The contribution of dominance and inbreeding depression in estimating variance components for litter size in Pannon White rabbits.

In a synthetic closed population of Pannon White rabbits, additive (VA ), dominance (VD ) and permanent environmental (VPe ) variance components as well as doe (bF d ) and litter (bF l ) inbreeding depression were estimated for the number of kits born alive (NBA), number of kits born dead (NBD) and total number of kits born (TNB). The data set consisted of 18,398 kindling records of 3883 does collected from 1992 to 2009. Six models were used to estimate dominance and inbreeding effects. The most complete model estimated VA and VD to contribute 5.5 ± 1.1% and 4.8 ± 2.4%, respectively, to total phenotypic variance (VP ) for NBA; the corresponding values for NBD were 1.9 ± 0.6% and 5.3 ± 2.4%, for TNB, 6.2 ± 1.0% and 8.1 ± 3.2% respectively. These results indicate the presence of considerable VD . Including dominance in the model generally reduced VA and VPe estimates, and had only a very small effect on inbreeding depression estimates. Including inbreeding covariates did not affect estimates of any variance component. A 10% increase in doe inbreeding significantly increased NBD (bF d  = 0.18 ± 0.07), while a 10% increase in litter inbreeding significantly reduced NBA (bF l  = -0.41 ± 0.11) and TNB (bF l  = -0.34 ± 0.10). These findings argue for including dominance effects in models of litter size traits in populations that exhibit significant dominance relationships.

Rapid genotyping of genetically modified laboratory animals from whole blood samples without DNA preparation.

A new, rapid method is described which permits the genotyping of genetically modified animals from a microlitre volume of whole blood samples via one step polymerase chain reaction amplification. The major advantage of the presented method is the exclusion of a DNA preparation step, which significantly reduces the time expenditure and work load of the genetic testing. Pilot studies indicate, that this method is efficient and applicable also on tissue biopsies and larger amount of blood providing a rapid and reliable new technique over conventional genotyping approaches.

Isoflurane causes neocortical but not hippocampal-dependent memory impairment in mice.

BACKGROUND: The aim of this study is to investigate the effect of general anaesthesia induced by isoflurane with buprenorphine on hippocampus-dependent and neocortex-dependent memory, respectively, in mice, and in addition, to compare the effects of such anaesthesia on these memory processes with the effects induced by lipopolysaccharide (LPS) administration on the same memory processes. METHODS: To assess hippocampus-dependent memory, isoflurane (for 15 min) after buprenorphine injection, or LPS 100 µg/kg (intraperitoneally) was administered 24 h before or after fear conditioning. The effect of these treatments on hippocampus-dependent memory was assessed using contextual fear-conditioning tasks at day 4. To assess neocortex-dependent memory, isoflurane anaesthesia or LPS was given 72 h after contextual fear conditioning. Neocortex-dependent memory assessment was performed at day 32. RESULTS: Unlike LPS injection, isoflurane with buprenorphine-induced anaesthesia does not impair freezing responses in hippocampus-dependent fear-conditioning memory tasks. On anterograde amnesia assessment: 49.67 ± 6.87% for the anaesthesia group and 54.5 ± 4.12% for the control group. On retrograde amnesia assessment: 47.16 ± 8.71% for the anaesthesia group and 54.5 ± 4.12% for control group; P > 0.05. Thus, neither isoflurane nor buprenorphine impair hippocampus-dependent memory. However, on the neocortex-dependent memory task, both isoflurane-induced anaesthesia and LPS-induced inflammation result in reduced freezing responses: 62.13 ± 5.80% for the anaesthesia group, 74.63 ± 5.69% for the LPS group, and 81.75 ± 3.26% for the control group; P < 0.05 compared with control group. CONCLUSION: General anaesthesia induced by isoflurane with buprenorphine may result in impairment of neocortex-dependent memory in mouse. However, general anaesthesia so induced does not impair hippocampus-dependent memory in mouse in our experimental conditions.

4He+ ion beam irradiation induced modification of poly(dimethylsiloxane). Characterization by infrared spectroscopy and ion beam analytical techniques.

In this study we investigated the chemical and surface wettability changes of poly(dimethylsiloxane) (PDMS) induced by a 2.0 MeV He(+) beam irradiation. The chemical changes created in PDMS were characterized by universal attenuated total reflectance infrared (UATR-FTIR) spectroscopy, while the changes of the wettability were determined by contact angle measurements. In a separate analysis, hydrogen depletion was also investigated with a 1.6 MeV He(+) beam by applying the elastic recoil detection analysis (ERDA) and Rutherford backscattering spectrometry techniques simultaneously. The ERDA results showed that the hydrogen content of PDMS decreased irreversibly, which means that volatile products were formed under radiolysis, such as hydrogen or methane. The results were completed with UATR-FTIR measurements. We propose a complete reaction mechanism for the processes taking place in PDMS. These ion beam induced processes, such as chain scissions, cross-linking, and depletion of small molecular weight fragments, lead to the formation of a silica-like final product (SiO(x)). The significant chemical changes at the surface influence the wettability of PDMS, making it considerably more hydrophilic. The penetration depth of the 2.0 MeV He(+) ions is significantly higher compared to that of other surface modification techniques, which makes the modified layer thick and homogeneous; on the other hand, it is easily controllable by the energy of the incident ions.

Effect of ambient temperature on the productive and carcass traits of growing rabbits divergently selected for body fat content.

Rabbits are particularly sensitive to heat stress which can affect productive performance, with rabbit breed/line possibly playing a role on the response to this condition. The study aimed at evaluating the effect of different ambient temperatures on the live performance and carcass traits of growing rabbits divergently selected for total body fat content. The two genetic lines (Lean and Fat) were selected based on the total body fat content estimated by computer tomography during five generations. From birth to slaughter (13 weeks of age), the rabbits were housed in two rooms where the temperature was controlled with air conditioners: in the control room the average ambient temperature was 20 °C and in the high temperature room it was 28 °C. After weaning (35 d), 60 Lean and 60 Fat rabbits/room were housed by two in wire-mesh cages and fed ad libitum with commercial pellets. The BW and feed intake (FI) were measured at 5, 7, 9, 11 and 13 weeks of age to calculate the daily weight gain (DWG) and feed conversion ratio (FCR). Mortality was recorded daily. At the end of the experiment, rabbits were slaughtered and carcass traits were measured. Mortality was independent of temperature and line. The temperature significantly influenced the FI, DWG, BW and the fat deposits: they were lower at higher ambient temperature. The effect of temperature differed according to the rabbits' total body fat content. At control temperature, the FI (165 vs 155 g/day; P < 0.05) and FCR (4.67 vs 4.31; P < 0.05) were higher in Fat rabbits, which also had more perirenal (36.2 vs 23.1 g; P < 0.05) and scapular fat (10.8 vs 7.1 g; P < 0.05). At high temperature, no differences in fat depots (14.5 vs 9.8 g; 5.3 vs 3.5 g) were found between the two lines. It can be concluded that temperature × genetic line interaction had an important role in productive and carcass traits, as the effect of temperature differs between Lean and Fat rabbits.

Intracellular Na+:K+ ratios in human cancer cells as revealed by energy dispersive x-ray microanalysis.

Intranuclear sodium, potassium, and chloride contents were measured by energy-dispersive x-ray microanalysis in freeze-fractured, freeze-dried, bulk-tumor samples taken from 10 patients suffering from invasive urogenital cancers. Human biopsies were carried out during the first diagnostic interventions before any cytostatic treatment had been applied. Pathohistological diagnosis established the malignancy in each case. The cancers were classified in three types: keratinizing, transitional cell, and hypernephroid carcinoma. More than 250 cell nuclei were measured from each type of cancer. The results were compared with those obtained in intact human urothelium taken from patients having no malignant processes. Proximal and distal tubular epithelial cell nuclei representing the origin of human hypernephroid cancer were also measured in rat kidney because corresponding healthy human material cannot be obtained. The analyses revealed, in all three types of cancer cells, that the average intranuclear sodium content increased more than three-fold, the potassium content decreased 32, 16, and 13%, respectively; meanwhile the chloride content increased, but to a lesser extent than did the sodium. The intranuclear Na+:K+ ratios were more than five-fold higher in the cancer cells on the average, and their distribution histograms were much broader than in the normal human urothelium and in the tubular cell nuclei of the rat kidney. The results obtained fit well with the theory of Cone, C. D., Jr. 1971. J. Theor. Biol. 30: 151-181 according to which the sustained depolarization of the cell membrane may be of mitogenic effect.

Coupling of conduction electrons to two-level systems formed by hydrogen: a scattering approach.

An effective Hamiltonian for a two-level system (TLS) which could model the interaction between a tunneling proton and the conduction electrons of a metal is investigated in a comparative way. In the conventional first-order Born approximation with plane waves, and for small-distance displacement of the tunneling particle, a simple correlation between the atomic motion and angular momentum change of the scattering electron is deduced. For such a displacement, and within a distorted wave Born approximation for initial and final states, the change in the scattering amplitude is expressed via bounded trigonometric functions of the corresponding difference of scattering phase shifts. The numerical value of this amplitude change is analyzed in the framework of a self-consistent screening description for an impurity embedding in a paramagnetic electron gas. The coupling thus obtained of the tunneling proton to a homogeneous electron gas is too weak to be in the range required for realization of the two-channel Kondo effect.

What causes hidradenitis suppurativa?

Hidradenitis suppurativa (HS)--a rather common, very chronic and debilitating inflammatory skin appendage disorder with a notoriously underestimated burden of disease--has long been a playground for the high priests of nomenclature: Ask a bunch of eminent dermatologists and skin pathologists to publicly share their thoughts on what causes HS, and they will soon get entrenched in a heated debate on whether this historical term is a despicable misnomer. Fortunately, the recently founded Hidradenitis Suppurativa Foundation (HSF; http://www.hs-foundation.org), to which EXP DERMATOL serves as home journal, has broken with this unproductive tradition and has encouraged publication of the current CONTROVERSIES feature. This is exclusively devoted to discussing the pathobiology of this chronic neutrophilic folliculitis of unknown origin. Although traces of terminological bickering remain visible, it does the HS experts in our virtual debate room credit that they engage in a constructive and comprehensive dissection of potential pathogenesis pathways that may culminate in the clinical picture we know under the competing terms HS or acne inversa. These experts sketch more often complementary than mutually exclusive pathogenesis scenarios, and the outlines of a conceivable consensus on the many open pathobiology questions begin to emerge in these CONTROVERSIES. Hopefully, this heralds a welcome new tradition: to get to the molecular heart of HS pathogenesis, which can only be achieved by a renaissance of solid basic HS research, as the key to developing more effective HS therapy.

Early solid additional feeding of suckling rabbits from 3 to 15 days of age.

Studies have shown that nutrient requirement of suckling kits is not satisfied, but they can be fed a double quantity of milk (double nursing) resulting in improved BW and weight gain. The aim of our trials was to give additional solid feed during the early suckling period (3 to 15 days of age) when rabbit kits drink exclusively milk. Two experiments were conducted with animals from Pannon Rabbit Breeding Program. In experiment 1 (n=77 does, 734 kits) the does received commercial feed (C) or C pellet supplemented with 0.2 g powdered thyme/kg (CT). Within both dietary groups of the does three groups of litters were formed: no additional solid creep feeding (N); soya bean-based pellet (S); S pellet with 1% added powdered thyme (ST). In group S and ST, cylinder-shaped solid pellets were made. At the beginning (3 days of age) two pieces of pellets were placed daily into the nestbox after nursing. Later on it was increased to six pellets till 15 days of age. The kits consumed the additional solid feed (S and ST), however, it did not affect the BW, weight gain or survival. In experiment 2 (n=30 does, 240 kits) all does consumed commercial feed. The additional feed for kits was based on commercial piglet feed. Three groups were formed: the litters in control group were fed no additional solid feed (K), kits were fed additionally with pellets (8 mm of diameter) based on piglet feed powder, pellet adhesive and water (PI), and extra glycerin powder was added to the mixture of piglet feed powder and water (PG). The experiment lasted from the age of 3 days till 21 days. At the beginning six pellets were placed on the nest material. Later on the amount was gradually increased to 24 pellets till age of 15 days. The kits consumed the pellets. The BW of PI group differed from group PG at age of 5, 9, 12 and 21 days by +7.3%, +6.5%, +5.9%, +4.8%, respectively (P<0.05) and from group K at age of 12 days by +5.9% (P< 0.05). The differences were more expressed at age of 16 and 19 days in favour of group PI (from K by +7.1%, +6.9% and from PG by +5.9%, +5 8%, respectively, P<0.01) and at 21 days of age (from K by +6.2%, P<0.01). To find appropriate composition of creep feed for kits further studies are needed.

The first characterization of a eubacterial proteasome: the 20S complex of Rhodococcus.

BACKGROUND: The 26S proteasome is the central protease of the ubiquitin-dependent pathway of protein degradation. The proteolytic core of the complex is formed by the 20S proteasome, a cylinder-shaped particle that in archaebacteria contains two different subunits (alpha and beta) and in eukaryotes contains fourteen different subunits (seven of the alpha-type and seven of the beta-type). RESULTS: We have purified a 20S proteasome complex from the nocardioform actinomycete Rhodococcus sp. strain NI86/21. The complex has an apparent relative molecular mass of 690 kD, and efficiently degrades the chymotryptic substrate Suc-Leu-Leu-Val-Tyr-AMC in the presence or absence of 0.05% SDS. Purified preparations reveal the existence of four subunits, two of the alpha-type and two of the beta-type, the genes for which we have cloned and sequenced. Electron micrographs show that the complex has the four-ringed, cylinder-shaped appearance typical of proteasomes. CONCLUSIONS: The recent description of the first eubacterial ubiquitin, and our discovery of a eubacterial proteasome show that the ubiquitin pathway of protein degradation is ancestral and common to all forms of life.

Taking the sting out of pain.

While the role of the brain kallikrein-kinin system in the development of various pathological processes, such as oedema formation following brain injury or induction of central hypertonia has generated major interest, the possible role of this system in nociceptive processing has received little attention. In their present paper, Mortari et al. (2007) show that bradykinin B2 receptor activation in the brain by the bradykinin analogue, Thr(6)-bradykinin, isolated from the venom of the social wasp, Polybia occidentalis potently reduces acute, noxious heat-evoked reflex responses in naive rats. The unknown underlying mechanism of this powerful antinociceptive effect reminds us that the supraspinal antinociceptive system is still a "black box" in many aspects and awaits thorough investigation.

[Transplantation of neural stem cells into the cochlea]. / Neurogene Stammzelltransplantation in die Kochlea.

BACKGROUND: Stem cell therapy is especially interesting for inner ear related diseases, since the hair cells are very sensitive and do not regenerate. Hair cell loss is therefore irreversible and is accompanied by hearing loss. In the last few years, different research groups have transplanted stem cells into the inner ear with promising results. In the presented study, our aim was to gain insight into how neuronal stem cells behave when they are transplanted, both in vitro and in vivo, into a damaged inner ear. METHODS: Neuronal stem cells from E9.5 day old mouse embryos were collected and infected with an adenoviral vector encoding green fluorescent protein (GFP). GFP+ cells were then transplanted into a damaged organ of Corti in vitro or into a damaged mouse inner ear in vivo. RESULTS: We were able to detect GFP+ cells close to the organ of Corti in vitro and in the organ of Corti in vivo. The GFP+ cells do not seem to be randomly distributed in either the in vitro or in vivo situation. Most interestingly, GFP+ cells could be detected close to places where hair cells had been lost in vivo. CONCLUSION: Neuronal stem cells are interesting candidates to replace lost hair cells. However, a great deal of research is still needed before they can enter clinical trials.

Alteration of activator protein 1 DNA binding activity in gentamicin-induced hair cell degeneration.

Sensorineural hearing loss is often associated with damage of cochlear hair cells and/or of the neurons of the auditory pathway. This damage can result from a variety of causes, e.g. genetic disorders, aging, exposure to certain drugs such as aminoglycosides, infectious disease and intense sound overexposure. Intracellular events that mediate aspects of aminoglycoside-mediated damage to hair cells have been partially unraveled. Several independent research groups have demonstrated a crucial role of mitogen-activated protein kinase signaling in aminoglycoside-induced ototoxicity. Mitogen-activated protein kinases are important mediators of signal transduction from the cell surface to the nucleus. Jun N-terminal kinases, members of the mitogen-activated protein kinase family, are strongly activated in cell culture conditions by stress inducing stimuli, including ultraviolet light, heat shock and tumor necrosis factor; therefore they are also referred to as stress-activated protein kinases. In hair cells aminoglycoside treatment was shown to activate the Jun N-terminal kinase signaling pathway. Activation of Jun N-terminal kinase leads to phosphorylation and thereby activation of transcription factors and consequently to altered gene expression. There are many nuclear Jun N-terminal kinase substrates including c-Jun, ATF-2, and Elk-1 proteins. One of the downstream targets of Jun N-terminal kinase is the transcription factor activating protein-1. Activating protein-1 is a dimeric complex composed of members of the Fos and Jun proteins. A variety of different stimuli is known to induce activating protein-1 activity. Induction of activating protein-1 is thought to play a central role in reprogramming gene expression in response to external stimuli. In this study we have analyzed the effect of gentamicin treatment on the downstream targets of Jun N-terminal kinase. Our results demonstrate that gentamicin treatment of explants of organ of Corti results in increased activating protein-1 binding activity. The main component of these activating protein-1 complexes is the c-Fos protein. Moreover, we show that the activating protein-1 induction is transient and occurs exclusively in hair cells of rat organ of Corti explants.

Correlation of malignancy with the intracellular Na+:K+ ratio in human thyroid tumors.

Energy-dispersive X-ray microanalysis was applied on human intraoperative biopsy materials of different thyroid tumors. To ensure suitability of these tissue pieces for quantitative microanalysis in freeze-fractured, freeze-dried bulk specimens, sampling was carried out with strictly defined criteria. Benign adenomas and differentiated and anaplastic carcinomas were selected for the studies on the basis of pathohistological investigations of the same specimen. The results of the tumor cells were compared to those obtained in apparently normal human epithelial cells. The number of normal cells analyzed was 349, whereas in the tumors 408, 423, and 891 cells were measured in the benign, differentiated, and anaplastic groups, respectively. Intracellular monovalent contents were calculated as percentage of cell dry mass; then, Na+:K+ molar ratios were calculated for each cell individually. Due mostly to the increase of Na+ content, the distribution histograms of the Na+:K+ molar ratio show an increase in the number of cells with a higher Na+:K+ ratio with increasing malignancy of the tumors studied. The differences proved to be statistically highly significant by the chi 2 test. Thus, in human thyroid, increasing malignancy is associated with increasing intracellular Na+:K+ ratio. The results give further support to the theory of C. D. Cone (J. Theor. Biol., 30: 151-181, 1971) according to which the sustained depolarization of the cell membrane results in an increased rate of cell division.

Similarities and differences between the responses of rat sensory neurons to noxious heat and capsaicin.

We have compared the membrane response of rat primary sensory neurons to capsaicin and noxious heat, using electrophysiological and ion flux measurements. Our aim was to determine whether, as recently proposed, the same molecular entity accounts for excitation by both types of stimulus. The properties of the ion channels activated by heat and capsaicin show many similarities but also important differences. The calcium permeability of heat-activated channels is lower than that of capsaicin-activated channels. Distinct single channels respond to heat or capsaicin, and only a few show dual sensitivity. At the whole-cell level, individual cells invariably show dual sensitivity, but the amplitudes of the responses show little correlation. We conclude that distinct molecular entities, which are both likely to be derived from the VR1 gene product, account for the membrane responses to heat and capsaicin.

Proteasomes and other self-compartmentalizing proteases in prokaryotes.

The proteasome represents the major non-lysosomal proteolytic system in eukaryotes. It confines proteolytic activity to an inner compartment that is accessible to unfolded proteins only. The strategy of controlling intracellular breakdown of proteins by self-compartmentalization is also used by different types of prokaryotic energy-dependent proteases. Genomic sequencing data reveal that various combinations of these energy-dependent proteases occur in prokaryotic cells from different lineages.

Growth-regulatory factors for normal, premalignant, and malignant human cells in vitro.

Normal human cells, cells from nonmalignant proliferative lesions, and primary and metastatic tumor cells can be maintained in vitro and analyzed for requirements for growth in chemically defined media. The human melanocytic cell system with normal melanocytes, precursor nevus cells, and primary and metastatic melanoma cells has been extensively studied for the phenotypic properties of the cells, including their requirements for exogenous growth factors and other mitogens. In high calcium-containing W489 medium, normal melanocytes require four supplements: IGF-I (or insulin); bFGF, TPA, and alpha-MSH. Nevus cells are largely independent of bFGF. Depletion of TPA from medium is not as detrimental to nevus cells as it is to melanocytes, but the phorbol ester is still essential for maintenance of the typical nevic phenotype. Primary melanoma cells require at least one growth factor, IGF-I (or insulin), for continuous proliferation. On the other hand, metastatic cells of melanoma as well as of carcinomas of colon and rectum, bladder, ovary, and cervix are able to proliferate after a short adaptation period in medium depleted of any growth factors and other proteins. Doubling times of metastatic tumor cells in protein-free medium are only 30-60% longer than in FCS-containing medium. The growth autonomy of human tumor cells is apparently due to the endogenous production of growth factors. Likely candidates for autocrine growth stimulation of human tumor cells are TGF-alpha, TGF-beta, and PDGF. Melanoma and colorectal carcinoma cells express functional EGF/TGF-alpha receptors, and produce TGF-alpha, indicating that this growth factor is produced for autocrine stimulation. In addition to the use of anti-growth factor antibodies, other strategies for the inhibition of autocrine growth stimulation include mAbs to growth factor receptors, soluble receptors, receptor-mimicking antiidiotype antibodies, and active immunization against growth factors. Whether any of these therapeutic approaches is clinically feasible will need to be determined in extensive preclinical investigations.

Formation of long-lived hydroxyl free radical adducts of proline and hydroxyproline in a Fenton reaction.

Proline and hydroxyproline when exposed to the hydroxyl free radical generating system of ADP-Fe(II)-H2O2 yielded long-lived free radicals. An analysis of the electron paramagnetic resonance spectra of the long-lived hydroxyl free radical adducts of proline and hydroxyproline is consistent with a free electron on a nitroxyl group interacting with the nitrogen atom as well as with three separate protons. In the case of proline, nitroxide formation was observed under the influence of tert-butyl-hydroperoxide, giving a similar EPR spectrum (Lin, J.S., Tom, T.C. and Olcott, H.S. (1974) J. Agr. Food Chem. 22, 526-528); however, the hydroxyl free radical adduct of hydroxyproline has not been described yet. In the case of the proline nitroxide radical, two of the three protons involved interact with the free electron equivalently. The coupling constants for the hydroxyl free radical adduct of proline are AN = 1.58 mT, AH1 beta = AH2 beta = 2.13 mT, AH3 beta = 1.77 mT and for hydroxyproline are AN = 1.54 mT, AH1 beta = 2.56 mT, AH2 beta = 2.03 and AH3 beta = 1.51. The data are consistent with the amine nitrogen of proline and hydroxyproline being oxidized to a nitroxyl group and the free electron of the nitroxyl interacting with the beta-protons of these amino acid hydroxyl free radical adducts.

Hydroxyl free radical reactions with amino acids and proteins studied by electron spin resonance spectroscopy and spin-trapping.

It has recently been shown that Fe(I) complexes of ADP or ATP generate OH radicals with H2O2 in a Fenton-type reaction. The OH radicals can be detected by using 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) as a spin trap in electron spin resonance spectroscopy. All the biologically occurring amino acids, some related compounds and several proteins (histone, bovine serum albumin, collagen) were tested as OH radical scavengers against DMPO. The tested compounds competed with DMPO in trapping OH radicals to various extents as shown by the decrease of signal intensity of DMPO-OH spin-adduct. The tested compounds did not oxidize Fe(II) itself, with the only exception being tyrosine, as revealed by properly designed ferrozine reaction. Some of the amino acids reacted also with the DMPO-OH spin-adduct to a certain extent, whereas others did not. The formation of carbon centered organic radicals of the amino acids could be detected under the influence of OH radicals by using the spin traps phenyl-tert-butylnitrone (PBN) and alpha-pyridyl-1-oxide-N-tert-butylnitrone (4-POBN). The proteins, however, did not react with these spin traps. One can conclude that the amino acids and proteins can be targets of OH radical damage even in vivo, and such phenomena may be of importance in the deterioration of the conformation of proteins, e.g., during aging or in some pathological processes.