed inhibition of the rate of protein synthesis in CEFs incubated in low Na+ medium was not caused by an unbalanced availability of intracellular amino acids. In addition, intracellular pH, as estimated by the measurement of the equilibrium distribution of benzoic acid, did not show any significant alteration in cells incubated in the presence of bicarbonate buffer and in low extracellular Na+. Moreover, the relationship between the rate of protein synthesis and the internal Na+ content was still observed in CEFs cultured in bicarbonate-containing media, but at lower or higher than physiological pH. Analysis by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) of the proteins synthesized by CEFs cultured at a reduced extracellular Na+ concentration showed that specific alterations of gene expression occurred.

Amino Acids↗
PubMed1988

Identification and characterization of tyrosine kinase activity associated with mitochondrial outer membrane in sarcoma 180 cells.

Tyrosine protein kinase activity has been detected in the mitochondrial fraction purified from sarcoma 180 tumor cells. Following hypotonic disruption of mitochondria, tyrosine kinase activity appeared to cosediment with monamine oxidase, marker enzyme of mitochondrial outer membrane; meanwhile, serine and threonine kinases were found to be associated with the inner membrane and matrix of mitochondria. Mitochondrial tyrosine kinase(s) showed thermosensitivity and Mn2+ dependence, useful properties for its characterization and separation from tyrosine kinases associated with other particulate fraction and from serine and threonine kinases associated with mitochondria. Following in vitro incubation of mitochondria with labelled ATP as substrate and analysis by PAGE, a complex pattern of phosphotyrosine containing proteins with a major band of 50-55 kilodaltons resulted.

Animals↗
PubMed1988

Enhancement of mitochondrial tyrosine kinase activity following viral transformation.

A tyrosine protein kinase activity has been detected in the mitochondrial fraction purified from normal and virus-transformed cultured cells. The addition of serum to cells whose growth was restricted by serum limitation induced a marked decrease of tyrosine kinase activity associated with the mitochondrial fraction. At all the culture conditions tested this enzyme activity always resulted several fold higher in the virus-transformed cells than in the normal parental cells.

Animals↗
PubMed1987

Multicomponent analysis of amino acid transport System L in normal and virus-transformed fibroblasts.

Amino acid transport System L in both normal Balb/c 3T3 cells and in those transformed with simian virus 40 (SV 3T3) was analysed kinetically under two different experimental conditions. Under 'zero-trans' conditions the results for both types of cell could be interpreted satisfactorily in terms of System L consisting of two components (L1 and L2) characterized by different Km values. This conclusion is in agreement with previous reports. However, under 'infinite-trans' conditions, the experimental data could not be accounted for in terms of only two components; the introduction of a third component (L3) was necessary to provide a satisfactory fit. Viral transformation affects only the L1 component, either by modification or by replacement, giving it a higher 'affinity' (lower Km) but a lower 'capacity' (lower Vmax).

Amino Acids↗
PubMed1987

Hyperosmolarity-induced stress proteins in chick embryo fibroblasts.

The effects of a short exposure of chick embryo fibroblasts to a hyperosmolar medium on monovalent cation content, rate of protein synthesis, and polypeptide pattern expression were studied. The hyperosmolar shock gave an immediate and pronounced inhibition of the protein-synthesis rate temporally related to a marked alteration of the intracellular Na+ content. Following the return of the cells to an osmolar environment, the internal Na+ content quickly resumed its previous level, while the recovery of the protein-synthesis rate was more gradual. During the recovery period, there was enhanced expression of at least 12 proteins. The 4 major induced proteins exhibited apparent molecular weights of 96, 87, 70, and 48 kDa. A reduction in the synthesis of five protein bands including three large polypeptides of 220, 160, and 140 kDa was also observed. A comparison with the 3 major proteins induced by a 44 degrees C heat shock indicated an apparent similarity with only two of the hyperosmolarity-inducible polypeptides. Moreover, evidence has been also obtained of the close similarity between the 96 and 75 kDa glucose-regulated proteins and the 96 and 75 kDa proteins inducible by a hyperosmolar shock or by a continuous hyperosmolar treatment, respectively. The kinetics of the stress-proteins appearance indicated nonsimultaneous induction. The presence of actinomycin D during the exposure of the cells to the stress and the recovery period suggested that the expression of some hyperosmolarity-enhanced proteins is regulated at the transcriptional level.

Animals↗