Evidence for the presence in calf thymus of a peptidic factor controlling DNA transcription in vitro.

A thymic factor causes a strong inhibition of the DNA-directed RNA polymerase reaction in vitro. The active factor was isolated from aqueous ultrafiltered thymus extracts and purified by means of chromatography on DEAE-cellulose and then on Dowex 50 WX2. The purified thymic factor was characterized as a peptide of low molecular weight (less than 5000). The biological activity of the thymic factor cannot be attributed to the presence of a nuclease or of a histone fragment. The RNA synthesis is controlled by this factor by means of electrostatic interactions between the peptide compound and DNA. Inhibitory activity on RNA synthesis was absent from kidney extracts.

Purification and characterisation of swine serum proteinase which hydrolyses epidermal inhibitory pentapeptide.

In this paper we describe the purification to molecular homogeneity of the enzyme that cleaves the synthetic epidermal mitosis-inhibiting pentapeptide (pyroGlu-Glu-Asp-Ser-Gly; EPP) from swine serum. Biochemical characterisation of the enzyme shows a glycoprotein with apparent molecular mass of 200 kDa. The Km and Kcat values for EPP hydrolysis are 0.624 mM and 694 s-1, respectively. Use of proteinase inhibitors shows the enzyme's metalloendopeptidase character. Moreover, captopril and lisinopril prevent the cleavage of EPP. The N-terminal amino-acid sequence of the purified protein corresponds to the N-terminal amino-acid sequence of swine kidney angiotensin-converting enzyme, a dipeptidyl carboxypeptidase (EC 3.4.15.1).

Small peptides controlling transcription in vitro are bound to chromatin DNA.

Low-molecular-weight peptides are linked to the chromatin DNA of several tissues, from which they can be dissociated by alkaline extraction at pH 9.5. The level of the active peptide fraction ranges between 10 and 35 micrograms/mg DNA. The removal of peptides from DNA causes a relevant amplification of DNA template capacity for prokaryotic and eukaryotic RNA polymerases. Gel filtration on Sephadex G-25 or BioGel P4 shows that the chromatin peptide fraction from purified DNA migrates as a sharp peak with an elution volume corresponding to a molecular weight of about 1000. The chromatin peptides are further purified by Sephadex G-10 and high-performance liquid chromatography. Four active fractions are isolated, one of which shows very high inhibition activity on the RNA synthesis in vitro. The amino acid analysis and the inhibition mechanism of the purified peptides are reported.

Acidic pentapeptide phosphorylated in vitro by calf thymus protein kinase NII binds to DNA in the presence of Mg2+ cations.

The pentapeptide pyroGlu-Ala-Glu-Ser-Asn has been synthetized and phosphorylated in vitro at level of serine by protein kinase NII isolated from calf thymus chromatin. It is noteworthy that the calf thymus kinase NII shows a remarkable affinity for this peptide. The [32P]peptide is able to bind to several DNAs in the presence of Mg2+ (lambda phage, calf thymus, pBR540 plasmid). This binding appears not specific with regard to the type of DNA and its base sequence. These data support the hypothesis that phosphorylated acidic domains of nuclear nonhistone proteins could bind directly to DNA in the presence of Mg2+ cations.

Regulatory activity of DNA-binding peptides on some metabolic pathways altered in aging.

The Smith theory, which describes aging as a consequence of damage at DNA transcription level, suggested to us the opportunity of studying the possible action of DNA-binding peptides from calf thymus on old rats. We previously demonstrated that this peptidic fraction exerts a regulative control on transcriptional activities of DNA in cell and cell-free systems. In order to verify the possible action of these low molecular weight peptides we chose a large range of metabolic and structural parameters which are altered in aging. The results obtained indicate the following conclusions. Lipids. The lipid levels of old rat liver and serum are altered compared with those of young rats; the administration of peptidic fraction to old rats reverses the lipid alterations observed. Glucides. In old rat liver the presence of glycogen is very scanty or completely absent; the animals treated with the peptides show an amount and distribution of glycogen similar to that of adult normal rats. ATP. The peptidic fraction causes in the old rats a marked increase of blood ATP, bringing the level in the range of values determined in young rats. DNA, RNA, proteins. The total synthesis rate of DNA, RNA and proteins in old rat liver is not influenced by the DNA-binding peptides. Vice versa the nucleic acids from liver nuclei of old rats given peptidic fraction contain a greater RNA component compared to control old rats. This result is confirmed by the strong increase of transcriptional activity of DNA for RNA polymerase caused by administration of peptidic fraction to old rats. This increased DNA transcription can be interpreted as a partial recovery of DNA transcriptional capacity which evidently might imply a restoration of impaired metabolic systems. The histochemical and stereological analyses of liver cell compartments confirm the biochemical data.

Presence of pituitary adenylate cyclase-activating polypeptide 38-immuno-like material in the brain and ovary of the female crested newt, Triturus carnifex: its involvement in the ovarian synthesis of prostaglandins and steroids.

The presence of pituitary adenylate cyclase-activating peptide (PACAP) 38-immuno-like material (PACAP 38-IL) in the brain and ovary of the crested newt, Triturus carnifex, and its action on ovarian steroidogenesis and prostaglandin synthesis were evaluated. The HPLC, brain and ovary extract peaks that eluted like PACAP 38 were considered PACAP 38-like material. The concentrations of PACAP 38-II in the HPLC extracts were measured by RIA. T. carnifex ovary was incubated with PACAP 38, brain and ovary PACAP 38-IL, and inhibitors of cyclooxygenase (COX), adenylate cyclase (AC) and phospholipase C (PLC) for 30 and 60 min. PACAI 38, and brain and ovary PACAP 38-IL increased prostaglandin E2 (PGE2) (30 and 60 min), and progesterone and corticosterone (60 min), but decreased oestradiol-17 beta (60 min). COX and PLC inhibitors counteracted the increases in PGE2, progesterone and corticosterone and the decrease in oestradiol-17 beta, and the AC, inhibitor also counteracted them except for PGE2. These results suggest that PACAP 38-IL, present in T. carnifex brain and ovary, acts on PLC, inducing the increase of PGE2 which, in turn, acting on AC, induces increases in progesterone and corticosterone and a decrease in oestradiol-17 beta.

Anesthesia with sevoflurane in bariatric surgery.

BACKGROUND: Sevoflurane is a good halogen agent for bariatric surgery anesthesia because of its physical and chemical characteristics and its repartition coefficient (blood/gas = 0.65). METHOD: From November 1997 to April 1998, 98 bariatric surgery procedures with sevoflurane anesthesia were done: 17 lipectomies, 71 vertical gastroplasties, and 10 biliopancreatic diversions in 71 women and 27 men, average age 30.3+/-8.3 years, with body mass index 43.9+/-5.7. The average operating time was 50+/-15 minutes for vertical gastroplasty, 160+/-20 minutes for biliopancreatic diversion, and 80+/-12 minutes for lipectomy. The technique of anesthesia was as follows: preanesthesia with atropine sulfate 0.01 mg/kg (dosage refers to ideal weight), ranitidine 50 mg, fentanyl 0.1 mg, ketorolac 60 mg; induction with propofol 0.5-1 mg/kg, succinylcholine 1 mg/kg; orotracheal intubation; maintenance with O2-N2O 50%, sevoflurane 1% to 1.5%, actracurium 0.5 mg/kg (dosage refers to ideal weight); awakening and decurarization with atropine sulfate 1 mg and prostigmine 2 mg. RESULTS: This method permitted correct control of the anesthesia, a quick awakening with a low incidence of nausea and vomiting, a prompt regain of physical and psychological functioning, an early discharge from the hospital, and a larger turnover of patients with lower costs. CONCLUSION: Sevoflurane balanced anesthesia seems to be the best anesthesiologic method for bariatric surgery.

Molecular models of small phosphorylated chromatin peptides. Structure-function relationship and regulatory activity on in vitro transcription and on cell growth and differentiation.

We previously reported the isolation of low molecular weight phosphorylated peptides from the chromatin of several tissues. The chromatin peptides show a regulatory activity on DNA in vitro transcription and on cell growth and differentiation. In this paper, we report a molecular model of the native peptides designed according to the structural information obtained by means of biochemical and mass spectrometry analysis: pyroGlu-Ala-Gly-Glu-Asp-Ser(P)-Asp-Glu-Glu-Asn. This or very similar sequences are present in many transcription factors; on the basis of the structural model we presented and of related protein sequences, we have synthesized the peptide pyroGlu-Asp-Asp-Ser-Asp-Glu-Glu-Asn. This peptide affects transcription rate in reconstituted systems in vitro and in isolated nuclei; moreover, it inhibits the growth of HL60 cells with a parallel stimulus of differentiation.

Synthesis and characterization of tetramethylrhodaminethiocarbamoyl-(Glu(1))-epidermal mitosis-inhibiting pentapeptide.

A fluorescent analog of epidermal mitosis-inhibiting pentapeptide (pGlu-Glu-Asp-Ser-Gly) was synthesized by reacting tetramethylrhodamine isothiocyanate with ring-opened epidermal mitosis-inhibiting pentapeptide. The ring-opening reaction of the pyrrolidone moiety was performed with mild acidic hydrolysis and the product purified by reversed-phase high-performance liquid chromatography. Tetramethylrhodaminethiocarbamoyl-(Glu(1))-epidermal mitosis-inhibiting pentapeptide was purified by chromatography on Sephadex G-25 and reversed-phase high-performance liquid chromatography. After characterization by amino acid analysis, the analog was incubated in presence of A431 cell line to visualize the cellular localization of the epidermal mitosis-inhibiting pentapeptide. The data gave negative results.

Degradation of thymic humoral factor gamma2 by human plasma: involvement of angiotensin converting enzyme.

The degradation of thymic humoral factor-gamma2 (THF-gamma2), an immunoregulatory octapeptide important for T-lymphocyte regulation, by enzymes present in human plasma, was investigated. THF-gamma2 was metabolized through two steps that involved the detaching of N-terminal amino acid leucine followed by hydrolysis of the Lys(6)-Phe(7) bond. The THF-gamma2 cleavages were sensitive to aminopeptidase and metalloproteinase inhibitors. The degradation was completely blocked by amastatin and specific inhibitors of angiotensin converting enzyme (ACE). The cleavages occurred independently, with two different kinetics, faster for the N-terminal hydrolysis than for that of the Lys(6)-Phe(7) bond. Purified human plasma ACE was used to characterize the hydrolysis of Lys(6)-Phe(7) bond. The K(m) and K(cat) values for THF-gamma2 hydrolysis were 0.273 mM and 107 s(-1), respectively. The optimum of chloride concentration was 300 mM, while that of pH was 7.6. The presence of ACE in circulating mononuclear cells raises the possibility that it may play a role in modulating the THF-gamma2 activity.

Unexpected finding of barium sulphate on the surface of a microspinal catheter.

During a study with a scanning electron microscope to evaluate the structure of microspinal catheter after its removal from subarachnoid space, we found an unusual case. The observation with the microscope of the tip of a catheter removed at the end of an operation for hip replacement in a old female showed the presence of grounded particles with a crystal shape covering the outer surface. Further analysis of this material with an Energy-Dispersive Spectrometer (EDS) showed that it was barium. The patient performed a large bowel barium enema 8 months earlier for a painful syndrome to the lower abdomen. Authors rule out the contamination from the skin and suggest two possible mechanisms of passage of barium from blood to cerebrospinal fluid (CSF) and so to the surface of the catheter.

Aortic arch surgery: retrospective analysis of outcome and neuroprotective strategies.

OBJECTIVE: To review intra- and postoperative data regarding surgical reconstruction of the aortic arch performed at our cardiosurgical centre during the past four years, and thus to deepen understanding of neurologic morbidity and of what constitutes the most effective neuroprotection. EXPERIMENTAL DESIGN: Retrospective study. SETTING: Regional University Hospital. PATIENTS: 29 patients who underwent reconstruction of aneurysm or dissection of the aortic arch. Intervention. Surgical replacement of the diseased aorta during deep hypothermia, alone or with selective cerebral perfusion (antegrade or retrograde). MEASURES: Overall mortality rate, neurologic morbidity rate, duration of extracorporeal circulation, of hypothermic circulatory arrest or of selective cerebral perfusion. Evaluation of the importance to neurological outcome of age, modality of operation (emergency or routine), biochemical parameters (glycemia, hematocrit) and perfusion technique. Recording of postoperative time of arousal, and possible correlation with length of selective cerebral perfusion. RESULTS: We observed a mortality rate of 39% (11 deaths) and a neurologic morbidity rate of 34%. Hypothermic circulatory arrest alone did not assure valid neuroprotection (5 cases, all with severe neurologic impairment), while better results were obtained with selective cerebral perfusion, especially antegrade (14 cases, with only 7% of neurologic morbidity rate). Hyperglycemia (>250 mg%) proved to be significantly associated (p=0.002) with increased incidence of adverse neurologic outcome, and the same association was observed between emergency status and adverse neurologic outcome (p=0.002). Moreover, we found an unexpected linear correlation between time of selective cerebral perfusion and postoperative time of arousal (r=0.728, p=0.000). CONCLUSIONS: Deep hypothermic circulatory arrest with selective cerebral perfusion currently represent a valid therapeutic option for brain preservation during reconstruction of the aortic arch in adults. It is mandatory to carry out a tight control of perfusion parameters (flow, pressures and temperature gradients) and biochemical variables (avoidance of hyperglycemia and modified ultrafiltration for fluid balance).

Incorporation of 14C-thymidine in human platelets: possible relation with a diffuse infection of micrococci in the L form.

Following the results of previous researches suggesting that platelets might carry microbial forms, the incorporation of 14C-thymidine in suspensions of platelets from 500 normal human subjects has been taken under examination. The results have always yielded positive data even though with marked differences of a quantitative order from a case to another. The hypothesis that such an activity might be the consequence of a synthesis of DNA in the mitochondria had to be excluded. The peculiar relations linking the incorporation rate to the number of platelets and to the presence of plasma or serum in given amounts and the strong inhibition exerted by oxytetracyclines suggest that the detected metabolic activity may be attributed to the presence of bacterial L-forms carried by platelets. The results of cultural, optical and electron microscopical investigations, which will be published elsewere, confirmed such interpretation.

Neuroendocrine thymus and beta-adrenergic responsiveness in aging mice.

It has previously been shown that thymus exerts a regulatory influence on the beta-adrenergic system, controlling, in particular, DNA synthesis in submandibular glands following injection with isoproterenol (IPR). A decreased peak of response found in old mice can be corrected by grafting a neonatal thymus into old animals. In order to clear whether its restoring action needs mutual interrelationships with other control systems or, alternatively, whether even some thymic factor can be effective, the IPR response mentioned above was also assayed in old animals treated with a thymic extract (TME). Among the numerous thymic factors, this extract was chosen due to the effect that it was prepared on the basis of non-immunological tests and had already been shown to be effective in correcting some alterations observed in old rats. Results show that TME is capable of partially restoring the impaired response found in old mice. The location of the peak of thymidine incorporation, however, is shifted towards the right with respect the peak time observed in young, old and thymus-grafted old mice. Without additional studies, it cannot be decided whether the shift and the only partial recovery is due to the particular dose and duration of the treatment or represents a weaker action of the extract with respect to that of thymic graft.

Purified angiotensin converting enzyme from Rana esculenta ovary influences ovarian steroidogenesis in vitro.

The aim of the present study was to purify and characterize angiotensin-converting enzyme (ACE) present in frog ovary (Rana esculenta). Detergent and trypsin-extracted enzymes were purified using a one-step process, consisting of affinity chromatography on lisinopril coupled to Sepharose 6B. The molecular mass was 150 kDa for both detergent-extracted and trypsin-extracted enzyme. The specific activity of detergent-extracted and trypsin-extracted ACE was 294 U mg(-1) and 326 U mg(-1) respectively. The optimum pH range was from 7-8.5 at 37 degrees C and the optimum temperature was 50 degrees C. Optimum chloride concentration was about 200 mM for synthetic substrate FAPGG (N-[3-(2-furyl)acryloyl] L-phenylalanyl glycyl glycine) and angiotensin I, and 10 mM for bradykinin. The Km and Kcat values for FAPGG were 0.608 +/- 0.07 mM and 249 sec(-1) respectively and I50 values for captopril and lisinopril, two specific ACE inhibitors, were 68 +/- 12.55 nM and 6.763 +/- 0.66 nM respectively. Frog ovary tissue from prereproductive period was incubated in vitro in the presence of frog ovary ACE (2.5 mU/ml), captopril (0.1 mM), and lisinopril (0.1 mM). Production of 17beta-estradiol, progesterone, and prostaglandins E2 and F2alpha was determined. The data showed a modulation of 17beta-estradiol, progesterone and prostaglandin E2 production by ovary ACE.

[Current views on the use of vasodilators in pulmonary hypertension]. / Attuali vedute sull'impiego dei vasodilatatori nell'ipertensione polmonare.

Vasodilators represent one of the main steps for the medical treatment of pulmonary hypertension; the rationale for their use is the reversibility of the pulmonary vasoconstriction, to be tested with a correct pharmacological trial. In this report the authors consider the use of calcium-channel blockers, prostaglandin and nitric oxide. Calcium blockers, the only drugs active when administered orally, provide a satisfactory clinical response in 25-30% of treated patients. Prostaglandins are active in a higher percentage of patients and can be infused in a domiciliary regimen with portable pumps even for long periods of time. Nitric oxide is the only selective pulmonary vasodilator; it is used in paediatric and adult cardiac surgery and in patients affected by respiratory distress syndrome, but its use is restricted to intensive care units and many cautions must be adopted. Finally some future therapeutic strategies are briefly reviewed: endothelin inhibitors, cGMP phosphodiesterase inhibitors etc.

Case reports in anesthesiology: their trend through 17 years.

UNLABELLED: The study objective was to delineate the trend of case reports (the simplest of the descriptive forms of study) in the anesthesiological literature by analyzing the frequency of publication, and citation, and especially of the place of citation of a sample of published case reports. It is our opinion that case report in anesthesia is particularly suitable for this specialty rather than for others and is often the first signal of a complication, an adverse event, an anesthetic problem in rare disease and alerts other anesthesiologists to the possibility of unexpected events. METHODS: We analyzed the case reports published on an Anesthesiological journal placed in the middle in term of Impact Factor, from January 1980 to December 1997. Citations of each case report were obtained using computer searches of the Science Citation Index (SCI). For each of these case reports we collected in a custom-designed data base the following data: year of publication, number of authors, number of citations per year, place of citation, type of article quoting the case report, number of self-citations, year of first citation. MAIN RESULTS: We identified 637 case reports and 1946 citations. The number of case reports increased through the years up to a peak in 1994-95 and the same trend was observed for citations and self-citations, the number of authors per case report was < or = 4 in 90.4%; 74.2% of total case reports cited were first cited within two years of publication, while 34.7% were never cited. The type of article quoting the case reports has been, in the majority of cases, an original article. CONCLUSIONS: The analysed case reports and the number of citations can give us information about the importance of a clinical situation at a particular time.

Small acidic peptides from wheat germ chromatin. II. Regulatory activity in specific transcription systems reconstituted in vitro.

A variety of evidence suggests that a family of chromatin peptides (CPs), characterized by 1000D molecular weight, a pH dependent association to DNA and a prevailing presence of acidic amino acids in their structure, is involved in the regulation of genes expression. Nevertheless their action mechanism is still unknown. In our in vitro specific RNA transcription systems the CPs affect the initiation and not the elongation. Furthermore they inhibit the RNA transcription by interaction with the DNA rather than with the enzyme. The phagic in vitro specific RNA transcription is less affected by CPs than the eubacteric system, suggesting a kind of selectivity for target DNA sequences involved in the initiation of transcription.

Small acidic peptides from wheat germ chromatin. I. Isolation and biochemical characterization.

RNA synthesis in cell and cell-free systems is inhibited by a family of acidic, low molecular weight chromatin peptides (CPs). These peptides were extracted from deproteinized DNA of prokaryotic and eukaryotic cells, but the low yield of purified material by this procedure hinders efforts aimed at understanding their action mechanism in gene regulation. In this report we describe two purification methods of CPs from an easily available source, wheat germ. A comparison is made between the method starting from deproteinized DNA and the method from purified chromatin. The biological effects (inhibition of L1210 cell growth and DNA in vitro transcription) of CPs from wheat germ together with their chemical characteristics (molecular weight, amino acid composition and presence of phosphoserine) show strong homology with those of CPs from other sources. These results suggest a possible role of these chromatin peptides in controlling gene expression.

Isolation and characterization of small phosphorylated peptides controlling transcription "in vitro" from trout testis chromatin DNA.

Low molecular weight peptides are linked to the chromatin DNA of several tissues, from which they can be dissociated by alkaline extraction. They show very high specific activity in the control of transcription "in vitro". In this work the biochemical properties of controlling transcription peptide effectors isolated from trout testis DNA are reported. The purified peptides prevailingly contain glutamic acid, serine, aspartic acid, glycine and alanine. Studies of the peptide structure by N-terminal analysis using the dansyl chloride procedure was unsuccessful, suggesting the presence of a blocked NH2 group. At the same time the active peptides cannot be digested by carboxypeptidases. The gel filtration of the chromatin peptidic fractions on Sephadex G-25, Trisacryl GF05 or Sephadex G-15 shows that the active peptides elute as a single major peak with an elution volume corresponding to a molecular weight of about 1000. The paper electrophoresis performed at different pH and ionic strength shows that the chromatin peptides are separated in two fractions. One of them is strongly acidic and migrates towards the positive pole until pH 1.9, indicating the presence of phosphoric residues which probably exert an important role in the control of transcription "in vitro". The chromatin peptides are further purified by Sephadex G-10 and high performance liquid chromatography. The amino acid analysis of the purified peptides are reported.