Synthesis of papain nanoparticles by electron beam irradiation - A pathway for controlled enzyme crosslinking.

Crosslinked enzyme aggregates comprise more stable and highly concentrated enzymatic preparations of current biotechnological and biomedical relevance. This work reports the development of crosslinked nanosized papain aggregates using electron beam irradiation as an alternative route for controlled enzyme crosslinking. The nanoparticles were synthesized in phosphate buffer using various ethanol concentrations and electron beam irradiation doses. Particle size increase was monitored using dynamic light scattering. The crosslinking formation by means of bityrosine linkages were measured by fluorescence spectra and the enzymatic activity was monitored using Na-Benzoyl-dl-arginine p-nitroanilide hydrochloride as a substrate. The process led to crosslinked papain nanoparticles with controlled sizes ranging from 6 to 11nm depending upon the dose and ethanol concentration. The irradiation atmosphere played an important role in the final bioactivity of the nanoparticles, whereas argon and nitrous oxide saturated systems were more effective than at atmospheric conditions in terms of preserving papain enzymatic activity. Highlighted advantages of the technique include the lack of monomers and crosslinking agents, quick processing with reduced bioactivity changes, and the possibility to be performed inside the final package simultaneously with sterilization.

Thermoresponsive poly[tri(ethylene glycol) monoethyl ether methacrylate]-peptide surfaces obtained by radiation grafting-synthesis and characterisation.

This report demonstrates the feasibility of radiation grafting for the preparation of polymer layers functionalised with short peptide ligands which promote cell adhesion. Thermoresponsive poly [tri(ethylene glycol) monoethyl ether methacrylate] (PTEGMA) layers were synthesised on a polypropylene substrate by post-irradiation grafting. A cell adhesion moiety, the CF-IKVAVK peptide modified with a methacrylamide function and a fluorescent label were introduced to the surface during the polymerisation process. The amount of CF-IKVAVK was easily controlled by changing its concentration in the reaction mixture. The changes in the surface composition, morphology, philicity and thickness at each step of the polypropylene functionalisation confirmed that the surface modification procedures were successful. The increase in environmental temperature above the cloud point temperature of PTEGMA caused a decrease in surface philicity. The obtained PTEGMA and PTEGMA-peptide surfaces above TCP were tested as scaffolds for fibroblast sheet culture and temperature induced detachment.

Polymer gel dosimetry close to an 125I interstitial brachytherapy seed.

Despite its advantages, the polymer gel-magnetic resonance imaging (MRI) method has not, as yet, been successfully employed in dosimetry of low energy/low dose rate photon-emitting brachytherapy sources such as 125I or 103Pd interstitial seeds. In the present work, two commercially available 125I seed sources, each of approximately 0.5 U, were positioned at two different locations of a polymer gel filled vial. The gel vial was MR scanned with the sources in place 19 and 36 days after seed implantation. Calibration curves were acquired from the coupling of MRI measurements with accurate Monte Carlo dose calculations obtained simulating the exact experimental setup geometry and materials. The obtained gel response data imply that while linearity of response is sustained, sensitivity (calibration curve slope) is significantly increased (approximately 60%) compared to its typical value for the 192Ir (or 60Co and 6 MV LINAC) photon energies. Water equivalence and relative energy response corrections of the gel cannot account for more than 3-4% of this increase, which, therefore, has to be mainly attributed to physicochemical processes related to the low dose rate of the sources and the associated prolonged irradiation time. The calibration data obtained from one 125I source were used to provide absolute dosimetry results for the other 125I source, which were found to agree with corresponding Monte Carlo calculations within experimental uncertainties. It is therefore suggested that, regardless of the underlying factors accounting for the gel dose response to 125I irradiations, polymer gel dosimetry of new 125I or 103Pd sources should be carried out as originally proposed by Heard and Ibbot (2004 J. Phys.: Conf. Ser. 3 221-3), i.e., by irradiating the same gel sample with the new low dose rate source, as well as with a well-characterized low dose rate source which will provide the dose calibration curve for the same irradiation conditions.

Three-dimensional dose verification of the clinical application of gamma knife stereotactic radiosurgery using polymer gel and MRI.

This work seeks to verify multi-shot clinical applications of stereotactic radiosurgery with a Leksell Gamma Knife model C unit employing a polymer gel-MRI based experimental procedure, which has already been shown to be capable of verifying the precision and accuracy of dose delivery in single-shot gamma knife applications. The treatment plan studied in the present work resembles a clinical treatment case of pituitary adenoma using four 8 mm and one 14 mm collimator helmet shots to deliver a prescription dose of 15 Gy to the 50% isodose line (30 Gy maximum dose). For the experimental dose verification of the treatment plan, the same criteria as those used in the clinical treatment planning evaluation were employed. These included comparison of measured and GammaPlan calculated data, in terms of percentage isodose contours on axial, coronal and sagittal planes, as well as 3D plan evaluation criteria such as dose-volume histograms for the target volume, target coverage and conformity indices. Measured percentage isodose contours compared favourably with calculated ones despite individual point fluctuations at low dose contours (e.g., 20%) mainly due to the effect of T2 measurement uncertainty on dose resolution. Dose-volume histogram data were also found in a good agreement while the experimental results for the percentage target coverage and conformity index were 94% and 1.17 relative to corresponding GammaPlan calculations of 96% and 1.12, respectively. Overall, polymer gel results verified the planned dose distribution within experimental uncertainties and uncertainty related to the digitization process of selected GammaPlan output data.

Dose verification of single shot gamma knife applications using VIPAR polymer gel and MRI.

This work describes an experimental procedure with potential to assess the overall accuracy associated with gamma knife clinical applications, from patient imaging and dosimetry planning to patient positioning and dose delivery using the automated positioning system of a Leksell Gamma Knife model C. The VIPAR polymer gel-MRI dosimetry method is employed due to its inherent three-dimensional feature and linear dose response over the range of gamma knife applications. Different polymer gel vials were irradiated with single shot gamma knife treatment plans using each of the four available collimator helmets to deliver a maximum dose of 30 Gy. Percentage relative dose results are presented not only in the form of one-dimensional profiles but also planar isocontours and isosurfaces in three dimensions. Experimental results are compared with corresponding Gammaplan treatment planning system calculations as well as acceptance test radiochromic film measurements. A good agreement, within the experimental uncertainty, is observed between measured and expected dose distributions. This experimental uncertainty is of the order of one imaging pixel in the MRI gel readout session (<1 mm) and allows for the verification of single shot gamma knife applications in terms of acceptance specifications for precision in beam alignment and accuracy. Averaging net R(2) results in the dose plateau of the 4 mm and 18 mm collimator irradiated gel vials, which were MR scanned in the same session, provides a crude estimate of the 4 mm output factor which agrees within errors with the default value of 0.870.

Polymer gel water equivalence and relative energy response with emphasis on low photon energy dosimetry in brachytherapy.

The water equivalence and stable relative energy response of polymer gel dosimeters are usually taken for granted in the relatively high x-ray energy range of external beam radiotherapy based on qualitative indices such as mass and electron density and effective atomic number. However, these favourable dosimetric characteristics are questionable in the energy range of interest to brachytherapy especially in the case of lower energy photon sources such as 103Pd and 125I that are currently utilized. In this work, six representative polymer gel formulations as well as the most commonly used experimental set-up of a LiF TLD detector-solid water phantom are discussed on the basis of mass attenuation and energy absorption coefficients calculated in the energy range of 10 keV-10 MeV with regard to their water equivalence as a phantom and detector material. The discussion is also supported by Monte Carlo simulation results. It is found that water equivalence of polymer gel dosimeters is sustained for photon energies down to about 60 keV and no corrections are needed for polymer gel dosimetry of 169Yb or 192Ir sources. For 125I and 103Pd sources, however, a correction that is source-distance dependent is required. Appropriate Monte Carlo results show that at the dosimetric reference distance of 1 cm from a source, these corrections are of the order of 3% for 125I and 2% for 103Pd. These have to be compared with corresponding corrections of up to 35% for 125I and 103Pd and up to 15% even for the 169Yb energies for the experimental set-up of the LiF TLD detector-solid water phantom.

L-745,870 suppresses the nighttime serotonin N-acetyltransferase activity in chick retina: in vivo evidence for agonist activity at D4-dopamine receptors.

This study examined the in vivo activity of L-745,870 at dopamine (DA) D(4) receptors, using the chick retina as a model system. In dark-adapted retinas of various vertebrates, including hen, DA acting via D(4) receptors suppresses melatonin content and activity of serotonin N-acetyltransferase (AA-NAT, a key regulatory enzyme in melatonin synthesis). Systemic administration to chicks of quinpirole (0.1 mg/kg), a high affinity agonist of D(3)/D(4)-DA receptors, potently decreased the nighttime AA-NAT activity of the retina. The quinpirole-evoked decline in the enzyme activity was attenuated by L-745,870 (0.1-10 nmol/eye). In addition to this action, L-745,870 given to chicks either directly into the eye (0.03-10 nmol/eye) or intraperitoneally (0.5-5 mg/kg) decreased the nighttime AA-NAT activity of the retina in a dose-dependent manner. The suppressive effect of L-745,870 on retinal AA-NAT activity was blocked by 2-chloro-11-(4-methylpiperazino)dibenz[ b, f]oxepin, an antagonist of D(4)-DA receptors, but was not affected by raclopride, an antagonist of D(2)/D(3)-DA receptors. Altogether these results indicate that in chicks L-745,870, the potent putative D(4)-DA receptor antagonist, behaves in vivo as a partial D(4) agonist.

Effects of cycloheximide and aminophylline on 5-methoxytryptophol and melatonin contents in the chick pineal gland.

The chick pineal gland rhythmically synthesizes two 5-methoxyindoles, melatonin and 5-methoxytryptophol. These rhythms are circadian in nature and have opposite phases. The aim of this study was to determine the effects of cycloheximide, a protein synthesis inhibitor, and aminophylline, an inhibitor of phosphodiesterase, on 5-methoxytryptophol content in the chick pineal gland and to compare this with the drugs' action on pineal melatonin production. Inhibition of melatonin biosynthesis by cycloheximide (1 mg/kg, i.p. ), revealed by a marked reduction in the nighttime activity of serotonin N-acetyltransferase (AA-NAT; a key regulatory enzyme in melatonin synthesis) and melatonin concentrations, was accompanied by a significant increase in 5-methoxytryptophol content. In contrast, administration of aminophylline (100 mg/kg, i.p.) to light-exposed chicks significantly increased pineal AA-NAT activity and melatonin levels and decreased 5-methoxytryptophol concentrations. It is concluded that in the chick the production of pineal 5-methoxytryptophol and melatonin is inversely correlated.

Near-ultraviolet radiation suppresses melatonin synthesis in the chicken retina: a role of dopamine.

Effects of near-ultraviolet radiation (UV-A; 325-390 nm, peak at 365 nm) on melatonin content and activity of serotonin N-acetyltransferase (AA-NAT; a key regulatory enzyme in melatonin biosynthesis) were examined in the retina of chickens. Acute exposure of dark-adapted animals to UV-A light produced a marked decline in melatonin content and AA-NAT activity of the retina. The magnitude of the observed changes was dependent upon duration of the light pulse and age of chickens, with 1-2-week old birds being more sensitive to UV-A action than 6-7-week old ones. The decrease in the nocturnal AA-NAT activity evoked by a 5-min UV-A pulse gradually deepened during the first 30 min after the return of chickens to constant darkness, then the enzyme activity began to rise, reaching nearly complete restoration within 2.5 hr. Systemic administration to chickens of alpha-methyl-p-tyrosine (an inhibitor of catecholamine synthesis; 0.3 g/kg) blocked the suppressive effect of UV-A light on retinal AA-NAT activity. Haloperidol, sulpiride (blockers of D2-family of dopamine (DA) receptors) and 2-chloro-11-(4-methylpiperazino)dibenz[b,f]oxepin (an antagonist of D4-DA receptors), given intraocularly (1-100 nmol/eye) prevented the UV-A light-evoked decrease in AA-NAT activity in the chicken retina in a dose-dependent manner, while raclopride (300 nmol/eye), an antagonist of D2/D3-DA receptors, was ineffective. In dark-adapted chickens exposure to UV-A light increased the DA content of the retina. It is concluded that UV-A radiation, similar to visible light, potently suppresses melatonin biosynthesis in the retina of chicken, with a D4-dopaminergic signal playing the role of an intermediate in this action.

Hydroxyl radical reaction with melatonin molecule: a computational study.

Theoretical calculations of the HO* radical reaction with the melatonin molecule were performed. Reaction pathways with C2, C3, C4, C6 and C7 as the target carbon atoms and corresponding radical adducts were studied. Low activation energies of all adducts suggest that these reactions should occur quite easily and with rather low selectivity. C2 carbon as the most probable site of attack and C3 as the least probable one are proposed.

[Cascade of biochemical events triggered by stimulation of adrenergic receptors in the rat pineal gland--from cell membrane to nucleus]. / Kaskada procesów biochemicznych uruchamiana przez pobudzenie receptorów adrenergicznych w szyszynce szczura--przeplyw sygnalu od blony komórkowej do jadra komórkowego.

Pineal glands of various vertebrate species synthesize melatonin in a circadian rhythm generated by an endogenous pacemaker. The levels of melatonin and activity of serotonin N-acetyltransferase (AA-NAT: a penultimate and key regulatory enzyme in melatonin biosynthesis) are low during the light phase and high during the dark phase of any natural or imposed light-dark illumination cycle. The expression of AA-NAT gene in rat pineal gland is regulated by a photoneural system that acts through the adrenergic-cAMP-related mechanisms in pinealocytes. Concomitant stimulation by noradrenaline of beta 1- and alpha 1-adrenergic receptors, in a mechanism of "AND gate" activation, results in a large, 60-100-fold increase in intrapinealocyte cAMP level. The role of cAMP-dependent transcription factors CREB, ICER and Fra-2 in turning on and off the AA-NAT gene expression is discussed.

The effects of near-ultraviolet light on serotonin N-acetyltransferase activity in the chick pineal gland.

Effects of near-ultraviolet light (UV-A; 325-390 nm, peak at 365 nm) on the activity of the pineal serotonin N-acetyltransferase (NAT; a key regulatory enzyme in melatonin biosynthesis) were examined in chicks. Acute exposure of dark-adapted animals to UV-A radiation produced a marked decline in NAT activity of the pineal gland. The magnitude of this suppression was dependent upon duration of the light pulse and the age of the animals. The decrease in the nighttime NAT activity evoked by a 5 min pulse of UV-A light applied during the fourth hour of the dark phase of the 12 hr light:12 hr dark cycle (LD) gradually deepened during the first 40 min after the return of animals to constant darkness, then the enzyme activity began to rise, reaching control values by 2 hr. Exposure of chicks to a 5 min pulse of UV-A light during the ninth hour of the dark phase produced a marked decline in pineal NAT activity, which was reversible after 15 min of darkness. Pretreatment of animals with an inhibitor of catecholamine synthesis, alpha-methyl-p-tyrosine (300 mg/kg, i.p.), or with a blocker of alpha2-adrenergic receptors, yohimbine (2 mg/kg, i.p.), antagonized the suppressive effect of UV-A light on nighttime NAT activity of the chick pineal gland. It is concluded that UV-A irradiation, similar to visible light, potently suppresses melatonin biosynthesis in the chick pineal gland, with an alpha2-noradrenergic signal playing the role of an intermediate in this action.

Effects of near-ultraviolet (UV-A) light on melatonin biosynthesis in vertebrate pineal gland.

The effects of near-ultraviolet (UV-A) irradiation on nocturnal activity of serotonin N-acetyltransferase (NAT; a key regulatory enzyme in melatonin biosynthesis) in the pineal gland of the rat and chick were investigated. Exposure of the animals to UV-A during the 4th or 5th hour of the dark phase of the 12:12 h light-dark (LD) cycle suppressed the night-driven NAT activity in a time-dependent manner, the effects being generally more pronounced in rats than in chicks. The UV-A-evoked suppression of the nocturnal NAT activity was completely restored within 2 h (chicks) or 3 h (rats) in animals which, after irradiation, were returned to darkness. When a short UV-A pulse was applied to the animals after midnight, it induced a decrease in the enzyme activity in both species; yet, the effect was readily reversible only in chicks. The results presented here, as well as other data, demonstrate that UV-A light is a powerful signal affecting the pineal melatonin-generating system both in mammals and avians, and that the involved mechanisms may differ in the tested species.

Effects of near-ultraviolet light on the nocturnal serotonin N-acetyltransferase activity of rat pineal gland.

Effects of near-ultraviolet (UV-A; 325-390 nm, peak at 365 nm) light on the activity of the pineal serotonin N-acetyltransferase (NAT; a penultimate and key regulatory enzyme in melatonin biosynthesis) were examined in rats. Acute exposure of dark-adapted animals to UV-A radiation produced a marked suppression of NAT activity of the pineal gland, the effect being dependent on exposure time. The decrease in the night-time NAT activity evoked by a 1-min pulse of UV-A light (as well as by a 15-s pulse of broad-band visible light) gradually deepened during the first 40 min of treatment of animals with constant darkness, then the enzyme activity began to rise reaching control values by 3 h. Treatment of rats with a protein synthesis inhibitor, cycloheximide, attenuated this night-driven reactivation of the pineal NAT activity. The presented results provide evidence that UV-A light is a powerful signal capable of controlling melatonin biosynthesis in rat pineal gland.

Suppressive effect of NMDA receptor antagonist MK-801 on nocturnal serotonin N-acetyltransferase activity in the rat pineal gland.

Treatment of rats at night with a non-competitive NMDA receptor antagonist MK-801 (0.025-1 mg/kg, i.p.) produced a dose-dependent decrease in the nocturnal activity of the pineal serotonin N-acetyltransferase (NAT), the rate limiting enzyme in melatonin biosynthesis. A maximal inhibition (by 67-75%) of the enzyme activity was observed after the drug doses of 0.1 mg/kg in female rats, and 1 mg/kg in male animals. The data suggest that the NMDA receptor-mediated glutamatergic neurotransmission is a step necessary for NAT induction (and melatonin biosynthesis) in the mammalian pineal gland.

[Analysis of seven year prophylactic examinations of uterine cervix carcinoma in women living in the town of Kielce]. / Analiza siedmioletnich badan profilaktycznych raka szyjki macicy u kobiet zamieszkujacych teren miasta Kielce.

The authors are presenting an analysis of the prophylactic examinations carried out within the period of seven years among women under the care of the Municipal Health Care Center in Kielce. They are also trying to evaluate the influence such examinations have in forming up the detectability of preneoplastic states and cancer of the uterine cervix. The studies were carried out from 1982-1988. A rule was adopted to repeat the examinations every two years. Owing to organizing difficulties, the first stage of the examinations took three years. The basis of the prophylactic examinations was cytologic examination. The detectability of intraepithelial neoplasm of the uterine cervix (CIN) in the three stages was 0.26%, 0.16%, and 0.07%. It was observed therefore, the gradual drop in the number of the detectable CIN among the population in the duration of the studies. A "shift" of detectable neoplasma changes in the uterine cervix in the direction of a higher percentage of preinvasive cancers and with an early invasion was observed. This shift, beside the stable decrement of the absolute number of detectable cancers is a favourable phenomenon and testifies the great usefulness and the effectiveness of the studies.