Stability determination of the formulations containing hyaluronic acid.

OBJECTIVES: The aim of this work was to investigate the long-term stability of four emulsions (creams and ointments) developed for carrying hyaluronic acid (HA) for dermal penetration. METHODS: The stability of obtained formulations was analysed by multiple light scattering and laser diffraction methods. RESULTS: The experimental results showed that the moisturizing creams were more stable than the ointments containing HA. The migration phenomenon of particles was observed in soft ointment with HA and the flocculation phenomenon was detected in ointment based on lanolin. The larger the water content, the more stable formulation can be, due to hygroscopic properties of HA. CONCLUSIONS: The identification of instability phenomena was shortened dramatically using optical method such as multiple light scattering. Even small changes in the stability of emulsion were determined very early. We suggest that the moisturizing creams may be more suitable than ointments to carry HA for dermal absorption. These studies are a significant first step towards further exploration into what form of HA would assure maximum effect, give consumers satisfaction and guarantee safety during application.

Anatomy of the occipital lobe using lateral and posterior approaches: a neuroanatomical study with a neurosurgical perspective on intraoperative brain mapping.

BACKGROUND: A major concern of occipital lobe surgery is the risk of visual field deficits. Extending anatomical occipital lobectomy to the functional requires awake conditions because the anterior resection border comprises language-, motor- and visuospatial function-related areas within the temporal and parietal lobes. This study investigated the lateral and posterior perspectives of the occipital lobe anatomy when approaching intraaxial occipital lobe lesions. MATERIALS AND METHODS: Ten adult cadaveric cerebral hemispheres were dissected after being prepared following the concept described by Klingler for the first time. RESULTS: The occipital lobe was located posteriorly to the parietotemporal line. Within the occipital lobe, the occipital horn of the lateral ventricle represented the only anatomical landmark. Laterally, optic radiation was identified as a part of the sagittal stratum. None of the intraoperatively identifiable tracts was found medial to the occipital horn. Language- and motor-related areas were identified anteriorly and should be actively identified when lobectomy based on function is planned. Subcortically, from a posterior perspective, the anterolateral border constituted the arcuate fascicle/superior longitudinal fascicle complex and was anteromedial to the thalamocortical tract. Remaining posterior to the line connecting the preoccipital notch with the superior Rolandic point avoided the cortical and white matter tracts related to language, motor and visuospatial function. CONCLUSIONS: Knowledge of occipital lobe anatomy and surrounding structures is essential to preoperatively assess the risk of the procedure and proper consultation of a patient in terms of the extent of resection, primarily concerning visual field deficits.

Heating and storage of structured acylglycerols with succinyl-linked stigmasterol residue does not cause negative chemical or biological changes.

Four structured acylglycerols with stigmasterol bonded by a succinyl linker were investigated and their stability were analyzed. Samples were heated to 60 °C and kept at that temperature to simulate storage, and to 180 °C to simulate frying conditions. The degradation of the synthesized compounds and formed derivatives was determined, and their cytotoxicity and genotoxicity on normal human cells from the digestive system was determined. Holding at 180 °C resulted in greater degradation of the compounds than holding at 60 °C. The most stable compound in each sample proved to be one with oleic acid in its structure-1,3-dioleoyl-2-stigmasterylsuccinoyl-sn-glycerol (DO2SSt) at 60 °C and 1,2-dioleoyl-3-stigmasterylsuccinoyl-sn-glycerol (DO3SSt) at 180 °C. These results indicate that the type of fatty acid in the molecule is more important than its position in the glycerol structure. None of the diacylmonostigmasterylsuccinoyl-sn-glycerols (DASStGs) before or after heating exhibited cytotoxic or genotoxic potential to small intestine and colon mucosa cells.

Thermo-oxidative stability and safety of new acylglycerols with stigmasterol residue: Effects of fatty acids saturation and position in the glycerol backbone.

The safety and thermoxidative stability of new diacyl-stigmasterylcarbonoyl-sn-glycerols (DAStGs) with two molecules of palmitic or oleic acids and one molecule of stigmasterol at the sn-2 or sn-3 position were studied. After heating to 60 °C, the compounds with stigmasterol at the sn-2 position were more stable than those with stigmasterol at the sn-3 position. The lowest level of degradation of stigmasterol after heating to 180 °C was detected for both compounds with oleic acid, followed by the samples with palmitic acid. The high content of SOPs, especially triolSt, as well as the high level of dimers showed the most effect on the cytotoxicity of DAStGs heated at both temperatures. DAStGs with oleic acid at sn-1,3 and stigmasterol at sn-2 position were the most stable compounds. Both oleic acid and the location of stigmasterol in the middle of the glycerol molecule play an important role in increasing the thermoxidative stability of stigmasterol.

Thermo-oxidative stability of asymmetric distigmasterol-modified acylglycerols as novel derivatives of plant sterols.

The study investigated the thermo-oxidative stability of distigmasterol-modified acylglycerols as a new structured acylglycerols. Samples were heated at 60 and 180 °C for 8 h. Their percentage degradation and products formed during heating were compared with free stigmasterol and stigmasteryl esters. The remaining of stigmasterol and fatty acid parts, the formation of stigmasterol oxidation products and the composition of polar and non-polar fractions were analysed using chromatographic methods. The cytotoxicity and genotoxicity were determined with the use of an MTT test and a comet assay, respectively. The highest stability during heating was observed for 2,3-distigmasterylsuccinoyl-1-oleoyl-sn-glycerol (dStigS-OA) and the lowest for 2,3-distigmasterylcarbonoyl-1-oleoyl-sn-glycerol (dStigC-OA). Data showed that the formation of thermo-oxidative degradation products is affected by the temperature and chemical structure of lipids present in the molecule. The dStigMAs bonded by a succinate linker and products formed during their thermo-oxidation showed no cytotoxic or genotoxic activity to normal human cells.

The level of antibody against E6 HPV 16 oncoprotein in blood sera of women with chronic HPV 16 infection and cervical cancer.

UNLABELLED: The aim of this study was to estimate of the role of chronic HPV 16 infection and the presence of anti E6 HPV 16 in the initiation of the cancerogenesis process of cervical cancer. MATERIAL AND METHODS: The study included two groups of patients. The first group comprised 323 women observed for three consecutive years (1998-2000), in whom the presence of HPV 16 viruses was estimated by PCR, and the level of anti E6 HPV 16 antibodies was estimated in the plasma with ELISA. A similar test was performed in a group of 46 patients with cervical intraepithelial neoplasia (CIN), 91 patients with invasive cervical cancer and 22 women after hysterectomy and RTG-therapy. RESULTS: In 32 patients, chronic HPV 16 infection showed a steady rise in the mean absorbance level of anti E6 HPV 16 antibodies from 0.04 in 1998 to 0.06 in 2000, while in HPV-negative women the mean absorbance value was 0.03-0.04. Mean absorbance value in patients with CIN III and invasive cancer rose with advancing stage of the cancer process and lowered after completion of oncological treatment. The values were 0.14, 0.33 and 0.13, respectively. CONCLUSION: The persistence of chronic HPV 16 infection and accompanying steady rise in absorbance index caused by an increase in the level of antiviral antibodies are a clear warning signal preceding in time the histological process of cancerogenesis.

Tumor necrosis factor alpha (TNF-alpha) modulates rat mast cell reactivity.

Nowadays there is growing evidence that some cytokines regulate biological functions of the mature mast cells. Therefore, we have studied whether TNF-alpha, the cytokine of multifunctional activities, could directly stimulate rat peritoneal mast cells to histamine secretion and whether it could modulate rat mast cell reactivity in anaphylactic (with ConA) and anaphylactoid (with compound 48/80) reactions. We have established that rat recombinant TNF-alpha does not activate rat mast cells to histamine release. However, TNF-alpha-treatment causes the decrease of spontaneous histamine release up to 85% (TNF-alpha concentration: 2 x 10(-9) M). Pretreatment of mast cells with TNF-alpha inhibits ConA-stimulate release of histamine with the percent release decreasing up to 33.7% of the control value (TNF-alpha concentration: 5 x 10(-9) M) and this decrease is statistically significant. Pretreatment of mast cells with TNF-alpha reduces compound 48/80-dependent histamine release as well and the percent release of histamine fell to 64.7% of the control value. We have concluded that TNF-alpha may play a significant role in regulation of mast cell secretory activity.

Detection of DNA, mRNA and early antigen of the human cytomegalovirus using the immunomax technique in autopsy material of children with intrauterine infection.

The present study focuses on the immunomax technique in association with the avidin-biotin-peroxidase complex (ABC) technique and a non-isotopic variation of in situ hybridisation (ISH) for optimal microscopical detection of human cytomegalovirus (HCMV). The studies were performed on an archival paraffin material originating from five children deceased due to intrauterine infection. The results of immunocytochemical and hybridocytochemical studies, with or without amplification using biotinylated tyramine, were compared with the routine histopathological results and results obtained using the polymerase chain reaction (PCR). Early antigen (EA)-HCMV was demonstrated in approximately twice as many cells as detected in the routine staining and also in cells that seemed morphologically intact. The hybridocytochemical studies confirmed the presence of HCMV DNA in cells that were positive in the immunocytochemical tests and, in addition (using the ISH-immunomax technique), in cell nuclei of intact myocardial myocytes. In general, fewer cells manifested the presence of HMCV mRNA than the presence of HCMV DNA. The immunomax technique was found to be more sensitive than the techniques of classical immunocytochemistry or of ISH. The former technique permitted the documentation of a higher number of HCMV replication sites than could be detected using the latter techniques. However, the clinical course of HCMV infection or the cause of death of the children was not directly related to the intensity of HCMV expression in tissues.

Effect of hyperosmolarity on recombinant protein productivity in baculovirus expression system.

A lot of strategies were applied to improve recombinant protein productivity in the baculovirus expression system. In this study we propose for foreign protein production fed-batch cultivation method at hyperosmotic environment induced by increased NaCl content. Obtained results suggested relatively high tolerance and adaptation abilities of Tn-5 insect cells exposed to hyperosmotic stress. The cells under hyperosmotic conditions increased the specific rate of glucose consumption and lactate production. The release of additional energy and precursors as a result of increased metabolism by osmotically stressed culture was involved in recombinant protein synthesis. Recombinant nucleoprotein productivity in nutritional feeding cultures exposed to hyperosmolarity was about 72% higher than that obtained in batch culture at physiological osmolarity, but 31% was a result of feeding and the rest 41% was a result of hyperosmolarity and increasing Na(+) concentration.

Can the 1,5-disubstituted tetrazole ring modify the co-ordinating ability and biological activity of opiate-like peptides?

The copper(II) complexing ability and the biological activity of beta-casomorphin-7 tetrazole analogues have been investigated. Potentiometric and spectroscopic (UV-Vis, CD and EPR) studies have been used to establish the thermodynamic stability, speciation and structure of Cu(II) complexes with YP-psi(CN4)-FPGPI-NH2 (1), YPF-psi(CN4)-AGPI-NH2 (2) and YPFP-psi(CN4)-GPI-NH2 (3). Comparison of the binding ability of the tetrazole analogues reveals that the most effective ligand for copper(II) is YPF-psi(CN4)-AGPI-NH2. The effectiveness of this ligand comes from its particular conformation suited for the Cu(II) 2N co-ordination mode in the physiological pH region. The ability of casomorphin tetrazole analogues to activate rat mast cells to histamine release in vitro in the presence of copper(II) has been studied.

Intestinal mucosa-associated bacteria modulate rat mast cell reactivity.

It is well known that in some conditions bacteria of physiological flora of gastrointestinal tract may become pathogenic. Each complaint which causes injury of gastrointestinal wall integrity permits bacteria to penetrate the tissues and affect the tissue cells. Since mast cells represent one of very important and numerous cellular elements of the gastrointestinal tract walls, bacteria can exert the effects on them. Therefore, the aim of our study was to examine the influence of four strains of intestinal mucosa-associated bacteria--Bacteroides thetaiotaomicron, Bacteroides fragilis, Bifidobacterium adolescentis and Escherichia coli on the mast cell reactivity. Our experiments were performed in vitro on isolated rat peritoneal mast cells and the reactivity of these cells was estimated on the basis of histamine release. We used the suspensions of whole bacteria, killed by heating at 65 degrees C. We have noticed that the magnitude of bacteria-induced histamine release from mast cells was very low (up to 6.0%) when compared with histamine release induced with Con A, compound 48/80 and TNF-alpha. However, all studied bacteria changed the reactivity of mast cells in anaphylactic (with ConA) and anaphylactoid (with compound 48/80) reactions. After 40 min preincubation with B. thetaioataomicron, B. fragilis, B. adolescentis or E. coli ConA-induced histamine release was diminished up to 25%, 71%, 58% and and 68% of maximal histamine release, respectively. Preincubation of rat mast cells with B. thetaioataomicron, B. fragilis, B. adolescentis or E. coli also changed their reactivity in anaphylactoid reaction with compound 48/80 (histamine release was diminished up to 70%, 63%, 63% and 60%, respectively).

Production of human papillomavirus type 16 early proteins in Bac-To-Bac Expression System (GibcoBRL).

Human papillomavirus type 16 (HPV16) is a major agent in cervical cancer etiology. Its early proteins are responsible for virus persistence, replication and initiation of neoplastic disease. In the present study we describe a use of baculovirus-insect cell expression system for production and study of HPV16 E2 and E4 proteins. The E2 protein binds specifically to viral DNA and E4 protein shows characteristic cytopathic effects on cells.

[Mastocyte proteinase]. / Proteinazy mastocytów.

Recent studies have led to a rapid expansion of knowledge concerning the structure and biology of mast cells neutral proteinases. Therefore, in this paper tryptases, chymases, carboxypeptidases and cathepsin G have been described and their biological roles have been discussed.

The role of sympathetic nervous system in the action of dopamine and dobutamine on the function of circulatory system.

To assess the role of peripheral structures of the sympathetic nervous system in the effects of dopamine and dobutamine on some functions of the cardiovascular system, experiments were carried out on anesthetized rabbits in which the blood flow in calf muscles was measured, during rest and during evoked twitches, using the method of 133Xe clearance. The arterial blood pressure and ECG were simultaneously recorded. A complete reversal of the action of dopamine on the arterial blood pressure and heart rate, after sympathetic nerve endings blockade, indicates an important role for the undisturbed neurotransmission in revealing hypotensive properties of this amine. In the action of dobutamine on blood pressure, heart rate and muscle blood flow, the blockade of peripheral structures of the sympathetic nervous system is of minor importance.

Monitoring the progress of infection and recombinant protein production in insect cell cultures using intracellular ATP measurement.

Several monitoring methods used to predict viable cell density have been the subject of extensive studies, including oxygen uptake rate, carbon dioxide evolution rate, optical density, NADH-dependent fluorescence and relative permittivity measurement. We propose intracellular ATP determination by bioluminescence assay to monitor the progress of baculovirus infection and recombinant protein production in insect cell cultures. We found that the ATP content in viable cells increased after virus addition. The increase in the ATP level was observed until the maximum recombinant protein accumulation was reached. At maximum product yield, the specific ATP content significantly decreased. Results obtained in both batch and fed-batch cultures demonstrated that the specific ATP level could be considered as a good indicator of recombinant protein productivity. Monitoring the cellular ATP content after viral infection makes it possible to define the optimum time for product harvest. The main advantage of applying the ATP assay as an index of the progress of infection and recombinant protein synthesis is its short time and sensitivity.

Synthesis and biological action of some derivatives of pyrido-[1,4]-diazepine and pyrido-[2,3-d]-pyrimidine.

The following novel compounds were synthesized: 1H-2, 3, 4, 9-tetrahydropyrido-[2, 3-e] [1, 4]-diazepino-2, 3, 9-trione 1a, 2-methyl-3, 4-dihydropyrido-[2, 3-d]-pyrimidin-4-one 2a, and their derivatives: N-methylmorpholino 1b and 2b, N-methylpiperidino 1c and 2c, N-(beta-hydroxy-gamma-morpholinopropyl) 1d and 2d, N-(beta-diethylaminoethyl) 1e and 2e, and 2-styryl 3a, 2-p-chlorotyryl 3b, and 3', 4', 5'-trimethoxystyryl 3c. The most potent hypotensive action was exerted by compound 2c (in a dose of 10 mg/kg), 2d (30 mg/kg), and 3a-3c (50 mg/kg).