Loggerhead sea turtle Caretta caretta plasma biochemistry and proteome profile modulation during recovery.

The aim of the study was to monitor and analyse injured and diseased loggerhead sea turtles (Caretta caretta) plasma proteome profiles and biochemistry parameters during their recovery period in rescue centre within different age and recovery period groups, and determine the potential biomarkers that can be used in diagnostics. The plasma biochemical parameters of total protein and glucose content, accompanied by aspartate aminotransferase (AST) and N-acetyl-cystein-activated creatinine kinase (CK-NAC) are highlighted as valuable and potential biomarkers of turtle's health status and condition. Using high throughput tandem mass tag (TMT)-based proteomic approach we identified 913 plasma proteins, 12 of which shown to be modulated in loggerheads age groups, and identified as a part of (i) platelet degranulation, (ii) neutrophil degranulation, and (iii) innate immune system pathways. The neurofascin (NFASC) is shown to be differentially abundant among all the age groups, and alpha-1-acid glycoprotein 2-like (ORM2) and alpha-1-antitrypsin-like (SERPINA1) proteins were recognized as members of all three above mentioned REACTOME pathways. Furthermore, 29 of plasma proteins were significantly differentially abundant in loggerheads age and recovery period groups. Out of 15 recognized pathways, those proteins were mostly included in three specific REACTOME pathways: (i) post-translational phosphorylation, (ii) regulation of Insulin-like Growth Factor (IGF) transport and uptake by Insulin-like Growth Factor Binding Proteins (IGFBPs), and (iii) platelet degranulation. The alpha-fetoprotein (AFP) was the only protein which showed statistically significant up-regulation patterns in all loggerhead age groups before release from the rescue centre, and the complement component 3 (C3) protein was the only protein modulated in all recovery period groups. Furthermore, C3 protein takes part in 9; and followed up with apolipoprotein A-I (APOA1) in 7; complement component 4 (C4), complement component 5 (C5) and kininogen-1 (KNG1) in 6 REACTOME pathways. Thereby, those proteins are highlighted and recommended as potential biomarkers of turtle's health status. Data are available via ProteomeXchange with identifier PXD029569. Finally, based on our results, we believe that comprehensive omics approach and routine plasma biochemical analysis, accompanied by proteins of acute phase, acid-base status and immune-response indicator analysis may significantly and reliably improve assessment of captive loggerheads rehabilitation and medication. SIGNIFICANCE: Monitoring and comparison of loggerhead sea turtles (C. caretta) blood plasma biochemistry parameters and plasma proteome profiles in relation to the age, and recovery period pointed out significantly differentially abundant proteins, along with certain biochemical parameter contents as potential biomarkers of turtle's fitness, health status and physiology.

Somatostatin-14 alters the thymus size and relation among the thymocyte subpopulations in peripubertal rats.

It is well known that somatostatin exerts a wide range of effects in the body, and acts as an autocrine or paracrine factor in the thymus. However, it has not been investigated yet whether somatostatin alters the thymus size and relation among the thymocyte subpopulations in the peripubertal rats. For this purpose, the peripubertal AO male rats were cannulated intracerebroventriculary and treated with repeated, low doses of somatostatin-14 (experimental group) or saline (control group). Twenty-four hours after the last treatment, we removed and prepared the thymuses for determination of thymocyte subpopulations by flow cytometry. After five days, animals were sacrificed and their thymuses taken for morphometrical analysis by stereological methods. We noticed that somatostatin-14 decreased volumes of thymus cortex and medulla, total number of thymocytes, number of thymocytes in the cortex and medulla and numerical density of thymocytes in deeper cortex. As a consequence of these changes, thymus size was also diminished. The phenotypic analysis of thymocyte subpopulations showed that somatostatin-14 decreased the percentage of CD4(+)CD8(+) cells with low level of TCR alphabeta expression, positively selected CD4(+)CD8(+)TCRalphabeta (high) cells and the most mature CD4(-)CD8(+)TCRalphabeta (high) cells, while the percentage of CD4(+)CD8(-)TCRalphabeta (high) thymocytes was slightly increased. Somatostatin-14 increased the relative proportion of the least mature CD4(-)CD8(-)TCRalphabeta (-/low), CD4(+)CD8(+)TCRalphabeta (-) cells and both of TCRalphabeta (-/low) single positive subpopulations. These results show that centrally applied somatostatin-14, induces hypotrophy of the thymus in peripubertal rats by changing the volumes and cellularities of the thymic compartments. Additionally, increased number of the least mature thymocytes and a deficiency of double positive cells indicate the involvement of somatostatin in the modulation of T cells maturation.

Induction of apoptosis in the blue mussel Mytilus galloprovincialis by tri-n-butyltin chloride.

Induction of apoptosis by tri-n-butyltin (TBT) in gill tissue of the mussel Mytilus galloprovincialis was investigated. The terminal dUTP nick-end labeling technique (TUNEL) was used to detect cells displaying DNA fragmentation within gill structures. Genomic DNA fragmentation was detected as characteristically ladder-like pattern of DNA fragments induced by single injection of different doses of TBT (1-5 microg/g) below the mantle, directly into the pallial fluid, after 24 h of incubation. DNA degradation of higher order DNA structure, as well as reduced G(0)/G(1) cell cycle region (the sub-G(1) region) was detectable after 1.5 h of TBT incubation. Presence of apoptotic cells in mussels' gills was indicated by the selective loss of G(2)/M cells concomitant with the appearance of cells with decreased DNA content in S and G(0)/G(1) cell cycle regions. The effect of the TBT on cell cycle in a mussel gill was a dose related and exposure time depending. The possible mechanism of induction of apoptosis in vivo in gill tissue of mussel treated with TBT is discussed.

Topographical characterization and surface force spectroscopy of the photochemical lignin model compound.

By combining the results from atomic force microscopy (AFM) and environmental scanning electron microscopy (ESEM), herein we investigate properties of photochemical lignin model compounds. We provide evidence that photochemical lignin forms random, probably non-functional structures. The topography of such structures is explored using ESEM. Non-functionality of such structures is proved by AFM and atomic force spectroscopy experiments wherein the photochemical lignin functionalized tip is approached to the substrate covered with photochemical lignin. There was no evidence of existence of any kind of host-guest interaction during the approach/retraction experiments. These results provide evidence for our previously stated hypothesis that photochemical lignin polymerization may be one of the degrading effects of UV radiation to the plant cell.

A Comparative Study of Enzymatically and Photochemically Polymerized Artificial Lignin Supramolecular Structures Using Environmental Scanning Electron Microscopy.

Environmental scanning electron microscopy images of the self-assembled structures of enzymatically (DHP) and photochemically polymerized (PCP) artificial lignin are herein presented. Differences in the structural organization between DHP and PCP polymer at the supramolecular level were reported. Based on topological information, we proposed a hypothesis about possible new physiological roles of lignin in live plant cells and the ecological significance of possible in-vivo photochemical lignin polymerization. Copyright 2000 Academic Press.

Visualization of artificial lignin supramolecular structures

In this paper we are presenting the results of our environmental scanning electron microscopy (ESEM) investigation of the lignin model compound--enzymatically polymerized coniferyl alcohol, also known as dehydrogenate polymer (DHP). The goals of this study were to visualize the supramolecular organization of DHP polymer on various substrates, namely graphite, mica, and glass, and to explore the influence of substrate surface properties and associated collective phenomena on the lignin self-assembled supramolecular structure. Based on results obtained with ESEM, combined with previously published results based on scanning tunneling microscopy (STM) and electron spin resonance (ESR) technique, we looked at lignin structure ranging from a monomer on a fraction of nanometer scale to a large aggregate on a fraction of millimeter scale, therefore using six orders of magnitude range of size. Herein, we are presenting evidence that there are at least four different levels of the supramolecular structure of lignin, and that its supramolecular organization is well dependent on the substrate surface characteristics, such as hydrophobicity, delocalized orbitals, and surface-free energy.

Castration of sexually immature rats affects sympathetic innervation of the adult thymus.

It has been hypothesized that maturational processes within the hypothalamo-pituitary-gonadal (HPG) axis and thymus are reciprocally regulated via neural pathways. To test this hypothesis, in the thymi of adult rats orchidectomized (ORX) at age of 1 (ORX-1), 7 (ORX-7) and 30 days (ORX-30): (i) noradrenaline (NA), dopamine (DA) and serotonin (5-HT) contents and acetylcholinesterase (AChE) activity were measured and (ii) the distribution of monoamine- and AChE-containing nerves and cells was examined by a sucrose phosphate glyoxylic acid (SPG) method and enzyme histochemistry, respectively. In all groups of ORX rats, the thymus weight was significantly increased over that in sham-ORX control rats. In the ORX-1 rats, the increase in the thymus weight was accompanied by a proportional increase in the content of both catecholamines and 5-HT; consequently the concentration of each of them remained unaltered. In these animals, the density of both SPG-stained thymus nerve fibers and cells also remained unchanged. In the ORX-7 rats, the rise in the thymus weight was followed by a proportional increase in the content of all monoamines, except for NA which was reduced. Therefore, in these rats neither the thymus concentrations of DA nor that of 5-HT differed from controls, while the concentration of NA was significantly decreased. The reduction in both NA content and concentration reflected a diminished density of SPG-positive nerve profiles. In the ORX-30 rats, the increase in thymus weight was neither paralleled by a proportional increase in the DA content nor in 5-HT, while the content of NA was decreased. Thus, in their thymi the concentration of both NA and DA, as well as that of 5-HT, were significantly reduced. In parallel with these changes, a decreased density of thymic SPG-positive nerve fibers and cells was found. In all ORX rats, the pattern of intrathymic distribution of SPG-positive fibers and cells remained unchanged. Orchidectomy affected neither the activity of AChE (expressed per gram of tissue) nor the density of AChE-positive nerves and cells in the thymus. As the changes in the density of adrenergic nerve fibers in the thymus from ORX rats were not followed by similar alterations in the density of AChE-containing nerve fibers, it does not seem likely that NA and AChE are colocalized in the thymus nerve fibers. The results also suggest that there is a critical period during ontogenesis when changes within the HPG axis evoked by orchidectomy can affect the sympathetic nerve input to the rat thymus and therefore, most likely, development and function of the organ.

Effects of estradiol and calcium on gonadotrophic cells in middle-aged female rats.

The effects of multiple treatment with estradiol dipropionate (EDP) or calcium glucoheptonate (Ca) or a combination of the two on gonadotrophic cells in the pituitary pars distalis of middle-aged female rats were examined. The animals were treated daily for two weeks with EDP (0.625 mg i.p./kg body weight) or Ca (11.4 mg/kg body weight) or EDP + Ca. Luteinising (LH) and follicle stimulating hormone (FSH)-producing cells were examined by immunohistochemistry using antisera to the specific (beta) beta-subunits of LH and FSH and a peroxidase-anti-peroxidase immunohistochemical procedure. Plasma levels of FSH and LH were measured by radio-immune assay. A stereological method for determining morphometric parameters in immunopositive FSH and LH cells was used. The number of gonadotrophs per unit area (mm2), their cellular volume and relative volume densities, as well as plasma levels of FSH and LH, were decreased in all treated females in comparison with the controls. The most significant decrease of these parameters was observed in EDP-treated animals. Such changes were also expressed in Ca-treated animals, but the alterations were less distinct. These results demonstrate that multiple EDP or Ca application to middle-aged female rats is able to inhibit, directly or indirectly, the morphofunctional state of gonadotrophic cells in the pituitary pars distalis.

Scanning electron microscopy studies of protein-functionalized atomic force microscopy cantilever tips.

Protein-functionalized atomic force microscopy (AFM) tips have been used to investigate the interaction of individual ligand-receptor complexes. Herein we present results from scanning electron microscopy (SEM) studies of protein-functionalized AFM cantilever tips. The goals of this study were (1) to examine the surface morphology of protein-coated AFM tips and (2) to determine the stability of the coated tips. Based on SEM images, we found that bovine serum albumin (BSA) in solution spontaneously adsorbed onto the surface of silicon nitride cantilevers, forming a uniform protein layer over the surface. Additional protein layers deposited over the initial BSA-coated surface did not significantly alter the surface morphology. However, we found that avidin-functionalized tips were contaminated with debris after a series of force measurements with biotinylated agarose beads. The bound debris presumably originated from the transfer of material from the agarose bead. This observation is consistent with the observed deterioration of functional activity as measured in ligand-receptor binding force experiments.

Orchidectomy affects the innervation of the rat thymus.

To assess a putative role of the neural pathways in transfer of information from the gonads to the thymus, adult AO rats were orchidectomized (ORX) or sham ORX (controls); sacrificed 1, 3, 7, or 15 days later and their thymi were analyzed for: (a) the concentrations of noradrenaline (NA), dopamine (DA) and serotonin (5-HT) and distribution of monoamine-containing nerve profiles and (b) the acetylcholinesterase (AChE) activity and distribution of AChE-containing nerve profiles. Three days after the castration, an elevation in the level of both catecholamines, reflecting an increase in the overall intensity of nerve fibers autofluorescence, was found. Seven days post castration neither NA nor DA concentration differed from the appropriate control values, while 15 days after the surgery the concentration of NA was lower than that in the controls, most likely, due to diminished density of noradrenergic nerve profiles. In both the rats sacrificed 7 and 15 days after orchidectomy the concentration of 5-HT was reduced as result of a decrease in the density of 5-HT-containing autofluorescent cells. The activity of AChE was depressed one day after the surgery; then increased, so that 3 days post castration its value was higher than that in the sham ORX. After this increase, AChE activity decreased being, at postoperative day 7 and 15, lower than that in the controls. It seems that this decrease in AChE activity reflected, not only a reduction in the density of AChE-containing nerve fibers, but also a decrease in the density of AChE positive cells. Thus, the results indicate that orchidectomy can evoke changes in the T-cell maturation altering modulatory influences on this process coming via neural route, as well as those coming from the mast cells and AChE positive epithelial cells which constitute important component of the thymus microenvironment.

Thyroid C cells of middle-aged rats treated with estradiol or calcium.

The structure and function of C cells of middle-aged female rats (14-months old) treated with estradiol dipropionate (EDP), calcium (Ca) or a combination of EDP+Ca were studied. A stereological method was used to determine the volume of calcitonin (CT)-immunoreactive C cells and their nuclei, and the relative volume density and mean number of the C cells per section were calculated. Serum levels of CT, osteocalcin, parathyroid hormone (PTH), and beta-estradiol were also measured. A significant decrease in body weight of the rats treated with EDP or EDP+Ca was observed. These treatments led to a significant decrease in cellular and nuclear volumes, relative volume density, and mean number of C cells per section, in comparison with the corresponding controls. A reduction of the serum level of CT, PTH, and osteocalcin was also recorded in EDP- and EDP+Ca-treated animals. No statistically significant differences between Ca- and vehicle-injected rats, with regard to all morphometric C cell parameters and biochemical values determined, were seen. However, a conspicuous degranulation of the C cells and decreased immunoreactivity for CT in the Ca-receiving group, which could be interpreted as the signs of increased activity of these cells, were noticed. This effect of Ca was also observed in rats injected with EDP and Ca in combination, when the inhibitory effect of EDP on C cell function was less noticeable than in the group treated with EDP alone.

[Diurnal rhythm of cortisol and aldosterone plasma levels in patients with chronic renal insufficiency]. / Ispitivanje dnevnog ritma nivoa kortizola i aldosterona u plazmi bolesnika s hronicnom insuficijencijom bubrega.

UNLABELLED: In patients with chronic renal failure (CRF) disturbances in cortical adrenal endocrine function were described previously. An increased plasma aldosterone level was documented in several studies [1-4]. The aim of this study was to investigate diurnal rhythms of plasma cortisol and aldosterone in CRF patients. In addition, in 3 patients on maintenance haemodialysis, the effect of dialysis procedure upon the plasma level of these hormones was studied. Ten patients with CRF of different etiologies, mean age 41.2 years, serum creatinine from 166-1336 mumol/L, creatinine clearance 4.2-29.2 ml/min, participated in the study. Blood samples were taken in the recumbent position at 6 am and at 6 h intervals thereafter for 24 h. Another group was made of 3 patients on maintenance haemodialysis for a mean 4.2 years. The effect of dialysis upon plasma cortisol and aldosterone levels was studied in the morning (7-11 h) and afternoon hours (14-18 h), with blood samples taken at one hour intervals. Patients were dialysed on cuprophan and polyacrilonitrile membranes. Plasma cortisol and aldosterone levels were determined using commercial RIA kits from Sorin Biomedica, Italy. The results obtained show that in 8 out of 10 patients with CRF in the predialysis period diurnal rhythm of cortisol was preserved. Investigation of the plasma aldosterone, with a 6 h sampling, however, shows in most of the patients studied no changes indicating a preserved diurnal rhythm. Investigation of the effect of haemodialysis treatment performed for 4 h on cuprophane as well as on PAN membrane, upon the plasma cortisol level has shown in morning hours the unchanged plasma cortisol level, compared to the same patients during the control study. Haemodialysis in the afternoon hours has produced, a small decrease of plasma cortisol during the first two hours, followed by an increase to the predialysis values. Plasma aldosterone levels during the morning and afternoon haemodialysis have shown variations similar to that observed in the same patients during the control study. CONCLUSIONS: This study has shown a preserved circadian rhythm of cortisol in kidney patients with CRF. A clear diurnal rhythm of aldosterone in these patients cannot be established. Investigation of the effect of haemodialysis procedure, performed on cuprophan and PAN, a more permeable membrane, upon the plasma levels of cortisol and aldosterone, has shown variations similar to that observed during the control study.

Stereological analysis of the sexually mature rat thymus after orchidectomy.

To assess the significance of gonadal function in the maintenance of thymic structure, male, sexually mature, 60 days old rats were bilaterally orchidectomized and the volumes of both the thymic compartments, total cellularity of each of them, as well as density (Nv) and mean diameter (D) of thymocytes within the specific thymic regions were stereologically analyzed 3, 7 and 15 days later. Three days after the orchidectomy a decrease in the thymic weight, and a reduction of both the cortical and medullary volumes, were observed. The reduction in cortical volume was followed by a decrease in overall number of the cortical thymocytes, suggesting an enhanced process of thymocyte death. In the same animals, the total number of the medullary thymocytes as well as D remained unchanged. Seven days after orchidectomy both volume of the cortex and total number of the cortical thymocytes reached the control values, while all the changes observed in the medulla of rats 3 days post castration, persisted. In addition, 7 days post castration, the volume of the interlobular connective tissue was increased and this persisted at day 15 post castration. The results indicate that orchidectomy can affect the thymic structure, and possibly the process of intrathymic T cell maturation; the extent of changes depending on the duration of the gonadal deprivation.

Prepubertal castration alters the phenotypic profile of adult rat thymocytes.

To assess the gonadal influence on the maturation of the thymus, rats were orchidectomized at different periods critical for programming of both gonadal and immune functions, and the composition of the intrathymic thymocyte population was determined in adults by flow cytometric analysis of the surface phenotype. The relative proportion of thymocytes expressing CD4, CD8 and T cell receptor (TCR)alpha beta was measured in the adult rats castrated at 1, 7 and 30 days. Castration performed at postnatal day 1 did not significantly affect expression of these molecules. However, in rats subjected to the surgery at day 7, a significant decrease in the proportion of CD4+CD8+ double-positive cells was found, in parallel with a proportional increase in the percentage of CD4+CD8- single-positive (SP) cells. In rats castrated at age 30 days, in addition to these changes, a small but significant increase in the percentage of CD4-CD8+ SP thymocytes was measured. Castration performed at age 7 and 30 days also caused an enrichment in the thymocyte population expressing TCR alpha beta, probably related to the increase of CD4+CD8- SP cells (7 days) and both CD4+CD8- and CD4-CD8+ SP cells (30 days). The total yield of thymocytes was increased in all experimental groups. We conclude that T cell maturation sequences in the adult thymus are altered if gonadal influence is removed during certain stages of thymus development.

Relationships between monoaminergic and cholinergic innervation of the rat thymus during aging.

The present study has been undertaken in order to investigate whether aging is accompanied by alterations in the thymic autonomic innervation. The results showed that in aged rats compared to young adult rats the density of monoaminergic histofluorescent nerve profiles decreased remarkably, while their pattern of intrathymic distribution remained unchanged. The thymic concentrations of noradrenaline (NA) and dopamine (DA) also significantly decreased between the age of 12 and 18 months. However, the density of thymic autofluorescent cells (afc) markedly increased over the same period, as well as the concentration of 5-hydroxytryptamine (5-HT). The aged rat thymus seemed to be able to maintain its cholinergic innervation in terms of density and pattern of distribution, while the density of cells with intracytoplasmic acetylcholinesterase (AChE) staining even increased. The neurochemical measurement showed an increase in the activity of AChE between the age of 9 to 18 months. The results indicate an altered relation between the components of thymic autonomic innervation of aged rats that might be related to the reduced immunocompetence of their T cells.

Comparative cephalometric analysis in patients with X-chromosome aneuploidy.

The influence of abnormal sex-chromosome constitution on cranial growth, shape and position of the craniofacial structures was analysed. Reduced cranial growth was found in women with Turner's syndrome (45 XO chromosome constitution) and in men with Klinefelter's syndrome (47 XXY chromosome constitution). Lack of or an extra X chromosome produced opposite effects on cranial-base flexion, jaw displacement and maxillary and mandibular inclination to the anterior cranial base. An extra X chromosome affected deviation in sagittal jaw relation while lack of an X chromosome influenced mandibular shape.

Effects of unfractionated and low molecular weight heparin on platelet-induced thrombin formation time and platelet adhesion.

Fraxiparin induced a significant inhibition of PITT 3 and 6 hours after the injection, and a nonsignificant moderate 25% prolongation after 9 hours. UFH inhibited PITT significantly 2 and 4 hours after the injection, and after 6 and 8 hours a nonsignificant 15 to 20% prolongation was observed. In hirudinized blood platelet adhesion was significantly prolonged 3 and 12 hours after the injection of Fraxiparin. If citrated blood was used to measure platelet adhesion a significant decrease was observed 3 to 18 hours after the Fraxiparin injection. UFH significantly inhibited platelet adhesion up to 8 hours after the injection. The long duration of the inhibition of platelet adhesion may possibly be correlated with the antithrombotic effect of heparin.

Effects of unilateral castration on the hypothalamic structures involved in the regulation of gonadal function in rat.

This study was undertaken to evaluate the effects of unilateral gonadectomy on the hypothalamic structures involved in the regulation of gonadal function in adult rats of both sexes. Unilateral gonadectomy was performed; 15 days later stereological parameters of cell activity of both the halves of hypothalamic preoptico-suprachiasmatic area (PO-SC) and arcuate nucleus (NA) were analyzed. Under the same experimental conditions the activities of the FSH and LH immunoreactive cells were analyzed separately in both the halves of the adenohypophysis. The results showed that in the rats of both sexes subjected to unilateral gonadectomy the mean diameter of cell nuclei of the contralateral half of PO-SC was significantly greater than that of the ipsilateral half. However, in the control intact or bilaterally gonadectomized rats, there were no significant differences in the values of the same parameter between two halves of PO-SC. On the other hand, neither in the unilaterally gonadectomized nor in the controls, the values of the mean diameter of NA cell nuclei differed significantly between the two halves of this structure. The FSH and LH pituitary cells behaved like NA cells. Therefore, since in the experimental animals compensatory function was developed, and since nervous signaling was different from the sides of the removed and intact gland, the present results suggest involvement of a pure nervous mechanism, besides hormonal control, in the regulation of the compensatory gonadal function. This mechanism seems to be functional in the rats of both sexes. These results also indicate that PO-SC is the anatomical structure involved in this regulation.

Clinical and meiotic studies in an infertile man with Y;13 translocation.

Clinical and meiotic studies were done on an infertile man with a translocation between Yq and 13q, who was identified through the birth of his son with partial trisomy 13q. Seminal plasma transferrin showed preserved Sertoli cell function while lactate dehydrogenase C4 indicated hypospermatogenesis. A quadrivalent in diakinesis and spermatogenic arrest in the second meiotic division was detected.