Introduction of axial chirality at a spiro carbon atom in the synthesis of pentaerythritol-imine macrocycles.

Novel chiral macrocyclic polyimines with spiro carbon atoms are described. The key feature of the synthesis is the formation of an axially chiral quaternary carbon atom having four constitutionally identical substituents. This is possible either by the freezing of the labile conformation of a spiro-diboronate moiety or by the diastereomeric fitting of a conformationally stable spiro-acetal moiety into a chiral framework. A general model for the description of this type of axial chirality is proposed.

Chiral, triformylphenol-derived salen-type [4 + 6] organic cages.

A one-pot synthesis of chiral [4 + 6] tetrahedral cage compounds containing a salen fragment on each face is presented. The formation of the [4 + 6] products remains in contrast to the reaction of 1,3,5-triformylphloroglucinol with chiral diamines where [2 + 3] keto-enamine pseudocyclophanes are formed exclusively. The presence of OH groups determines the structural and spectroscopic properties of these cage compounds while a change in the reaction conditions facilitates the isolation of the microcrystalline products of the specific surface area varying from 5 to 578 m(2) g(-1).

Binding of fibrinogen molecules to pig platelets and their membranes.

Following addition of ADP, 125I-labelled fibrinogen binds specifically to pig platelets. This binding is completely inhibited by the unlabelled fibrinogen. Quantitative analysis indicates the presence of 12,400-25,000 molecules of fibrinogen which can be bound with an association constant of 5 . 10(8) M-1 to platelets. Fibrinogen receptors were found to be active in the isolated platelet membranes as well. Quantitative analysis of the saturable binding of fibrinogen to the platelet membranes showed that these receptors react with the same affinity with fibrinogen molecules. In contrast to the intact platelets, the platelet membranes can specifically bind fibrinogen in the absence of ADP. We conclude that a specific receptor for fibrinogen is exposed on the surface as a result of cell damage which is the first step of the platelet membrane isolation.

Reactivity of fibrinogen derivatives with antisera to human fibrin D-dimer and its gamma-gamma chain remnant.

Highly purified D-dimer was obtained from plasmin digest of human cross-linked fibrin. After reduction of its disulfide bonds, the gamma-gamma chain remnant, containing cross-linking site, was then isolated by ion-exchange chromatography on CM-cellulose. Antisera obtained by immunizing rabbits with D-dimer and its gamma-gamma chain remnant contained a small population of antibodies which specifically reacted with D-dimer. Thus, a specific radioimmunoassay system allowing detection and quantitation of D-dimer in the presence of fibrinogen and monomeric fragment D was made possible.

Tubulin is not phosphorylated in resting and thrombin-activated platelets.

We have investigated tubulin phosphorylation in human platelets, in order to evaluate whether it might be involved in the microtubular marginal band reorganization during platelet activation. Tubulin was identified with the use of specific monoclonal antibodies directed against alpha and beta subunits of tubulin. After metabolic 32P-labeling of platelets and analysis of separated proteins from whole cells, no phosphorylation of tubulin could be detected on autoradiography of platelet proteins either in resting platelets or during thrombin-induced activation. We also analyzed tubulin-enriched cytoskeletal fractions of resting or thrombin-stimulated platelets prepared in the presence of taxol, in comparison with tubulin-deprived cytoskeletal fractions prepared in the absence of this microtubule-stabilizing drug. Neither polymeric tubulin, assembled in microtubules and belonging to the platelet cytoskeleton, nor dimeric soluble tubulin showed significant 32P labeling. Finally, no tubulin was recovered among tyrosine-phosphorylated platelet proteins immunoprecipitated with a specific anti-phosphotyrosine protein monoclonal antibody. Thus, human platelet tubulin is not phosphorylated either in unstimulated platelets or in thrombin-stimulated platelets. The fact that both alpha and beta subunits are involved appears to be a unique feature of platelets in comparison with other cells. Microtubule-associated proteins are more likely to be involved in the unbundling of the platelet marginal band.

Modification of fibrin network ultrastructure by Fab fragments specific for different domain of fibrinogen.

Kinetics of inhibition of fibrin monomer polymerization produced by Fab fragments prepared from immunochemically purified monospecific antibodies to the surface epitopes of different domains of fibrinogen molecule has been correlated with electron microscopic observations of resulting specimens. Fab fragments prepared from anti FgD antisera were the most efficient inhibitors of thrombin-catalysed conversion of fibrinogen to fibrin; polymerization of fibrin monomers as detected spectrophotometrically was abolished at 2:1 molar ratio of anti FgD Fab fragments to fibra monomer. These Fab fragments acting as a steric hindrance of polymerization sites inhibited the first stage of fibrin monomer aggregation. Interaction of Fab fragments derived from antibodies specific for alpha 239-476 with corresponding segment of fibrinogen molecule resulted in a weak inhibition of fibrin monomer polymerization. However, fibrin obtained in the presence of these Fab fragments was significantly modified and showed no periodicity. This observation may suggest that anti alpha 239-476 Fab impaired the course of the second stage of fibrin monomer polymerization, i.e. lateral association of fibrin fibrils.

The amino acids that constitute sequence gamma 268-282 of fibrinogen are not involved in fibrin monomer polymerization.

Congenitally abnormal fibrinogens with impaired fibrin monomer polymerization have been described to contain single amino-acid substitutions localized in certain positions of the gamma 275-330 peptide region. To evaluate the role of the amino-acid sequence in the vicinity of Arg275 in fibrin monomer polymerization, the peptide fragment corresponding to gamma 268-282 was synthesized and used to obtain peptide-specific antibodies. These antibodies, when purified immunochemically on the immobilized peptide, bound to the intact fibrinogen and fibrin monomers with the same binding affinity. However, they did not recognize the gamma 268-282 epitopes on the denatured and reduced fibrinogen molecules. The lack of influence of antipeptide antibodies on fibrin monomer polymerization indicates that the gamma 268-282 peptide is not directly involved in the structure of the polymerization site in the D domain of fibrinogen. It is suggested that substitution of Arg275 either by His or Cys in abnormal fibrinogens results probably in conformational changes which disturb a proper orientation of the polymerization site and reduce its expression.

The expression of proliferating cell nuclear antigen (PCNA) in leukemia cell lines HL-60 and K-562 at the light and electron microscope level.

PCNA antigen was localized at the light and electron microscopes level in two human leukemia cell lines HL-60 and K-562. PCNA expression was used to discriminate cycling from non-cycling cells. PCNA protein at the level of the light microscope was present in 70% of the cell in HL-60 cell line and in 65% of the cells in K-562 line. Streptavidin immunogold method was used for localization of PCNA expression at the ultrastructural level. Positive staining for this protein was seen as granular pattern in the nucleus and in the cytoplasm. In the nucleus the gold particles were seen to be associated with heterochromatin and euchromatin of the leukemia cells. In cytoplasm it was found on the endoplasmic reticulum and associated with ribosomes. Controls of the leukemia cells incubation with normal mouse serum showed no labelling at the light and electron microscope level.

Prognostic factors in the operative and palliative treatment of pancreatic cancer.

Recent studies have emphasized the importance of patient selection for the surgical resection of pancreatic adenocarcinoma based on reproducible prognostic factors. The aim of the study was to investigate the prognostic factors affecting long-term survival in patients with resectable and nonresectable pancreatic cancer and to evaluate their prognostic value. Forty six patients (25 women, 21 men, aged 44-80) with ductal adenocarcinoma of the pancreas were reviewed. Primary tumor size and regional enlargement of lymph nodes was assessed with enhanced CT scan. 13 patients were treated conservatively, 9 with standard Whipple procedure (pancreatoduodenectomy) and 24 - with palliative surgery. Survival probabilities were computed using univariate Kaplan-Meier analysis. Log-rank test was used to compare survival between groups. Overall median survival was 6 months with a 4 years survival of 2.2%. There was no difference in survival time (ST) between patients aged 65 years or younger and older (p=0.71). MeanST in patients after Whipple procedure was 10.3, after palliative surgery - 9.4 and after conservative treatment - 4.4 months (p<0.05). Thirty-day surgical mortality was 9.4%. ST was significantly longer in patients with tumors 3 cm or less of diameter compared with larger ones (p<0.05). Presenting signs and symptoms, like jaundice, diabetes, alkaline phosphatase, aspartate and alanine aminotransferase elevation and history of cholecystectomy did not have any significant impact on survival. The only significant independent factors improving survival were: operative treatment and tumor size smaller than 3 cm.

Immunogold labelling of PCNA and Ki-67 antigen at the ultrastructural level in laryngeal squamous cell carcinoma and its correlation with lymph node metastasis and histological grade.

Streptavidin-gold was used for the immunolocalization of PCNA and Ki-67 antigen at the ultrastructural level with a postembedding technique in biopsies of 15 patients with laryngeal squamous cell carcinoma. Positive immunoelectron staining was obtained in 9 cases for PCNA (60%) and in 8 cases for Ki-67 (53%). PCNA was predominantly found in heterochromatin of the nucleus of laryngeal carcinoma cells in a granular pattern. Positivity for PCNA was not found in nucleoli. In 4 cases, positive staining was observed both in nucleus and cytoplasm. In the cytoplasm, it was found to be present on the endoplasmic reticulum and on ribosomes throughout the cytoplasm. Ki-67 antigen was localized in the nucleus where it was associated with heterochromatin and euchromatin. It was also observed in nucleoli in all cases. Cytoplasmic localization of Ki-67 antigen was similar to that of PCNA. All 8 cases that were positive for Ki-67 were also positive for PCNA. Control incubations did not result in labelling with steptavidin-gold particles for both antigens. A significant correlation between PCNA and Ki-67 expression in association with pathological characteristics such as nodal status and histological grade was not found. Our data indicate that Ki-67 antigen staining correlates with PCNA labelling, whereas a relationship between proliferation markers and tumour progression was not found.

Immunoelectron microscopical identification of the c-erbB-2 oncoprotein in patients with laryngeal squamous cell carcinoma.

The peroxidase-antiperoxidase (PAP) method was used for localization of the c-erbB-2 oncoprotein at the electron microscopical level in 15 patients with laryngeal squamous cell carcinoma. It was found that c-erbB-2 oncoprotein was present in 7 of 15 samples. Electron microscopical examination revealed expression of the c-erbB-2 oncoprotein both on the membrane of individual cells and in the cytoplasm. In 5 of the 7 cases of positive labeling, it was observed only on the plasma membrane of cells whereas in 2 cases, there was also cytoplasmic staining. Reaction product was associated with endoplasmic reticulum, and the nuclear envelope and was scattered throughout the cytoplasm on ribosomes. Control incubations using normal rabbit serum instead of the primary antibody showed no labeling. A significant correlation between c-erbB-2 oncoprotein and pathological characteristics such as nodal status and histological grade was not found.

[The role of nutrition on the treatment of septic syndrome and multiple organ failure]. / Rola zywienia w leczeniu zespolu septycznego i niewydolnosci wielonarzadowej.

Basing on current literature the issues regarding nutrition in patients with septic syndrome and multiple organ failure were discussed. It was emphasised that patient feeding along with providing metabolic substrates also serves as a treatment method. Diet ingredients in specific carbohydrate, protein and fat metabolism disorders were analysed. We focused upon the role of emulsion of fat, especially medium chain triglycerides (MCT) and -3-linolenic acid (fish oil). The unique role o f glutamine, arginine and branched chain amino acids (BCA) was highlighted. Patient nutrition with accordance to the presented new methods has become an efficient routine of treatment in septic syndrome. It may constitute an efficient prophylaxis against multiple organ failure.

Exercise effect on the circadian rhythm of left ventricular systole in healthy men with higher and lower physical fitness.

The purpose of the present work was to investigate the effect of exercise on the diurnal changes of electromechanical systolic time (QS2) in healthy men with different physical fitness (N = 20, PWC150-804.3 +/- 114.6 kpm/min), and with higher (N = 12, PWC150-881.3 +/- 71.8 kpm/min) and lower (N = 8, PWC150-688.8 +/- 46.0 kpm/min) physical fitness. The study on 20 male volunteers aged 19-21 at 6 times of 24-hours period in intervals (4-hours) was performed. The policardiograms according to Blumberger at rest and after 10 minutes of exercise were obtained. The individual level of exercise at test PWC150 was determined. The values of QS2 from policardiograms according to Weissler's method were read and for heart rate were corrected. The mathematical analysis by the method of Halberg was made. It was found that in healthy men at rest the circadian rhythm of QS2 existed. The effect of exercise on the circadian rhythm of QS2 was dependent on level of physical fitness. After exercise in human with higher physical fitness the circadian rhythm of QS2 was presented, whereas in human with lower physical fitness it was not evident.

Mechanism of antifungal action of kanosamine.

The antibiotic kanosamine inhibited growth of Saccharomyces cerevisiae and a range of human pathogenic fungi, including Candida albicans. Kanosamine was transported into C. albicans cells by the glucose transport system and subsequently phosphorylated. The product of its intracellular metabolism, kanosamine-6-phosphate, was an inhibitor of the enzyme glucosamine-6-phosphate synthase. Inhibition was competitive in respect to one of the substrates, D-fructose-6-phosphate, with Ki = 5.9 mM, and was non-competitive in respect to the second substrate, L-glutamine. On the other hand, kanosamine-6-phosphate had no effect on the enzyme catalysing the next metabolic step, namely glucosamine-6-phosphate N-acetylase. The action of kanosamine on C. albicans cells resulted in profound morphological changes, inhibition of septum formation and cell agglutination. Experiments with S. cerevisiae mutants showed that the presence of the Cdr1p drug efflux pump did not affect the antifungal activity of kanosamine.

[The number and affinity of fibrinogen receptors in blood platelets of patients with ischemic stroke]. / Liczba i powinowactwo receptorów dla fibrynogenu w krwinkach plytkowych chorych na niedokrwienny udar mózgu.

Parameters of fibrinogen binding with blood platelets (number of receptors and their affinity) have been studied in patients with ischemic stroke. Due to the increased platelet ability to aggregate in the ischemic diseases such studies seem helpful. The studies involved 13 patients with ischemic stroke. Blood platelets collected from younger patients (under 50 years) possessed significantly higher number of receptors binding fibrinogen than blood platelets of healthy individuals (p less than 0.02). These receptors significantly more strongly bound ligand than those in the control group (p less than 0.05), and in the group of older patients with stroke (less than 0.05). Fibrinogen binding to blood platelets in patients over 50 years of age did not differ significantly from that in the control group. These results may indicate, that the increased platelet aggregation might be a significant pathogenic factor of the stroke in younger patients.

Adenine nucleotides and 2,3-dophosphoglycerate in the erythrocytes during physical exercise and restitution in healthy subjects.

The reported investigations were carried out in 6 young (20 years old) healthy males who performed a 10-minute exercise on a Monark cycle ergometer at workloads increasing the heart rate to 170/minute. Before the exercise, after its termination, and after 30 minutes of restitution the concentrations of adenosine-5' triphosphate, adenosine-5' diphosphate, adenosine-5' monophosphate, 2,3-diphosphoglycerate, nicotinamide-adenine dinucleotide and nicotinamide-adenine dinucleotide phosphate were assayed in erythrocytes. After the end of exercise a non-significant increase in ATP concentration and a non-significant decrease of 2,3-DPG were found in the erythrocytes. The concentrations of ADP and AMP were lower than at rest, both, after the end of exercise and after 30 minutes of restitution (p less than 0.05). The concentrations of nicotinamide-adenine dinucleotide and nicotinamide-adenine dinucleotide phosphate decreased after exercise (p less than 0.05) fell even more after 30 minutes of restitution (p less than 0.05).

Elevated plasma gastrin, CEA, and CA 19-9 levels decrease after colorectal cancer resection.

BACKGROUND AND AIMS: Gastrin stimulates mucosal growth of much of the gastrointestinal tract and has also been implicated in promoting growth of colonic tumors, but its role in colorectal carcinogenesis remains controversial. This study determined fasting serum gastrin levels before and after surgery for colorectal cancer (CRC) and the relationship to the clinical stage of the disease to investigate it possible prognostic role. PATIENTS AND METHODS: Fasting radioimmunoassay gastrin, CA 19-9, and CEA levels were measured before and after surgery for CRC. Helicobacter pylori status was also assessed since it causes significant hypergastrinemia. RESULTS: Mean fasting plasma gastrin level was significantly higher in CRC patients than in controls before surgery but not 59 days after surgery. Mean CEA and CA 19-9 levels were significantly higher in patients with CRC before surgery than after tumor resection. There was a significant positive correlation between the plasma gastrin, CEA, and CA 19-9 levels and the CRC stage (Dukes' classification). CONCLUSION: The significance of gastrin as a marker for diagnosis or prognostic purposes in colorectal cancer needs to be further examined.

Usefulness of p16 and K-ras mutation in pancreatic adenocarcinoma and chronic pancreatitis differential diagnosis.

BACKGROUND: The differentiation of chronic pancreatitis (CP) from pancreatic adenocarcinoma (PA) remains a great challenge. The purpose of the study was to compare the prevalence of p16 and K-ras mutation in PA and CP in order to evaluate their usefulness in differential diagnosis of those diseases. METHODS: The study included 44 patients who underwent Whipple resection or distal pancreatectomy for PA (23 subjects) or CP (21 subjects). DNA from pancreatic tissue was analysed for K-ras mutation (codon 12) and p16 mutations with PCR amplifications. RESULTS: The K-ras gene mutation has been shown in 17 (73,9%) cases with pancreatic adenocarcinoma which was significantly more often than in chronic pancreatitis - 9 (42,8%) (p<0,01). Prevalence of p16 mutations in patients with PA was 18 (78,3%) and with CP - 7 (33,3%) (p<0,01). K-ras and p16 mutations together have been observed in 16 (69,6%) cases in patients with PC and only in 3 (14,3%) - with CP (p<0,01). No statistically significant association between K-ras or p16 mutations and tumor size, sex or patient age has been observed. CONCLUSION: It is suggested that simultaneous measurement of K-ras and p16 mutations may provide an additional tool in differential diagnosis of chronic pancreatitis and pancreatic adenocarcinoma.