Biochemical assessment of growth factors and circulation of blood components contained in the different fractions obtained by centrifugation of venous blood.

The aim of this study was to evaluate a biochemical marker with different elements of a normal blood serum and centrifuged blood serum after a different rotation system. For this technique, we used five fractions of a blood Concentrated Growth Factors system (bCGF) and a particular device for the different rotation program. Blood samples were collected from 10 volunteers aged between 35 and 55 in the Operative Unit of the “Sapienza” University of Rome with only a fraction of different biochemical elements. Through an individual blood phase separator tube of venous blood, active factions of serum and 4 fractions of red buffy coat were taken. The biochemical markers with 14 elements were examined at times: P1-11 minutes, P2-12minutes, P3-15 minutes. Exclusively biological materials which are normally applied in the regeneration techniques for different defects and lesions were used with this technique. After specific rotation programs, a different result was obtained for each cycle: P1, P2, P3. In test tubes obtained by separated blood, we observed a higher concentration of proteins, ions, and other antigens compared to normal blood plasma. Examining the biochemical results of different elements, we observed an increase (P≤0,01). Since each person’s DNA is different, we could not have the same results in 5 fractions of blood concentration, we did, however, find a good increase in only a fraction of proteins, immunoglobulin and different ions. We obtained five fractions after centrifugation, and we had an increase in different biochemical elements compared to normal blood (P≤0,01) which is significant at different times. These biochemical elements were stimulated by different growth factors, which are used by the immune system, and they induced the formation of hard and soft tissues and good regeneration.

Sodium iodide associated to salicylic acid in the topical management of chronic oral candidiasis: a randomized trial.

Candidiasis is a relevant problem in oral medicine practice. We compared the antimycotic activity of nystatin with a solution of sodium iodide associated to salicylic acid (SISA) in the topical management of chronic candidiasis. Consecutive patients affected by chronic candidiasis were randomly allocated to SISA (group A) or nystatin (group B). VAS and swab scores were recorded at the beginning and at the end of the study while the healing index was evaluated at the end of the study only. Data were analyzed by STATA 10 MP. Forty patients (20 male, 20 female) were randomized. SIAS was as effective as nystatin in affecting VAS (p greater than 0.05) and swab score (p greater than 0.05). A statistically significant reduction (p less than 0.05) of healing index was observed in both groups. No side effects were reported. SISA topical application, shows a comparable efficacy to the nystatin in the management of chronic oral candidiasis. Its use could represent an adequate alternative to the nystatin above all in the cases of drug-resistance. Further large scale randomized trials are warranted to confirm these preliminary findings.

[The "damage control" in severe hepatic injuries: our experience]. / Il "damage control" nel trattamento di gravi lesioni epatiche: nostra esperienza.

PURPOSE: To evaluate our 12-year experience in the treatment of complex hepatic injuries with periepatic packing and damage control priciples. METHOD: A retrospective review was conducted of 21 Patients with grade IV-V injuries of the liver and severe haemorrage induced hypothermia and acidosis admitted to the Ospedale Maggiore Trauma Center in Bologna from 1989 to 2001 RESULTS: All the Patients had major blunt trauma. Mean age was 39.6; mean ISS 41.5; mean RTS 4.13; extimated loss of blood was greater than 5300 ml. Packing provide definitive control of bleeding in 16 Patients but 10 had recurrent bleeding or bleeding from different injuries such as bone fractures and required further surgery or arterial embolization. 12 Patients died (57.2%). Survival was strongly associated with the ISS, GCS, the loss of blood and acidosis. CONCLUSION: The authors concluded that in selected circumstances the traditional approach to hepatic injuries is not appropiate. In this situation, alternative and aggressive treatment--damage control--has been recommended as the procedure of choice.

Sulfatides and arylsulfatase A activity in major salivary glands of hamster (Mesocricetus auratus) after adenocarcinoma induction in oral cavity.

A biochemical study of sulfatides and arylsulfatase A (ASA) was carried out in the submandibular and sublingual glands of the male and female hamster Mesocricetus auratus after experimental induction of oral adenocarcinoma by 7,12-dimethylbenzanthracene (DMBA). Hamster experimental groups included control animals, animals treated with beta-carotene, animals treated with DMBA, and animals treated with DMBA plus beta-carotene. Oral cavity treatment with DMBA induced carcinogenesis in the buccal mucosa, but not in the major salivary glands, where nevertheless, the morphology and expression of both parameters examined changed. In fact, sulfatide concentrations and enzyme activity increased significantly, while in control and beta-carotene-treated hamsters they were similar in both glands and sexes. After administration of DMBA plus beta-carotene, sulfatide concentration decreased, as did ASA activity, slightly in the submandibular gland and remarkably so in the sublingual one of female hamsters. Thin-layer chromatography (TLC) analysis of lipid patterns, after DMBA treatment, revealed considerable differences, not only in sulfatides, but also in other lipid fractions, as well as between the two glands and two sexes. These findings show that oral cavity treatment with DMBA is not able to induce carcinogenesis in the major salivary glands examined; however, it does cause considerable metabolic changes.

Nephroprotective effect of treatment with calcium channel blockers in spontaneously hypertensive rats.

The influence of hypertension and of treatment with some dihydropyridine-type Ca(2+) channel blockers and with the nondihydropyridine-type vasodilator hydralazine on the morphology of kidney was investigated in 26-week-old spontaneously hypertensive rats (SHR) and in age-matched Wistar-Kyoto rats. Fourteen-week-old SHR were treated for 12 weeks with a nonhypotensive dose of lercanidipine or with equihypotensive doses of lercanidipine, manidipine, nicardipine, and hydralazine. In control SHR, systolic pressure values were significantly higher in comparison with Wistar-Kyoto rats. Treatment with the low dose of lercanidipine did not reduce systolic blood pressure in SHR, whereas the higher dose of lercanidipine or other compounds tested significantly decreased systolic pressure values. Glomerular hypertrophy accompanied by signs of glomerulosclerosis, increase of mesangial cells, and convoluted tubules degeneration were observed in control SHR. Hypotensive doses of Ca(2+) antagonists countered glomerular injury, the increase of mesangial cells, the reduction of capsular space, and tubular degeneration. Hydralazine, in spite of its hypotensive activity, displayed a slight nephroprotective action. The nonhypotensive dose of lercanidipine countered in part glomerular injury, narrowing of capsular space, and tubular degeneration, and decreased mesangial cell augmentation in SHR. These results suggest that treatment with dihydropyridine-type Ca(+2) antagonists counters hypertensive glomerular and tubular changes occurring in SHR. The demonstration of nephroprotection by the nonhypotensive dose of lercanidipine suggests that the renal effects of the compound may be in part unrelated to its hemodynamic activity.

Sulphatides in the brain of spontaneously hypertensive rats.

Sulphatides were assayed in preparations of frontal cortex, neostriatum and hippocampus of 6-month-old male spontaneously hypertensive rats (SHR, systolic pressure 215 +/- 6 mmHg) and age-matched normotensive Wistar-Kyoto (WKY) rats (systolic pressure 143 +/- 6 mmHg) by thin layer chromatography associated with spectrophotometry and histochemistry. The volume of gray and white matter of the above areas was also measured by microanatomical techniques associated with image analysis. Sulphatide levels were unchanged in the frontal cortex and neostriatum and decreased in the hippocampus of SHR in comparison with WKY rats. No changes of metachromatic sulphatide staining were found in the different brain areas investigated of SHR, whereas a decrease of positive metachromatic areas was noticeable in the frontal cortex and neostriatum, but not in the hippocampus of SHR. A reduction of volume of frontal cortex gray and white matter as well as of striosomes and of gray matter of hippocampus was found in SHR. No changes in the total volume of neostriatum and in the volume of white matter of hippocampus were observed between SHR and normotensive WKY rats. These findings, which are consistent with recent evidence of the occurrence of atrophic changes in the brain of SHR, showed that sulphatide levels were decreased in the hippocampus of SHR. In this area no reduction of white matter was observed. Sulphatide concentrations are thought to reflect the status of brain myelinated fibers. The not parallel decrease of sulphatide levels and white matter volume in the majority of brain areas investigated suggests the occurrence in SHR of sulphatide changes not corresponding simply to a reduction of myelinated pathways.

Forebrain white matter in spontaneously hypertensive rats: a quantitative image analysis study.

The volume and the morphology of brain white matter as well as the number and the size of glial fibrillary acidic protein (GFAP)-immunoreactive astrocytes were investigated in 6-month-old spontaneously hypertensive rats (SHR) and age-matched normotensive Wistar-Kyoto (WKY) rats. The volume of frontal and occipital cortex and of hippocampus was decreased in SHR in comparison with normotensive rats, whereas the volume of neostriatum was unchanged. A remarkable decrease of the volume of internal capsule and striosomes, a moderate reduction of that of corpus callosum and no changes of the volume of external capsule and of white matter of hippocampus were also observed in SHR. In SHR the number of astrocytes was higher in the frontal and occipital cortex and in the white matter of the CA1 and CA3 subfields of the hippocampus, but not in the corpus callosum or in the grey matter of the CA1 and CA3 subfields. Staining for myelin did not reveal alterations in single fibre sheath morphology. These findings indicate the occurrence of changes of forebrain white matter in SHR, consisting in the reduction of it without qualitative modifications of myelinated fibres. The development of gliosis apparently not related with changes of volume of white matter was also found.

Dopamine D2-like receptors in the kidney of spontaneously hypertensive rats: a radioligand binding assay and light microscope autoradiography study.

1. Dopamine D2-like receptors were investigated in sections of kidney from male spontaneously hypertensive rats (SHRs) at 6 and 14 weeks of age using radioligand binding assay and autoradiographic techniques with [3H]-spiperone as a ligand. 2. Systolic blood pressure values were slightly higher in 6-week-old SHRs in comparison with age-matched normotensive Wistar-Kyoto (WKY) rats and considerably higher in 14-week-old SHRs in comparison with the other groups investigated. Renal dopamine levels were higher in SHRs aged 6 and 14 weeks in comparison with age-matched WKY rats. Noradrenaline concentrations were similar in 6-week-old SHRs and normotensive WKY rats, and increased slightly in SHRs aged 14 weeks. 3. The density of [3H]-spiperone binding sites was similar in SHRs and WKY rats at 6 weeks of age, and decreased in SHRs at 14 weeks in comparison with age-matched normotensive animals. Light microscope autoradiography revealed the accumulation of silver grains in the tunica adventitia, in the adventitia-media border of intrarenal arteries and within cortical tubules. A few specific silver grains were also developed in the glomerular tuft. No changes in the density and pattern of silver grains were noticeable between SHRs and WKY rats at 6 weeks of age, whereas a reduction in silver grains largely affecting vascular binding sites was observed at 14 weeks of age. 4. Renal denervation considerably decreased the density of [3H]-spiperone binding sites in sections of rat kidney, with an almost complete loss of vascular binding sites. 5. The above findings indicate the occurrence of a decrease of dopamine D2-like receptors in the kidney of SHRs with the progress of hypertension. Dopamine D2-like receptors which are mainly prejunctional are involved in the modulation of sympathetic neurotransmission in the kidney. The loss of these receptors in SHRs may contribute to the pathophysiology of hypertension.

Pharmacological characterisation and autoradiographic localisation of a putative dopamine D3 receptor in the rat kidney.

The pharmacological profile and the microanatomical localisation of a putative dopamine D3 receptor in the rat renal cortex were investigated using radioligand binding assay and light microscope autoradiography techniques. [3H]7-hydroxy-N,N-di-n-propyl-2-aminotetraline ([3H]7-OH-DPAT) was used as a ligand. [3H]7-OH-DPAT was bound specifically to sections of renal cortex. The binding was time-, temperature- and concentration-dependent, of high affinity and guanine nucleotide-insensitive. The dissociation constant (Kd) value was 0.57 +/- 0.02 nM and the maximum density of binding sites (Bmax) was 62.4 +/- 3.5 fmol/mg tissue. The pharmacological profile of [3H]7-OH-DPAT binding to sections of rat renal cortex suggests the labelling of a dopamine D3 receptor. Light microscope autoradiography revealed the accumulation of the radioligand primarily within cortical tubules and to a lesser extent in the glomerular tuft. In glomeruli, binding sites were found mainly in mesangium and mesangial cells. The demonstration of a putative dopamine D3 receptor in slide-mounted sections of rat renal cortex suggests that appropriate radioligand binding assay techniques combined with autoradiography, may contribute to characterise peripheral dopamine receptor subtypes.

Arylsulphatase A activity and sulphatide concentration in the female rabbit oviduct are under physiological hormonal influence.

Oviduct samples of female rabbits in different phases of the reproductive cycle were analysed to establish the role of sex steroid hormones in the regulation of sulphatide concentration and arylsulphatase A activity. In addition to biochemical procedures, histochemical techniques were used to localize both enzyme activity and the natural substrate. The plasma concentrations of progesterone and 17 beta-oestradiol were determined by radioimmunoassay (RIA). The findings show that the parameters examined undergo considerable changes during the different phases of the reproductive cycle. Oestrogens exert an inducing action on arylsulphatase A activity, while progesterone inhibits it. Fluctuations of the catabolic arylsulphatase activity condition the sulphatide concentration, which reaches maximum values at the eighth post-ovulatory day when progesterone dominance is consolidated. In this phase of the reproductive cycle, thin-layer chromatography confirms the presence not only of larger quantities of sulphatides, but also of all other lipid fractions.

Fluctuations in levels of various gangliosides in the different tracts of the oviduct of the frog (Rana esculenta) during the reproductive cycle.

The ganglioside content and pattern have been followed in the different tracts (rectus, convoluted and uterine) of the frog oviduct during the reproductive cycle. The main variations we observed are: a) average higher levels of ganglioside sialic acid in the preovulatory phase, with two peaks in March and April for the convoluted and rectus tract, respectively, and a more homogenous behaviour for the uterine tract; in all three tracts of the oviduct a minimum coincident with the ovulation has been found; b) a balanced presence of sulfolipids and gangliosides in the uterine tract: in fact sulfolipids, whose variations have been determined in a previous work, are higher when gangliosides are lower and vice versa, maintaining nearly constant the total negative charge due to these glycolipids; c) an alternate fluctuation of monosialo- and disialo-gangliosides in the preovulatory phase and a net trend toward the increase of monosialo- and the decrease of disialogangliosides in the postovulatory phase; trisialo-gangliosides are in general less represented and show less marked variations; d) the presence of particular gangliosides in particular moments of the reproductive cycle: Fuc-GM1, a fucosylated ganglioside, is higher than the more represented GM1 during the ovulation, while GD1 alpha, a ganglioside with a sialic acid residue linked to GalNAc, is steadily present in all three tracts after ovulation.(ABSTRACT TRUNCATED AT 250 WORDS)

A rapid procedure for elimination of non-polar lipids hampering the usual polar lipid extraction and TLC separation.

A new and rapid method is proposed for extraction of non-polar lipids from tissues where they are present as abundant components which can interfere with the usual procedures of lipid extraction and TLC separation, and hamper, in particular, sulphatide visualization. A solvent more hydrophobic than chloroform, i.e. n-hexane, was utilized to remove the neutral lipids from samples of female rabbit parotid gland, and the n-hexane phase was used for TLC which showed considerable amounts of cholesterol esters, in addition to triglycerides, diglycerides and monoglycerides. The methanol phase, now devoid of non-polar lipids, was utilized to prepare TLC plates in order to separate and visualize the polar lipid fractions, in particular the sulphatides.

Expression of acidic glycosphingolipids and arylsulphatase A activity in human pathological endometrium.

Endometrium biopsic samples from women with cystic hyperplasia or adenocarcinoma were analysed by biochemical procedures to verify fluctuations in the acidic glycosphingolipid (sulphatide) concentration and arylsulphatase A (ASA) activity. Comparing the values of the considered parameters with those obtained in normal subjects, it was observed that ASA activity significantly increased in both pathologies; in contrast, sulphatide concentration underwent a non-significant decrease in hyperplasia and a statistically significant increase in neoplasia. The thin-layer chromatography (TLC) images revealed not only quantitative, but also qualitative differences in the lipid fractions. In fact, compared with controls, the sulphatides showed one more marked fraction in the neoplastic endometrium, and two fractions with different Rf values in the hyperplastic one. Moreover, two new unknown fractions also appeared in some subjects with cystic hyperplasia. The findings suggest the lipid metabolism undergoes considerable changes under the pathological conditions examined. The fluctuations observed, in particular, in the sulphatide concentration are believed to be related to changes in the biosynthetic and catabolic activities of the key enzymes directly involved in their metabolism, i.e. arylsulphatase A and sulphotransferase, which are regulated by sex hormones.

Comparative analysis of lysosomal hydrolases and natural glycoprotein substrates in the rat major salivary glands.

1. The activities of some lysosomal hydrolases and the concentrations of their natural substrates were studied in the submandibular and sublingual glands of male and female rats using biochemical procedures. 2. In sublingual gland enzyme activities and substrate concentrations show the highest values. 3. The enzyme activities appear, in general, lower and the natural substrate concentrations higher in the females with respect to males. 4. In both glands beta-galactosidase shows the highest activity and beta-glucosidase the lowest. 5. These findings suggest that metabolic turnover of glycoproteins is slower in females than in males, probably because the oestrogens control the activity of lysosomal hydrolases.

Changes induced by progesterone treatment in the sulphatides of the frog oviduct.

Sulphatides have been studied by histochemical and biochemical procedures in the oviduct of the frog in different experimental conditions. In ovariectomized or hypophysectomized animals, compared to sham-operated, an increase in sulphatides was observed. The progesterone treatment did not significantly modify this lipid fraction in ovariectomized frogs, while in hypophysectomized frogs it induced a further increase. Densitographic profiles of the sulphatides, obtained by thin-layer chromatography (TLC) and also recorded by Tesak equipment, were similar in ovariectomized or hypophysectomized frogs following hormone treatment because they showed three distinct fractions in both experimental groups of animals. The appearance of a third fraction never previously observed was probably induced by the progesterone treatment. Moreover, under the effects of this hormone, the phospholipid fractions (phosphatidylcholine and phosphatidylethanolamine) also showed different densitographic profiles.

Sialic acid and neuraminidase activity in the frog oviduct: comparative biochemical investigation in the different tracts during the reproductive cycle.

1. A biochemical study was carried out on the protein-bound and lipid-bound sialic acid, and neuraminidase activity in the different tracts of the oviduct of the frog Rana esculenta during the reproductive cycle. 2. Plasma sexual steroids were also investigated by RIA. 3. Fluctuations in neuraminidase activity are related to that of glycoprotein sialic acid and plasma estradiol. Glycolipid sialic acid does not have a close relationship either with neuraminidase or plasma estradiol. 4. Very high plasma concentration of progesterone before ovulation and, on the contrary, its drop after ovulation were observed. 5. The results are discussed and hypotheses advanced to explain fluctuations of the studied parameters during the reproductive cycle.

Arylsulphatase activity in the oviduct of the frog Rana esculenta. II. Progesterone-induced changes following ovariectomy and hypophysectomy.

The effects of progesterone treatment on arylsulphatase activity were studied histochemically and biochemically in the frog oviduct under different experimental conditions. In ovariectomized animals, the hormone induced a large increase in enzyme activity, while in hypophysectomized ones there was a large decrease in this activity. These results indicate that the facilitatory and inhibitory effects of progesterone on arylsulphatase activity are influenced by the presence in situ of the gonad. Hypotheses are advanced to explain different effects of the progesterone treatment.

Arylsulphatase activity in the oviduct of the frog Rana esculenta. I. Oestradiol-induced changes following ovariectomy and hypophysectomy.

The effects of oestradiol treatment on arylsulphatase activity in the frog oviduct are reported. Oestradiol-induced changes were also investigated in ovariectomized and hypophysectomized animals. Under all the experimental conditions, hormonal treatment causes an increase in enzyme activity. This can be observed biochemically and also histochemically on frozen sections. Hypotheses are advanced to explain fluctuations in arylsulphatase activity.

Arylsulphatase activity and cerebroside sulphates in the frog oviduct during the reproductive cycle.

The presence of arylsulphatase A and cerebroside sulphates in different tracts of Rana esculenta oviduct during different phases of the reproductive cycle were investigated by histochemical and biochemical procedures. The results indicate that enzyme activity shows seasonal fluctuations connected with the phase of the sexual cycle. The concentrations of cerebroside sulphates (the natural substrates of arylsulphatase A) is related to the activity of this hydrolytic enzyme. The role of arylsulphatase A activity in regulating the substrate concentration, and particularly that of sulphatides, is discussed.