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Biomedical subjects

G Serafini

Publications and source records attributed to G Serafini.

At least 55 records · Page 3Linked to original sources
PubMed1994

The soluble but not mitochondrially bound hexokinase is a substrate for the ATP- and ubiquitin-dependent proteolytic system.

Intracellular protein degradation is highly selective, however, the mechanism(s) underlying this selectivity are not fully understood. We have previously shown that purified rabbit hexokinase type I, an enzyme present in mammalian brain both in soluble and mitochondrial bound form, is conjugate to ubiquitin and then degraded by a rabbit reticulocyte fraction II. In the present study we report that the mitochondrial bound hexokinase is stable for several hours in the same proteolytic system both in the presence or absence of ATP. E1, E2 and E3, the enzymes of the ubiquitin conjugating system, are able to incorporate 125I- or biotin-labelled ubiquitin in an ATP-dependent manner in soluble hexokinase as well as in a number of mitochondrial proteins. Furthermore, the mitochondria by themselves have a pronounced ATP-dependent ability to conjugate 125I-ubiquitin. However, Western blotting experiments, using a specific antibody against hexokinase, or against ubiquitin, showed that the mitochondrial bound enzyme is neither ubiquitinated nor degraded. This result has been confirmed by purification of bound hexokinase from the brain mitochondrial fraction or following the incubation of intact mitochondria with ATP, 125I-ubiquitin and E1, E2 and E3. Thus, mitochondrial bound hexokinase is not recognized by the ubiquitin conjugating system while the soluble enzyme is conjugate to ubiquitin and then degraded. Furthermore, the soluble hexokinase from rabbit brain was isolated by immunoaffinity chromatography and shown to be recognized by an anti-ubiquitin antibody. These results suggest that the intracellular distribution of protein is an important feature of a protein which determines its susceptibility to ubiquitin-dependent degradation.

Adenosine Triphosphate↗
PubMed1994

Auditory evoked potentials at 40 Hz (SSR40Hz) in post-trauma coma patients.

The authors evaluated auditory evoked steady state rest potentials at 40 Hz (SSR40Hz) in 21 post-trauma coma patients and compared their predictive value for prognosis of survival and quality of life with the results of the electroencephalogram (EEG), the computed tomography (CT) scan, and the Glasgow Coma Scale. SSR40Hz potentials recorded immediately after trauma had a maximum (100%) sensibility and specificity for a fetal prognosis and a high value for life prognosis. Consequently they appear to be a useful parameter for deciding on organ explant procedures. The EEG was more reliable in predicting a quality of life prognosis.

Adult↗
PubMed1994

[Influence of antihypertensive therapy on the "white coat effect"].

The aim of this study was to evaluate the alarm reaction when hypertensive patients either receiving or not receiving drug therapy have their blood pressure measured (the so-called "White coat effect"). A group of 64 patients (38 males and 26 females, mean age 52 +/- 11 years, OMS stage I-II) was studied. Twenty-three patients were not receiving antihypertensive therapy, 41 patients were regularly taking antihypertensive therapy prescribed by their own doctors. Non-invasive monitoring of arterial pressure (AP) and heart rate (HR) was carried out in all patients. The following parameters were examined in this study: mean of 3 measurements of AP and HR before monitoring (CAP, CHR), daily mean of AP and HR monitoring (MAP, MHR). The following results were obtained: 1) non-treated patients, CAP 157/108 +/- 19/11 mmHg, CHR 83 +/- 11 b/min, MAP 155/100 +/- 20/7 mmHg, MHR 76 +/- 7 b/min; 2) treated patients, CAP 151/96 +/- 21/16 mmHg, CHR 73 +/- 16 b/min, MAP 142/86 +/- 15/12 mmHg, MHR 70 +/- 10 b/min. The two-tailed "t"-test for paired data showed a statistically significant difference (p < 0.001) between diastolic CAP and diastolic MAP in both groups of patients. Systolic CAP was significantly greater than systolic MAP in treated patients (p < 0.01), whereas CHR was significantly higher than MHR in non-treated patients (p < 0.001). These data demonstrate that the alarm reaction to measuring blood pressure is present in both treated and non-treated hypertensive patients. Antihypertensive treatment appears to lessen the chronotropic but not the pressure response to measurement of AP in a hospital setting.

Antihypertensive Agents↗
PubMed1994

[Doppler color-echo and flowmetric evaluations in ovarian tumors in postmenopausal patients].

The use of color Doppler techniques in transvaginal sonography (US) permitted the identification of some typical flowmetric patterns of malignant ovarian cancers, because of such characteristics of neoplastic vessels as no media in the vessel wall, the presence of A-V shunts, and so on. These features reduce vascular impedance, from which low pulsatility index and resistance index values result. Eighty-one patients with ovarian masses were examined: in 19 of them histopathology revealed malignant lesions in different stages. Eighteen of those lesions exhibited suspect transvaginal patterns; in one case only the US patterns suggested a benign lesion, which was a serous cystadenocarcinoma with thin and regular septa. In all the malignant lesions flow could be sampled in six sites at least; the lowest pulsatility index values, obtained by multiple samplings in the same lesion, were considered as the most representative of the histologic features of the lesion. The average of the lowest pulsatility index values in 18 of 19 malignant lesions was 0.75. In one case only (the only false negative in the series) the lowest pulsatility index value was 1.20. Doppler signals were detected in 58 of 62 benign lesions only and the average of the lowest pulsatility index values was 1.80. The only kind of condition with low pulsatility index was the inflammatory disease (all the 6 sampled cases). If we consider a pulsatility index < 1 as the cut-off value for malignant lesions, the sensitivity and the specificity of color Doppler US were 95% and 91%, respectively, in our series. The positive predictive value of the exam was 75% and its negative predictive value was 95%.

Color↗
PubMed1993

Intracellular distribution of hexokinase in rabbit brain.

Hexokinase in mammalian brain is particulate and usually considered to be bound to the outer mitochondrial membrane. Investigation of rabbit brain mitochondria prepared either by differential centrifugation and discontinuous density gradient centrifugation has provided evidence that this particulate fraction also contains endoplasmic vesicles and synaptosomes. Solubilization of the bound hexokinase by different combinations of detergents and metabolites has proved the existence of different hexokinase binding sites. Electron microscopic examination of hexokinase location by immuno-gold labelling techniques confirmed that hexokinase is indeed predominantly bound to mitochondria but that a significant proportion is also bound to non-mitochondrial membranes. Attempts to quantify this distribution were unsuccessful since different figures were obtained using anti-hexokinase IgG affinity purified on immobilized native or denatured hexokinase. Binding studies of the purified rabbit brain mitochondrial hexokinase to rabbit liver mitochondria and microsomes confirmed that in addition to a binding site on mitochondria there is another binding site on microsomes. The N-terminal sequence of hexokinase has been shown to be important for mitochondria binding and also for microsome binding. These results suggest that the intracellular localization of hexokinase in rabbit brain is not exclusively mitochondrial and that the metabolic role of this enzyme should be reconsidered by including a binding site on the endoplasmic reticulum.

Animals↗
PubMed1993

Acoustic structure of vocalization and stapedius muscle activity during vocal development in chickens (Gallus gallus).

The link between stapedius muscle activity and acoustic structure of vocalization was analysed in cocks of age 20-30 to 90-100 days old. The results show that stapedius muscle activation depends on the acoustic structure of vocalization and changes during vocal development. This dependence was observed in spontaneous calls and in vocalizations elicited by stimulating the mesencephalic "calling area". In 30-day-old cocks stapedius muscle EMG response is never associated with vocalizations with an acoustic energy content which is always distributed at frequencies higher than 2000 Hz. The coupling between vocalization and stapedius muscle activity begins later, when birds produce vocalizations with acoustic energy shifted towards lower frequencies. Overall, stapedius muscle activity is related to a bird's production of high amplitude low frequencies. These results support the hypothesis that the primary role of the stapedius muscle during normal vocal development is to dampen the amplitude of low frequency energy that reaches the cochlea during vocalization.

Aging↗
PubMed1993

SSR-40Hz electrogenesis: results of clinical investigation.

This study attempts to provide clinical evidence on the origin of SSR-40Hz auditory evoked potential studying posttrauma coma patients. The SSR-40Hz present two types of morphology: an acute narrow wave, defined as "sharp", and a slow wide curve defined as "rounded". In constant recording of auditory brainstem responses (ABR) the "sharp" wave correlated with the absence of middle latency responses (MLR), and the "rounded" with their presence. In some patients the morphology changed passing grom a "sharp" to a "rounded" wave and a tendency towards widening preceded the appearance of middle latency responses. Perhaps the functional electrophysiologic block may have diminished in sub-cortical neuronal areas not involved in MLR genesis.

Adolescent↗
PubMed1993

[The role of echography in osteolytic tubercular abscesses].

Tubercular abscesses are relatively common complications of tubercular spondylodiscitis. Fifty-one patients with suspected abscesses were selected from a group of 97 patients with tubercular spondylodiscitis and submitted to US. In 10 cases CT was performed before US and detected 7 abscesses, all of them confirmed by US. In the extant 41 cases, CT followed US; in 13 cases only US poorly visualized ilio-psoas muscles. As for the group of 23 patients who underwent both CT and US, if the former method is assumed as the reference gold standard, overall US sensitivity is 97% (1 false negative) and its specificity is 100%. In all cases where US findings were accurate and specific enough, CT was not performed; the patients were followed every seventh month and no abscesses found. US showed abscesses in the iliac fossa in 20 cases, along the psoas fascia in 6 and in the thighs in 3 cases. Two cases of gluteal localization were observed, together with 1 Grynfelt's triangle abscess, 1 Petit's triangle and 1 Scarpa's triangle abscesses; finally, 1 abscess was found in the knee. The most common appearance of tubercular abscesses is a hypoechoic and inhomogeneous pattern; sometimes caseum makes the abscess solid and hyperechoic. Calcifications were unusual in our series. All patients were submitted to percutaneous drainage under US guidance. The results proved US to allow the early and unquestionable diagnosis of tubercular abscesses and to confirm clinical suspicion. Moreover, US is also useful to guide percutaneous drainage and to follow the patients after drainage. As for CT, it remains the method of choice to depict vertebral involvement, but, in our series, it exhibited no significant advantages over US in the study of abscessual lumbar collections.

Abscess↗
PubMed1992

Carotid body tumors: US evaluation.

The ultrasound (US) findings in 20 patients with 23 carotid-body chemodectomas were reviewed. Twenty-two of 23 tumors could be seen at US; the remaining lesion could not be differentiated from surrounding enlarged lymph nodes resulting from thyroid cancer. The lesions were solid, slightly heterogeneous masses that ranged in size from 1.2 to 5.0 cm and were located within the carotid bifurcation. Pulsed Doppler analysis of blood flow within the tumor mass was possible in eight patients with nine chemodectomas, and low-resistance waveforms were obtained from multiple sites within the mass in all cases. The diagnostic possibility of a chemodectoma has to be considered when a solid mass is detected within the carotid bifurcation. On the basis of these findings, as US diagnosis was possible in 18 of 20 patients in the authors' series. Doppler analysis of the mass to evaluate intratumor blood flow is helpful in differentiating chemodectomas from other solid, nonhypervascular masses.

Adult↗
PubMed1991

Evidence for a particulate location of ubiquitin conjugates and ubiquitin-conjugating enzymes in rabbit brain.

Conjugate ubiquitin was previously found in the nucleus, cytoplasm, and membranes of eukaryotic cells while the enzymes of the ubiquitin-conjugating system appear to be cytoplasmic. We have prepared the mitochondrial fraction from rabbit brain by discontinuous density gradient ultracentrifugation and by Western blotting, using a specific antibody against conjugate ubiquitin, showing that it contains ubiquitin conjugates in a very wide molecular weight range. Electron microscopy and measurement of specific enzyme markers show that this fraction not only contains mitochondria but also some endoplasmic reticulum vesicles. Immunostaining with anti-ubiquitin IgG followed by immunodecoration with colloidal gold particles provides evidence for the presence of conjugate ubiquitin both in mitochondria and in the endoplasmic reticulum. Furthermore, this "mitochondrial fraction" shows a pronounced ATP-dependent ability to conjugate 125I-ubiquitin into a number of endogenous proteins as evidenced by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. Addition of E1, E2, and E3, the enzymes of the ubiquitin conjugating system purified from rabbit reticulocytes, does not further increase this ubiquitination nor incorporate 125I-ubiquitin into additional protein bands. The same mitochondrial fraction is not able to carry out any ATP-dependent degradation of 125I-albumin; however, it contains an isopeptidase activity able to release the covalently incorporated 125I-ubiquitin and is also able to conjugate 125I-ubiquitin to exogenous proteins as oxidized RNase. By affinity chromatography on ubiquitin-agarose of fraction II of a crude Triton X-100 extract of the mitochondrial fraction, several proteins corresponding in Mr to the E1 and E2s enzymes were obtained. These proteins were also able to form specific ubiquitin-thiol ester bounds on sodium dodecyl sulfate-polyacrylamide gels and to support 125I-ubiquitin conjugation to oxidized RNase. Detergent fractionation of the mitochondrial fraction provided evidence for a possible localization of the ubiquitin conjugating activity in the mitochondrial external membrane and endoplasmic reticulum. The presence of an active ubiquitin protein conjugating system in mitochondria and endoplasmic reticulum may be related to the turnover of organelle proteins as well as to specific cell functions such as import of proteins into mitochondria and ubiquitination of externally oriented membrane-bound proteins.

Animals↗
PubMed1991

Human hexokinase type I microheterogeneity is due to different amino-terminal sequences.

Human placenta hexokinase type I was previously shown to be present in two subtypes with similar isoelectric points but different molecular masses of 112 and 103 kDa, respectively. In order to exclude that these subtypes arise by artifact(s) occurring during the protein purification, we have developed a single-step immunoaffinity chromatography for the isolation of microgram quantities of hexokinase. The results obtained confirmed the presence of both hexokinase subtypes in human placenta. By Northern blot analysis a single mRNA species that hybridized with a hexokinase-I cDNA was found to be present in human placenta. Furthermore, in vitro translation of placenta mRNA in a rabbit reticulocyte lysate followed by hexokinase immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography showed that only one hexokinase with apparent molecular mass of about 112 kDa is expressed in this tissue and suggests a post-translational modification as a probable cause of hexokinase I microheterogeneity. To further investigate this point we have purified the high and low Mr hexokinase and determined their NH2-terminal sequences. The results obtained show that when compared with the amino acid sequence deduced from a cDNA the high Mr hexokinase starts at amino acid 11 while the low Mr hexokinase starts at amino acid 103. Since the first 10 amino acids are involved in the binding of hexokinase to mitochondrial porin these data provide an explanation both for the inability of these hexokinases to bind to mitochondria and for their differences in Mr.

Amino Acid Sequence↗
PubMed1991

Whole-blood filterability in sudden deafness.

Sixteen patients with sudden deafness (SD), diagnosed on the basis of a battery of audiometric tests, but with no other medical or surgical pathology requiring drug treatment, underwent monitoring of their hemorheological profiles to see whether disturbances in the microcirculation could be linked to SD. Plasma viscosity, the filterabilities, (using a low-shear positive pressure system) through 5-microns-diameter pore Nuclepore filters, of whole blood and red and unfractionated white cells were monitored in 16 SD patients and 32 controls matched for age, sex and socioeconomic status. Whole blood filterability and the filterability of the red blood cells were significantly impaired in the SD patients, which suggests that alterations in the microcirculation are linked, in some way, to sudden deafness.

Adult↗
PubMed1990

Preparation and characterization of monoclonal antibodies to human hexokinase type I.

1. Three different immunization protocols and several screening procedures were used to prepare seven mouse monoclonal antibodies to human placenta hexokinase type I. None of these monoclonals were able to recognize the native enzyme but all detected hexokinase when adsorbed onto polystyrene plates or on immunoblots after SDS/polyacrylamide-gel electrophoresis. 2. All seven monoclonals recognize the two different subtypes of human hexokinase I equally well. Limited tryptic digestion of hexokinase followed by Western blotting and immunodetection show that these monoclonals recognize epitopes that lie in different tryptic peptides. 3. Comparative ELISA studies showed that human hexokinase types I and II have great immunological similarities while hexokinase I from different mammalian species and yeast hexokinase are recognized with different affinities.

Animals↗