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The reaction of 4-phenyl-1,2,4-triazolin-5-one [Ia] and 3,4-diphenyl-1,2,4-triazolin-5-one [Ib] with 1-bromo-3-chloropropane was carried out. The obtained compounds [IIa, b] were subjected to the reaction with secondary amines and ethylenediamine with resulted in 1-(3-aminopropyl)-1,2,4-triazolin-5-one derivatives.

In the reaction of 4-phenyl-1,2,4-triazolin-5-one [Ia] and 3,4-diphenyl-1,2,4-triazolin-5-one [Ib] with 1-chloro-2,3-epoksypropane, the respective derivatives of 1-(2,3-epoksypropane)-4-phenyl-1,2,4-triazolin-5-one [IIa] and 1-(2,3-epoksypropane)-3,4-diphenyl-1,2,4-triazolin-5-one [IIb] were obtained. Then these compounds were converted into the corresponding aminoalkanol derivatives of 1,2,4-triazolin-5-one [IIIa, b-VIIIa, b] in reaction with secondary amines. The new compounds affected significantly the central nervous system of mice.

T cells are involved in the pathogenesis of nephrotic syndrome (NS). The aim of the study was to determine whether the activity of T-helper-1 (Th1) and T-helper-2 (Th2) cells and the distribution of the lymphocyte subsets, namely CD45RA+CD4+ ("naive" helper T cells, suppressor-inducer), CD45RA+CD8+ ("naive" suppressor T cells, suppressor-effector), CD45RO+CD4+ ("memory" helper T cells), are predictive for steroid sensitivity in children with primary NS. These parameters were assessed at the onset of disease, before initiation of steroid therapy. Two groups of NS children were retrospectively formed according to steroid sensitivity (SS) or resistance (SR). The activity of Th1 and Th2 cells was defined by the production of interleukin-2 (IL-2), interferon-gamma, IL-4, and IL-10 in the supernatants of CD4+ T cell cultures activated with autologous monocytes presenting tetanus toxoid (TT). Peripheral lymphocyte subsets were determined using double- or triple-color flow cytometry. In SS children with NS we found a decreased proliferative response of CD4+ T cells to TT stimulation, cytokine synthesis indicating the predominance of Th2 activity, and an increased percentage of activated suppressor-inducer (CD45RA+ CD4+CD25+, 5.18+/-0.8, P<0.001) and suppressor-effector (CD45RA+CD8+CD25+, 2.05+/-0.6, P<0.01) cells, with the concomitant reduction of activated memory cells (CD45RO+CD4+CD25+, 0.2+/-0.1, P<0.001). In children with SRNS we found an increased proliferative response of CD4+ T cells to TT, a rise in activated memory (CD45RO+CD4+CD25+, 3.82+/-0.7, P<0.01) and suppressor-inducer peripheral T cells (CD45RA+ CD4+CD25+, 3.85+/-0.6, P<0.01), but a low percentage of activated suppressor-effector (CD45RA+CD8+ CD25+, 0.5+/-0.2, P<0.05) T cells. We conclude that prior to treatment the distribution of lymphocyte subpopulations in peripheral blood together with Th1 and Th2 cell activity provides a useful tool for evaluating the likelihood of steroid sensitivity in patients with primary NS.

In the reaction of phenylacetic acid hydrazide or malonic acid hydrazide with isocyanates, new derivatives of semicarbazide were obtained [I-VIII]. Cyclization of these derivatives with a aqueous 2% sodium hydroxide led to the formation of new derivatives of delta 2-1,2,4-triazolin-5-one [IX-XVI]. Some of them were investigated pharmacologically on the central nervous system in mice.

T cells are involved in the pathogenesis of nephrotic syndrome (NS). The aim of the study was to determine whether the activity of T-helper-1 (Th1) and T-helper-2 (Th2) cells are predictive for steroid sensitivity in children with primary NS. These parameters were assessed at the onset of disease, before initiation of steroid therapy. Two groups of NS children were retrospectively formed according to steroid sensitivity(SS) or resistance(SR). Activity of Th1 and Th2 cells was defined by the production of IL-2, IFN-gamma and IL-4, IL-10 (ELISA), respectively, in the supernatants of the culture of CD4+ T cell cultures activated with autologous monocytes presenting tetanus toxoid (TT). Peripheral lymphocyte subsets were determined using double or triple colour flow cytometry. In SS children with NS we found the cytokine synthesis indicating the predominance of Th2 activity. We conclude that prior to treatment the Th1 and Th2 cell activity provides a useful tool to evaluate the probability of steroid sensitivity in patients with primary NS.

Ehtyl esters of 4-phenyl-1,2,4-triazolin-5-one-1-acetic acid and 3,4-diphenyl-1,2,4-triazolin-5-one-1-acetic acid were transformed into the corresponding amides [Ia, b]. The aminomethylation reactions afforded Mannich bases [IIa, b-VIa, b] exhibiting the expected pharmacological effects.

The aim of the study was to investigate the impact of L-arginine (nitric oxide donor), L-NNA (NO synthase inhibitor), heparin and procaine on the pancreas' microcirculation, serum interleukin 6 (IL-6) level, and microscopic alterations of the pancreatic gland in acute pancreatitis (AP) in rats. AP was induced by 4 i.p. injections of cerulein (15 micrograms/kg/h). Microcirculatory values of the pancreas were measured by means of laser Doppler flowmetry 5 h after the first cerulein injection. Remarkable morphologic changes in the pancreas, including parenchymal necrosis, an elevation of serum IL-6 activity, and significant drop of pancreatic capillary perfusion was observed in rats with NO synthase inhibition. L-arginine improved the pancreatic microcirculation but worsened the microscopic alterations within the pancreas. Heparin had a beneficial effect on the microcirculatory values, serum IL-6 activity, and morphologic changes. Procaine had no effect on the course of AP. Authors conclude that heparin, improving the pancreatic capillary blood perfusion, may be considered as a promising therapeutic agent in acute pancreatitis.

Allele frequencies are clinally distributed for many protein polymorphisms in Europe, suggesting that the current populations are derived from an ancestral group that expanded from the Near East. It is not yet fully established whether that expansion took place during the Neolithic or earlier or whether the detectable protein variation faithfully reflects the underlying molecular variation. In this study we address the latter question by describing geographic patterns of genetic diversity at seven highly polymorphic DNA markers. Two of these markers are minisatellites, four are microsatellites, and the seventh is a locus of the HLA system. By analyzing a database of 304 samples, with more than 130,000 chromosomes, we found evidence for a major clinal component of genetic variation. At most loci spatially close populations resemble each other genetically, and the degree of genetic similarity, as measured by spatial autocorrelation statistics, decreases at increasing distances. The observed patterns of molecular variation do not seem to differ qualitatively from those identified for protein polymorphisms. This suggest that low levels of population structuring, described in some mitochondrial DNA studies, may reflect different evolutionary histories for nuclear and maternally inherited markers or, alternatively, that spatial patterns of mitochondrial DNA variation may need more sensitive statistical methods to be recognized.

The aim of this study was to investigate the impact of L-arginine (nitric oxide synthase substrate), L-NG-nitro-L-arginine (nitric oxide synthase inhibitor), and heparin on the pancreas microcirculation, serum IL-6 level and microscopic alterations of the pancreas in acute pancreatitis in rats. Acute pancreatitis was induced by 4 i.p. injections of cerulein (15mg/kg). Microcirculatory values were measured by means of laser Doppler flowmetry 5 h after the first cerulein injection. Remarkable histopathological changes in the pancreas, including parenchymal necrosis, an elevation of serum IL-6 level, and a significant drop of pancreatic capillary perfusion was observed in rats with nitric oxide synthase inhibition. L-arginine improved the pancreatic microcirculation but worsened the microscopic alterations within the pancreas. Heparin had a beneficial effect on the microcirculatory values, serum IL-6 concentration, and morphologic changes. Authors conclude that inhibition of nitric oxide synthase aggravates acute pancreatitis. L-arginine treatment improves pancreatic perfusion but potentiates morphological alterations. Heparin, improving the microcirculation and inflammatory changes within the pancreatic gland, may be considered as a promising therapeutic agent in acute pancreatitis.

It is generally held that there are genetic factors for dizygotic (DZ) twinning, but the pattern of inheritance is far from clear. We report on a rare pedigree with three sets of spontaneous DZ female twin births in three successive generations. To our knowledge no similar pedigrees have been reported in literature. Zygosity determination in three pairs of twins was performed with ABO, MN and Rh blood groups from erythrocytes and with STR analysis from samples of saliva. In our pedigree, the direct transmission of the DZ twinning through the maternal line seems show an autosomal dominant pattern of inheritance. We believe that the inheritance of DZ twinning appears to be recessive in most case and autosomal dominant only in rare instances. It is plausible with the hypothesis that the expression of the dizygotic twinning trait in a mother shows a genetic heterogeneity.

Local microcirculatory dysfunction within the pancreatic gland might be an important factor in the conversion of oedematous to necrotizing pancreatitis. Therapeutic agents, improving the pancreatic blood flow, might be valuable in acute pancreatitis treatment. An influence of nitric oxide, heparin and procaine treatment on microcirculatory values in acute pancreatitis (AP) in rats was investigated. Acute pancreatitis was induced by i.p. injection of cerulein in four doses of 15 microg kg-1 each at 1-h intervals. The rats with pancreatitis were divided into five groups, 12 animals each. One group remained without treatment, four groups were treated i.p. either with NO synthase inhibitor L-NNA (2x25 mg kg-1 or heparin 2x2.5 mg kg-1 or L-arginine 2x100 mg kg-1 or procaine 2x25 mg kg-1. Five control groups, ten animals each, received saline, L-NNA, heparin, L-arginine or procaine only. Five hours after the first ceruleine injection microcirculatory values within the pancreas were measured by means of laser Doppler flowmetry. Acute pancreatitis caused a significant drop of microcirculatory value to 37% of the basal value. The L-NNA administration resulted in a further insignificant reduction of the pancreatic blood flow to 34%. An improvement of microcirculation was observed in rats with pancreatitis receiving heparin (76%) and L-arginine (72%). Procaine had no effect on microcirculatory disturbances within the pancreas in rats with pancreatitis. Cn-induced acute pancreatitis (AP) causes microcirculatory deterioration within the pancreas. Heparin and nitric oxide donor, L-arginine, might be considered as therapeutic agents, improving the diminished pancreatic tissue perfusion observed in acute pancreatitis. Procaine does not improve the pancreatic blood flow in acute pancreatitis., (Copyright 1997 The Italian Pharmacological Society.)

A prospective randomized trial involving 30 patients was performed to assess whether second-look endoscopy could improve the efficacy of injection therapy for bleeding peptic ulcers. The inclusion criteria were the presence of active arterial bleeding or a non-bleeding visible vessel at emergency endoscopy. All the patients received emergency injection of thrombin with adrenaline solution and were subsequently randomized (15 patients in each group). All the patients receive a second elective endoscopy within the first 24 hours with repeated injection of thrombin-adrenaline solution (Group I) or fibrin sealant-Beriplast-(Group II). A tendency towards better results was noted in the Group II. Recurrent bleeding was observed in 6.6% versus 26.6% patients in Group I. The need for emergency surgery occurred in (6.6% in Group I versus 0 in Group II) and length of hospital stay was (13.2 in Group I versus 7.4 in Group II [p < 0.05]). No mortality was observed in both of the groups. In conclusion, our data suggest the possibility of a small benefit with second-look endoscopy with repeated injection of fibrin sealant.

Microsatellite analysis, based on fluorescein labeling and reading through a semiautomatic single wavelength sequencer, is described. Pairs of labeled polymerase chain reaction (PCR) samples, mixed in equimolar proportion, were electrophoresed and the specific peaks read in a single gel lane. Identity was asserted when peaks overlapped in a unique fluorescent signal which, compared with individual sample profiles, had a twofold intensity. Classification was achieved by blending individual PCR products to 'locus specific allelic ladders' (composite samples containing a repertory of fragments allelic to a given locus) and by noticing the specific peak enhancement. The resulting protocol of analysis assigned no size and classified allelic forms by tandem repeat number. Applied to a large repertory of PCR products and compared with manual electrophoresis, this protocol proved to be reliable and reduced times and costs of genotype analysis. Analysis of comigrating peak profiles is highly objective and provides convincing evidence for diagnostics and identity tests.

One hundred and ninety-one patients who had emergency endoscopy performed for acute gastroduodenal haemorrhage and were found to be bleeding actively (N = 141) or to have an ulcer containing a non-bleeding visible vessel (N = 50) were studied. Patients with active bleeding were divided into two groups: group I included 47 patients in whom diagnostic endoscopy was performed and conventional management applied; and group II contained 94 patients in whom, apart from conventional treatment, endoscopic thrombin injections could also be offered. Fifty patients with non-bleeding visible vessels were admitted to a prospective randomised trial and assigned to either endoscopic injections (group A, N = 25), or no primary endoscopic treatment (group B, N = 25). The introduction of the injection therapy in patients who were actively bleeding resulted in a significant reduction of the emergency operation rate (70.2% v. 12.8%; P < 0.001), mortality rate (21.3% v. 8.5%; P < 0.05 and blood transfusion requirements (4,220 +/- 1,840 ml v. 1,980 +/- 980 ml; P < 0.05). Endoscopic thrombin injections of non-bleeding visible vessels reduced the rebleeding rate (40% v. 4%; P < 0.01) and blood transfusions (2,939 +/- 1070 ml v. 1,390 +/- 552 ml; P < 0.05).

The main subject of considerations is acute necrotizing pancreatitis, based on the authors opinion and literature. Frequency of the necrosis in acute pancreatitis is established in between 7-15%. Because of very severe and often lethal complications these patients need special surgical attention. Enhanced CT is the most valuable diagnostic procedure to visualize the extend of pancreatic necrosis. There is no common agreement upon the mode of treatment except from that infected necrotizing pancreatitis should be operated. Indications for surgical treatment with particular analysis to necrosectomy and laparostomy is discussed.

The accuracy of procedures for sizing hypervariable restriction fragments by Southern blot analysis (SBA) has been tested under three different experimental conditions: (i) intrablot serial analyses: three heterozygous DNA profiles were tested 14 times each in the same gel electrophoresis; (ii) intralaboratory analyses: we replicated three profiles (six autoradiographic bands) in over 100 SBA experiments; (iii) interlaboratory analyses: 15 serial measurements produced in a recent collaborative study (Forensic Sci. Int. 1991, 49, 1-15) were taken into account. In these three cases, a typical U-shaped correlation curve between molecular size and coefficient of variation was found. We explain decrease of accuracy at both extremities of the gels in terms of: (i) enlarged shapes of bands and mean electrophoretic resolution at the cathode; (ii) diffusion and blurring of bands at the anodal edge. The three populations of data were subjected to a chi 2 test and to the Kolmogorov-Smirnov test in order to verify their compliance with normal distribution. Twenty-three out of 27 tests indicated no significant deviation from the assumption of Gaussian distribution. We recommend the adoption of tests for normality to validate the use of symmetric confidence intervals for calculating gene frequencies and asserting a match between adjacent bands.

In reaction of oxalic acid dihydrazide with isothiocyanates were obtained the respective thiosemicarbaside derivatives (Ia-IXa). Their cyclization in alkaline media have given the corresponding 4,4'-substituted-5,5'-mercapto-3,3'-bi-1,2,4-triazole (Ib-IXb), in acidic media however were formed the 2,2'-amino-substituted-5,5'-bi-1,3,4-thiodiazole (Ic-VIIIc).

In the reaction of 4-ethoxycarbonylmethyl-1,2,4-triazoline-5-thione derivatives (Ia,b) with the primary amines the corresponding amides (IIa,b-IVa,b) have been formed. These amides were then undergone to reduction with LiAlH4 leading to the formation of corresponding amines (Va,b-VIIa,b). Then by means of the reaction with acetic anhydride these amines were transformed into corresponding acetylamine derivatives of 1,2,4-triazoline-5-thione (VIIIa,b-Xa,b).

Acute pancreatitis was induced in 19 anesthetized dogs by retrograde injection of bile mixed with trypsin into the pancreatic duct. Two groups, of six animals each, were treated with intravenous infusion of synthetic antiproteases: gabexate mesilate and nafamostat mesilate in doses of 1 mg/kg per hr. One group of seven animals remained untreated. Two untreated dogs died during the experiment. All the treated dogs survived. Hemodynamic data were monitored hourly during a 6-hr observation period. In the untreated animals, cardiac output, mean arterial pressure, and left ventricular stroke volume decreased rapidly; an increase of pulmonary vascular resistance and systemic vascular resistance was observed. Synthetic antiproteases, given as a therapy, improved the hemodynamic parameters significantly and prevented the animals from developing shock. Gabexate mesilate and nafamostat mesilate seem to be of value in the treatment of experimentally produced acute pancreatitis in dogs.

A miniature, vertical electrophoretic system is described that allows quick elution and efficient harvesting of double-stranded, high molecular weight DNA fragments from agarose gels. A gel slice containing the band of interest is excised from the agarose gel after submarine electrophoresis and placed into an electrophoresis cell, assembled by inserting a truncated micropipette tip into a 2.0 mL polypropylene tube. The tip supports the agarose slice and is connected to dialysis tubing. Platinum wire electrodes are placed over the tip and at the bottom of the test tube. DNA molecules are quickly eluted into the dialysis bag (60 s to 10 min for bands ranging from 2 to 23 kb) and then easily recovered. The method is highly efficient and allows considerable time saving. Of special interest is its applicability to small starting amounts of DNA (less than 0.5 micrograms per band). Purity of the DNA is comparable, if higher, to that obtainable by conventional electroelution on dialysis bags and DEAE-cellulose membrane electrophoresis. The system is simple enough to be used as a routine method for eluting multiple bands in short times.

Acute pancreatitis was induced in 13 anesthetized dogs by retrograde injection of bile mixed with trypsin into the pancreatic duct. Six animals were treated with intravenous infusion of new synthetic antiprotease. Nafamostat Mesilate, at a dose of 1 mg/kg/h. Four out of seven untreated animals died during the experiment. All the treated dogs survived. Hemodynamic data were regularly monitored during a ten-hour observation period. Cardiac output, mean arterial pressure and left ventricular stroke volume decreased rapidly in the untreated animals. An increase in systemic vascular resistance and pulmonary vascular resistance was observed in dogs without treatment. Nafamostat Mesilate given as therapy significantly improved the hemodynamic parameters, and prevented the animals from developing shock. The study demonstrates an advantageous influence of synthetic antiprotease Nafamostat Mesilate on the course of acute experimental pancreatitis.

Transferrin allele (TF) distribution was investigated in three Bantu and one Babenga Pygmy groups of Congo, by isoelectric focusing on polyacrylamide (PIEF). In comparison with other Black populations, the four groups had a twofold higher TF*D frequency. Three different TF*D mutants were observed, the commonest type of which was identical by PIEF to a *D1 Caucasian allele. The classification of the other two is discussed. The homozygous TF D1 pattern was present in Babenga. The polymorphism of transferrin confirmed the uniqueness of anthropological traits in such Congo populations.

Several procedures for casting 100 microns ultrathin immobilized pH gradients are described. When acrylate/glass molding cassettes (Pascali et al., Electrophoresis 1987, 8, 371-373) are used two main problems are encountered: (i) a tendency of polymerization solutions, at the beginning of delivery, to spread across the glass surfaces with troublesome effects on the gradient stratification, and (ii) the raising of steep menisci at both extremities of the pH intervals, originating from capillarity phenomena and resulting in nonuniform gradients with bowed electrophoretic patterns. The first shortcoming was acceptably solved by increasing the density of sucrose gradients, and pouring them into prewarmed molding cassettes. The detrimental effect of menisci could be overcome by using a 'squeezing-sealing mold' technique. A molding cassette was endowed with a continuous, squared spacer frame, the upper side being open by inserting a wedged clip. A slight excess of polymerization solution was first dispensed into the cassette and squeezed away on removal of the clip. By completely excluding air from the molding cassette, uniform and well reproducible ultrathin gels could be cast. A major advantage of ultrathin immobilized pH gradient gels is the drastically shorter focusing time.

A neutral haemoglobin variant was identified by the combined use of different analytical methods. Isoelectric focusing on immobilized pH gradients (under denaturing conditions) allowed to detect and alpha chain variant. HPLC of tryptic digest showed that its amino-acid variation resided on T alpha 6 peptide (position 41-56). In the fast-atom bombardment (FAB) positive mass spectrum of the tryptic digest, the protonated molecular ion of the T alpha 6 peptide occurred 48 mass units lower than the normal T alpha 6 fragment, corresponding to a Phe----Val substitution. To a partial sequence determination (aminopeptidase digestion of the T alpha 6 peptide, followed by amino acid determination and FAB-MS analysis of the digestion-generated mixture) the substitution appeared to be on Phe43 (CE1). This variant is already known as Hb Torino. The procedure here described proved to be fast and simple, and feasible whenever neutral variants are supposed to occur.

The polymorphism of transferrin (Tf) is currently being studied by isoelectric focusing in carrier ampholyte-generated pH gradients, carrier ampholyte-separator pH gradients or in immobilized pH gradients. Details for obtaining reproducible results with each of the three procedures are outlined. The effectiveness of pretreatment of serum samples with ferrous/ferric salts is discussed, and incubation times optimized after spectrophotometric measurement of the monoferric Tf conversion. Most of the presently available commercial batches of carrier ampholytes do not reliably discriminate the six common TfC subtypes. Resolution of C1, C3 and C2 was achieved by adding 20 to 90 mM HEPES slab gels prepared with various carrier ampholytes. Isoelectric focusing in carrier ampholyte-separator pH gradients cannot be recommended as a standard typing procedure because the results strongly depend on the batch of carrier ampholytes. Tf subtype resolution was only achieved by using isoelectric focusing in immobilized pH gradients with pH slopes reliably reproducible from one experiment to another. Two major shortcomings of immobilized pH gradients are a marked tendency to protein precipitation at the application site and an interaction of proteins with the charged matrix. A protocol for Tf subtyping in immobilized pH gradients is described, based on prior desialylation of samples instead of pretreatment with iron. Sample entry into the matrix was optimized by addition of 5 mM Tris to the gels, and initially running them at low voltage. Recommendations are provided for the application of Tf typing for paternity testing.

Acute pancreatitis was induced in ten anesthetized dogs by retrograde injection for bile mixed with trypsin into the pancreatic duct. Five animals were treated with i.v. infusion of gabexate mesilate in a dose of 1 mg/kg per hour. Hemodynamic data were regulary monitored during a 10-h observation period. Cardiac output (CO), mean arterial pressure (MAP), and left ventricular stroke volume (LVSV) decreased rapidly in untreated animals. An increase of systemic vascular resistance (SVR) and pulmonary vascular resistance (PVR) was observed in dogs without treatment. Gabexate mesilate given as a therapy significantly improved the hemodynamic parameters. The study demonstrates an advantageous influence of synthetic antiprotease gabexate mesilate on the course of acute experimental pancreatitis.