Applicability of 2,4-dinitrophenylhydrazine (DNPH) method of protein oxidative damage measurement in the seminal plasma of canine ( Canis lupus familiaris) and stallion ( Equus caballus).

Seminal plasma (SP) proteins are responsible for sperm functional quality. Developing a reliable method to determine the degree of oxidative damage of these proteins is important for establishing semen fertilizing ability. The main aim of the study was to verify the applicability of protein carbonyl derivatives measurement in the SP of canine and stallion, using a method with 2,4-dinitrophenylhydrazine (DNPH). The research material consisted of ejaculates obtained from eight English Springer Spaniels, and from seven half-blood stallions during the breeding and non-breeding season. The content of carbonyl groups in the SP was measured on the basis of the reactions with DNPH. The following reagent variants were used to dissolve protein precipitates: Variant 1 (V1) - 6M Guanidine solution and Variant 2 (V2) - 0.1M NaOH solution. It has been shown that to obtain reliable results for the measurement of protein carbonylated groups in the dog and horse SP, both 6M Guanidine and 0.1M NaOH may be used. A correlation was also found between the number of carbonyl groups and the total protein content in the canine (V1: r = -0.724; V2: r = -0.847) and stallion (V1: r = -0.336; V2: r = -0.334) SP. Additionally, the study showed a higher content (p≤0.05) of protein carbonyl groups in the stallion SP in the non-breeding season compared to the breeding season. The method based on the reaction with DNPH, due to its simplicity and cost effectiveness, appears to be suitable for large-scale application in the determination of the SP proteins oxidative damage in dog and horse semen.

Transcriptome analysis of boar spermatozoa with different freezability using RNA-Seq.

Semen freezability is associated with genetic markers, and there is a diverse set of sperm transcripts that have been attributed to various cellular functions. RNA-Seq was performed to compare the transcript profiles of spermatozoa from boars with different semen freezability. We examined ejaculates from the Polish large white (PLW) boars that were classified as having good and poor semen freezability (GSF and PSF, respectively; n = 3 boars per group) by assessing post-thaw motility characteristics, mitochondrial membrane potential, plasma membrane and acrosome integrity. Total RNA was isolated from fresh spermatozoa from boars of the GSF and PSF groups and subjected to RNA-Seq (Illumina NextSeq 500 platform). Transcript abundance was assessed with the DESeq2, DESeq, and EdgeR Bioconductor R packages, and varying numbers of differentially expressed gene (DEG) transcripts were detected in the spermatozoa of each boar. Using RNA-Seq, we identified several genes associated with inflammation and apoptosis (FOS, NFATC3, ITGAL, EAF2 and ZDHHC14), spermatogenesis (FGF-14 and BAMBI), autophagy (RAB33B), protein phosphorylation (PTPRU and PTPN2) and energy metabolism (ND6 and ACADM) that were predominantly up-regulated in poor freezability ejaculates. Quantitative reverse transcription polymerase chain reaction (RT-qPCR) validated the transcript expression levels detected by RNA-Seq and thus confirmed the reliability of this technique. Subsequent validation with western blotting showed that the expression of three proteins was in accordance with the transcript abundance. Overall, we demonstrated that the up-regulation of the DEG transcripts in spermatozoa was associated with poor semen freezability. We suggest that spermatozoa transcriptome profiling provides a foundation to further elucidate the relevance of sperm-related transcripts on cryo-survival. The sperm-related transcripts, namely FOS, NFATC3, EAF2, BAMBI, PTPRU, PTPN2, ND6 and ACADM, are potential markers for predicting the freezability of boar semen.

Identification of proteoforms of albumin and kallikrein in stallion seminal plasma and their correlations with sperm motility.

The aim of this study was to identify the proteoforms of albumin and kallikrein in stallion seminal plasma (SP), and to determine their correlations with sperm motility parameters. The experimental material consisted of ejaculates from 8 stallions, which were collected during the breeding and non-breeding seasons (BS and NBS, respectively). SP proteins were identified by 2-D PAGE and mass spectrometry (MALDI TOT/TOF MS). Sperm motility parameters were analyzed using the CASA system. Protein expression (integrated optical density-IOD) of albumin proteoforms 1 (ALB 1) and 2 (ALB 2) and kallikrein proteoforms 1 (KAL 1) and 2 (KAL 2) was correlated (p⟨0.05) with sperm motility parameters (total motility and progressive motility) during the BS. No significant correlations were found between the expression of albumin or kallikrein and sperm motility parameters during the NBS. The presence of correlations between the expression of ALB 1, ALB 2, KAL 1, KAL 2 and selected sperm motility parameters could suggest that the analyzed components of the SP belong to the group of fertility-associated proteins (FAPs).

Total RNA quality in boar spermatozoa with different freezability.

In this study the quality of total RNA, isolated from fresh spermatozoa, was compared between boars with good and poor semen freezability (GSF and PSF, respectively). Semen from 3 boars with GSF exhibited significantly higher total motility, mitochondrial function, plasma membrane integrity and reduced lipid peroxidation compared with 3 boars with PSF after cryo- preservation. There were variations in the quality of RNA isolated from spermatozoa of boars with GSF and PSF. Boars with GSF exhibited mainly full-length, intact RNA, whereas substantial amounts of degraded RNA were detected in spermatozoa from boars with PSF. Further under- standing of the biological relevance of RNAs in sperm function is critical to improve the freezabil- ity of boar semen.

Osteoglycin, a novel coordinator of bone and glucose homeostasis.

OBJECTIVE: The skeleton, which is strongly controlled by endocrine factors, has recently been shown to also play an active endocrine role itself, specifically influencing energy metabolism. However, much less is known about this role. Therefore, we sought to identify novel endocrine factors involved in the regulation of both bone mass and whole-body glucose homeostasis. METHODS: We used transcriptomic and proteomic analysis of Y1 receptor deficient osteoblasts combined with the generation of a novel osteoglycin deficient mouse model and performed comprehensive in vivo phenotype profiling, combined with osteoglycin administration in wildtype mice and human studies. RESULTS: Here we identify a novel role for osteoglycin, a secreted proteoglycan, in coordinating bone accretion with changes in energy balance. Using an osteoglycin knockout mouse model, we show that at a whole body level, osteoglycin acts to suppress bone formation and modulate whole body energy supplies by altering glucose uptake through changes in insulin secretion and sensitivity, as well as by altering food intake through central signaling. Examining humans following gastric surgery as a model of negative energy balance, we show that osteoglycin is associated with BMI and lean mass as well as changes in weight, BMI, and glucose levels. CONCLUSIONS: Thus, we identify osteoglycin as a novel factor involved in the regulation of energy homeostasis and identify a role for it in facilitating the matching of bone acquisition to alterations in energy status.

The benefits of cooling boar semen in long-term extenders prior to cryopreservation on sperm quality characteristics.

This study investigated the effects of long-term extenders on post-thaw sperm quality characteristics following different holding times (HT) of boar semen at 17 and 10°C. Sperm-rich fractions, collected from five boars, were diluted in Androhep(®) Plus (AHP), Androstar(®) Plus (ASP), Safecell(®) Plus and TRIXcell(®) Plus (TCP) extenders. The extended semen samples were held for 2 hr at 17°C (HT 1) and additionally for 24 hr at 10°C (HT 2), after they were evaluated and frozen. CASA sperm motility and motion patterns, mitochondrial membrane potential (MMP), plasma membrane integrity (PMI) and normal apical ridge (NAR) acrosome integrity were assessed in the pre-freeze and frozen-thawed semen. The Vybrant Apoptosis Assay Kit was used to analyse the proportions of viable and plasma membrane apoptotic-like changes in spermatozoa. Results indicated that boar variability, extender and HT significantly affected the sperm quality characteristics, particularly after freezing-thawing. Differences in the pre-freeze semen were more marked in the sperm motion patterns between the HTs. Pre-freeze semen in HT 2 showed significantly higher VCL and VAP, whereas no marked effects were observed in the sperm membrane integrity and viability (YO-PRO-1(-) /PI(-) ) among the extenders. Post-thaw sperm TMOT and PMOT were significantly higher in the AHP and ASP extenders of HT 2 group, whereas VSL, VCL and VAP were markedly lower in the TCP extender. Furthermore, spermatozoa from the AHP- and ASP-extended semen of HT 2 group were characterized by higher MMP, PMI and NAR acrosome integrity following freezing-thawing. In most of the extenders, the incidence of frozen-thawed spermatozoa with apoptotic-like changes was greater in HT 1. The findings of this study indicate that holding of boar semen at 10°C for 24 hr in long-term preservation extenders modulates post-thaw sperm quality characteristics in an extender-dependent manner. These results will further contribute to the improvement in the cryopreservation technology of boar semen.

Age and seasonal-dependent variations in the biochemical composition of boar semen.

This study investigated the effect of age- and seasonal-related variations in the composition of boar semen over a 3-year period. At the onset of 8 months of age, ejaculates were collected from four boars and allocated into three groups: 8 to 18, 19 to 30, and 31 to 42 months and were divided into two seasonal periods: autumn-winter and spring-summer. Boar variability had a significant effect on most of the analyzed semen parameters. Significantly, higher ejaculate volumes were observed in the boars older than 18 months of age during the autumn-winter period. Sperm concentration was higher in boars less than the age of 18 months of age, whereas the total sperm numbers were significantly higher during the autumn-winter period, regardless of the age group. Seasonal effects in sperm motility were more marked in boars at the age of 19 to 30 months, being significantly higher during the autumn-winter period. The proportions of spermatozoa with intact, normal apical ridge acrosome, and osmotically tolerant acrosomal membranes were markedly higher in boars at the age of 19 to 30 months during the autumn-winter period. Spermatozoa harvested during the spring-summer period were more susceptible to lipid peroxidation, irrespective of the age group. Significantly, higher levels of protein content and concentrations of nonthiol-containing antioxidant components of the seminal plasma (SP) were detected in boars less than 18 months of age during the autumn-winter period. Seasonal effects on the pH and proteinase inhibitory activity in the SP were more marked in boars less than 18 months of age, whereas alkaline phosphatase activity was greater in boars at the age of 19 to 30 months during the autumn-winter period. Substantial amounts of the thiol-containing antioxidants of the SP were detected in boars older than 18 months of age during the spring-summer period. Neither osmolality nor total antioxidant status was affected by differences in the seasonal periods or age groups. The findings of this study indicate that age- and seasonal-related variations affect the reproductive tract functions in the boar, resulting in marked changes in the biochemical composition of the semen.

A new look at the shoulder anterior capsuloligamentous complex complementing the insertion of the subscapularis tendon-Anatomical, histological and ultrasound studies of the lesser tuberosity enthesis.

PURPOSE: The aim of this study was to examine the relation between the anterior capsuloligamentous complex (ACLC - recognized as a single structure composed of the anterior capsule and its ligaments: medial glenohumeral ligament and anterior bundle of inferior glenohumeral ligament) and the subscapularis tendon with their footprints on the lesser tuberosity. METHODS: In this study, 19 fresh cadaveric shoulder specimens were examined: 13 in morphometric measurements and 6 were examined in a histological study. The subscapularis tendon and the ACLC were dissected until their insertion onto lesser tuberosity. Measurements of both dissected structures and their footprints on the lesser tuberosity were taken with a standard caliper. Six shoulders for histological examination were dissected without separation of the subscapularis tendon from the ACLC and longitudinal and transverse section samples were taken. Additionally, two of these six shoulders underwent ultrasound assessment before final sample preparation. RESULTS: Two well-isolated structures were clearly identifiable: muscle with its tendinous chords and the ACLC, forming together the anterior wall of the joint. The ACLC insertion complemented the tendon insertion - superiorly the thickest part of the tendon stayed in contact with the thinnest part of the ACLC and inferiorly the relation was opposite - the ACLC insertion reached its maximum transverse length. This reciprocal relation was similar to superior and posterior rotator cuff tendon-capsule complex. The footprint on the lesser tuberosity, being purely fibrocartilage enthesis, was composed in 45% of the ACLC insertion. Also the fibers run of the tendon and the ACLC were different: histologic assessment confirmed the tight fusion of the tendon and the ACLC, though those two layers were easily identified as their fibers never mixed. CLINICAL RELEVANCE: Better understanding of the anterior shoulder compartment anatomy simplifies an understanding of some arthroscopic procedures. The shape of the footprint, an importance of a broad superior part of the subscapularis tendon and its relation to the ACLC could be an anatomic proof and explanation for already existing surgical activities: subscapularis release and repair and soft tissue or bony procedures (Latarjet) in anterior shoulder instability. We declare that the experiments comply with the current law of the country in which they were performed (i.e. Polish law).

Proteomic Characterization of Zinc-Binding Proteins of Canine Seminal Plasma.

The zinc-binding proteins (ZnBPs) of the seminal plasma are implicated in different processes related to sperm-egg fusion. The aim of this study was to characterize the ZnBPs of canine seminal plasma using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and mass spectrometry. The ZnBPs were isolated from the ejaculates of five dogs by affinity chromatography and subjected to 2D-PAGE analysis. The acquired spots, detected across the gels, were analysed by mass spectrometry. Using 2D-PAGE analysis, it was shown that canine seminal plasma comprised about 46-57 zinc-binding polypeptides, with molecular mass ranging from 9.3 to 138.7 kDa and pI at pH 5.2-10.0. It was found that zinc-binding polypeptides of low molecular masses (9.3-19.0 kDa and pI at pH 6.1-10.0) were predominant in the seminal plasma, and seven polypeptides, with molecular masses ranging from 11.7 to 15.4 kDa and pI at pH 6.8-8.7, were characterized by high optical density values. In addition, analysis with mass spectrometry (LC-MS-MS/MS) revealed that the identified seven polypeptides are canine prostate-specific esterase (CPSE), which is the main proteolytic enzyme of the seminal plasma. The findings of this study indicate an important regulatory role of seminal plasma zinc ions in the functional activity of CPSE, which is of great significance for maintaining the normal function of canine prostate and the spermatozoa functions.

Prevalence of ankylosing spondylitis in Poland and costs generated by AS patients in the public healthcare system.

The aim of the research was to analyse the prevalence of ankylosing spondylitis (AS) in Poland and to assess the costs generated by AS patients in the system of public health care. The database of national payer-National Health Fund (NHF)-has been analysed. For the analysis, the information has been extracted from IT system about each treated patient with ICD-10 = M45 code as main or coexisting diagnosis included in the mandatory reports from entities of public healthcare service. In the years 2008-2013, from 28,800 to 32,800 persons diagnosed with AS as main or coexisting diagnosis have been registered in NHF database. In 2013, the prevalence amounted to 7.48 for 10,000 persons of general population-the highest in Kujawy-Pomerania province (10.92 per 10,000 inhabitants), Silesia (10.04) and Swietokrzyskie province (9.81). In male patients, this coefficient amounted to 8.91 per 10,000 inhabitants, whereas in female-to 6.15. In 2013, the prevalence for men was the highest in the age group of 60-64 years and for women in the group of 65-69 years. The healthcare expenses related to AS financed by the NHF increased from 13,200 million PLN (6.3 million USD) in 2008 to 72,600 million PLN (21,900 million USD) in 2013. The increase in healthcare expenses related to AS patients in the public healthcare system is significant and noticeable. High regional diversity is also a vital issue. It is necessary to carry out further research on the incidence rate of AS in Polish population.

Prostasomes of canine seminal plasma - zinc-binding ability and effects on motility characteristics and plasma membrane integrity of spermatozoa.

Prostasomes are small lipid membrane-confined vesicles that are involved in various fertilization-related processes. The aim of this study was to demonstrate canine seminal plasma prostasomes' ability to bind zinc ions, as well as examining their effects on sperm motility characteristics and plasma membrane integrity during cold storage. Ejaculates, collected from five cross-bred dogs (n = 50), were subjected to ultracentrifugation followed by gel filtration (GF) on a Superose 6 column. Prostasomes appeared as a single fraction in the elution profile. Transmission electron microscopy (TEM) analysis of canine prostasomes revealed the presence of membrane vesicles with diameters ranging from 20.3 to 301 nm. The zinc-affinity chromatography on a Chelating Sepharose Fast Flow - Zn(2 +) showed that from 93 to 100% of the prostasome proteins bind zinc ions (P(+) Zn). SDS-PAGE revealed that canine P(+) Zn comprised four protein bands, with low molecular weights (10.2-12 kDa). We have also shown a positive effect of prostasomes (p < 0.05), especially variant B (2% of total seminal plasma protein) on canine sperm motility parameters after 2 h storage at 5°C (TMOT%, 44.75 ± 5.18) and PMOT%, 12.42 ± 1.59) and VAP, VSL, VCL, when compared with Control (TMOT%, 7.30 ± 1.41 and PMOT%, 1.70 ± 0.42). Higher percentage of spermatozoa with intact plasma membrane (SYBR/PI dual staining) and intact acrosome (Giemsa stained), after 2 h storage at 5°C, was showed, in variant A (1.5% of total seminal plasma protein) and B, when compared with Control and variant C (2.5% of total seminal plasma protein). The prostasomes' effect on motility and plasma membrane integrity of canine cold-stored spermatozoa may be related to their ability to bind zinc ions and regulate their availability to the sperm.

Effects of storage in different semen extenders on the pre-freezing and post-thawing quality of boar spermatozoa.

The aim of this study was to investigate the effects of storage of semen in different commercial extenders on the pre-freezing and post-thawing quality of boar spermatozoa. Semen was diluted in BTS, Androhep (AH) and Gedil (GD), stored for 24 h at 17°C, and then frozen in accordance with the cryopreservation protocol. Analyses of the quality of spermatozoa included: motility, normal apical ridge (NAR) acrosome, plasma membrane integrity (PMI), mitochondrial membrane potential (MMP), measurements of ATP content and activity of superoxidase dismutase (SOD) and glutathione peroxidase (GPx). Prior to the freezing process, no significant effect of the extender on the sperm quality parameters was noted. After thawing the spermatozoa it was demonstrated that the type of extender used influenced PMI, MMP, ATP content and activity of GPx. In the AH extender the percentage of spermatozoa with PMI and ATP content in spermatozoa was significantly higher (P<0.05) as compared to the BTS or GD extenders. In addition, semen stored in the AH was characterised by a statistically higher (P<0.05) percentage of spermatozoa with MMP and increased activity of GPx as compared with the BTS. The results obtained indicate that for the cryopreservation process, boar spermatozoa stored for 24 hours in liquid state can be used. However, the type of extender used prior to freezing may have a significant effect on the post-thawing quality of the spermatozoa. The AH extender better secured the quality of thawed boar spermatozoa as compared with the BTS or GD.

Semen quality assessments and their significance in reproductive technology.

Semen quality assessment methods are very important in predicting the fertilizing ability of persevered spermatozoa and to improve animal reproductive technology. This review discusses some of the current laboratory methods used for semen quality assessments, with references to their relevance in the evaluation of male fertility and semen preservation technologies. Semen quality assessment methods include sperm motility evaluations, analyzed with the computer-assisted semen analysis (CASA) system, and plasma membrane integrity evaluations using fluorescent stains, such as Hoechst 33258 (H33258), SYBR-14, propidium iodide (PI), ethidium homodimer (EthD) and 6-carboxyfluorescein diacetate (CFDA), and biochemical tests, such as the measurement of malondialdehyde (MDA) level. This review addresses the significance of specific fluorochromes and ATP measurements for the evaluation of the sperm mitochondrial status. Laboratory methods used for the evaluation of chromatin status, DNA integrity, and apoptotic changes in spermatozoa have been discussed. Special emphasis has been focused on the application of proteomic techniques, such as two-dimensional (2-D) gel electrophoresis and liquid chromatography mass spectrometry (LC-MS/MS), for the identification of the properties and functions of seminal plasma proteins in order to define their role in the fertilization-related processes.

Bariatric surgery, bone loss, obesity and possible mechanisms.

Bariatric surgery remains the most effective treatment for severely obese patients. However, the potential long-term effects of bariatric surgical procedures on health, including bone health, are only partially understood. The goal of this review was to present data on the impact of bariatric surgery on bone metabolism and to analyse possible reasons for the loss of bone mass that frequently occurs after bariatric surgery. Such factors include nutritional deficiencies, rapid weight loss per se, effects of fat-derived adipokines and gut-derived appetite-regulatory hormones. However, the relative roles of these factors in skeletal regulation and the mechanisms by which they work are not yet fully defined. Our review was focussed on the complex relationship between body weight, fat mass and bone mass, as well as peripheral and central mediators potentially involved in the dual regulation of both energy and bone homeostasis. We also review the data on the inverse relationship between central obesity, bone marrow fat and osteoporosis. As the number of bariatric operations increases, it is imperative to recognize mechanisms responsible for bariatric surgery-induced bone loss, with careful monitoring of bone health including long-term fracture incidence in patients undergoing these procedures.

Isolation and characterization of zinc-binding proteins of canine seminal plasma.

Zinc-binding proteins from seminal plasma (ZnBPs) originate in the secretions of different accessory sex glands and are implicated in key events associated with sperm-egg fertilization processes. This study describes the isolation and characterization of the ZnBPs of canine seminal plasma. Ejaculates were collected from three crossbred dogs for a 2-week period. The ZnBPs as well as non zinc-binding proteins (nZnBPs) were isolated by zinc-dependent affinity chromatography. The isolated fractions were subjected to native gel electrophoresis (one-dimensional polyacryamide gel electrophoresis, PAGE) and sodium dodecyl sulphate polyacryamide gel electrophoresis (SDS-PAGE), using denaturing and reducing conditions. Zinc-elution profile using affinity chromatography displayed two protein fractions represented by the nZnBPs and ZnBPs, respectively. Using native gel electrophoresis, it was found that both the nZnBPs and ZnBPs occurred in their native state as aggregates, ranging from 140 to 669 kDa. The nZnBPs were disaggregated into 8 protein bands, with molecular weights ranging from 10.7 to 79.7 kDa, following SDS-PAGE analysis. By contrast, SDS-PAGE analysis of the ZnBPs revealed 13 protein bands, with molecular weights ranging from 11.6 to 152.3 kDa. Densitometric analysis showed that 46-48% of nZnBPs could be accounted by protein fractions with molecular weights of 10.7 and 14.2 kDa. Also, 2 protein fractions with molecular weights of 11.6 and 14.3 kDa, were predominant in ZnBPs, accounting for approximately 28-30% of the total proteins. These results demonstrate the zinc-binding capacity of proteins secreted by the canine prostate. The findings of this study indicate that ZnBPs of canine seminal plasma comprise several protein fractions, which might be implicated in the reproductive processes in the dog.

Characteristics of selected seminal plasma proteins and their application in the improvement of the reproductive processes in mammals.

Understanding the biochemical processes associated with ovum fertilization and knowledge about the structure and function of individual substances participating in these processes is crucial for the development of biotechnological methods to improve reproduction of animals and humans. Among many components of seminal plasma, proteins and peptides play a specific role in regulation of the fertilization process, particularly through their ability to bind various types of ligands such as polysaccharides, lipids and ions. Heparin-binding proteins regulate capacitation and acrosome reaction processes. Affinity of plasma proteins to mannans of the fallopian tube epithelium facilitates formation of spermatozoa reservoirs in the female reproductive tract. Ability to bind phosphorylcholine is one of the conditions for the coating of the seminal plasma proteins on the sperm membrane and also determines the formation of oligomeric forms of certain proteins. Zinc binding by seminal plasma proteins regulates sperm chromatin condensation state. It also affects motility of these cells and acrosome reaction. The interspecies analysis indicates significant structural and functional similarities, especially for the proteins with low molecular weight. Fertility associated proteins (FAPs) have been determined in the bull, stallion, boar, ram and dog. The contents of these proteins correlate with the indicators of the fertilizing abilities of sperm. In humans, several seminal plasma proteins were found which serve as diagnostic markers of spermatogenesis, seminiferous epithelium state, and azoospermia. To determine the semen ability for preservation, measurement of some seminal plasma protein content may also be used. Addition of specific plasma proteins to a spermatozoa solution undergoing the process of preservation may be used to retain the features of the cells responsible for efficient fertilization.

Hypercalcaemia caused by a carcinoid tumour.

Humoral hypercalcaemia resulting from carcinoid tumours is uncommon. We report a case of hypercalcaemia because of excessive secretion of parathyroid hormone-related protein (PTHrP) in a 77-year-old woman with an advanced carcinoid tumour. Fibroblast growth factor 23 levels were also elevated. The hypercalcaemia responded to adjunctive therapy with long-acting octreotide analogue therapy, bisphosphonates and steroids. The role of PTHrP in humoral hypercalcaemia of malignancy, its association with neuroendocrine tumours, as well as the therapeutic use of somatostatin analogues are reviewed.

Gas chromatographic investigation of phenylpropenoid glycerides from aspen (Populus tremula L.) buds.

20 phenylpropenoid glycerides were synthesized by esterification of glycerol by p-coumaric, ferulic and caffeic acids. Main diagnostic ions in mass spectra and linear temperature programmed retention indices (LTPRI) of trimethylsilyl derivatives of these compounds were determined by GC/MS analysis. On the basis of these analytical parameters 39 in various degree substituted phenylpropenoid glycerides were for the first time identified in diethyl ether and ethyl acetate extracts from aspen buds.

In favour or against euthanasia--dentistry students' opinion.

Euthanasia and a doctor-assisted suicide are not only practiced worldwide, but also legalized in some countries. In Poland they are broadly discussed. The purpose of this paper was to define dentistry students attitude towards human right to ask for death and euthanasia. In the research 148 students of both sexes were questioned. Generally students were against euthanasia in the world as well as in Poland, although men were in majority for euthanasia. Believers were against it due to ethically-religious reasons, while non-believers were for euthanasia motivating it by rational reasons.