Ageing in men with normal spermatogenesis alters spermatogonial dynamics and nuclear morphology in Sertoli cells.

BACKGROUND: Ageing in men is believed to be associated with fertility decline and elevated risk of congenital disorders for the offspring. The previous studies also reported reduced germ and Sertoli cell numbers in older men. However, it is not clear whether ageing in men with normal spermatogenesis affects the testis and germ cell population dynamics in a way sufficient for transmitting adverse age effects to the offspring. OBJECTIVES: We examined men with normal spermatogenesis at different ages concerning effects on persisting testicular cell types, that is the germ line and Sertoli cells, as these cell populations are prone to be exposed to age effects. MATERIAL AND METHODS: Ageing was assessed in testicular biopsies of 32 patients assigned to three age groups: (i) 28.8 ± 2.7 years; (ii) 48.1 ± 1 years; and (iii) 70.9 ± 6.2 years, n = 8 each, with normal spermatogenesis according to the Bergmann-Kliesch score, and in a group of meiotic arrest patients (29.9 ± 3.8 years, n = 8) to decipher potential links between different germ cell types. Besides morphometry of seminiferous tubules and Sertoli cell nuclei, we investigated spermatogenic output/efficiency, and dynamics of spermatogonial populations via immunohistochemistry for MAGE A4, PCNA, CREM and quantified A-pale/A-dark spermatogonia. RESULTS: We found a constant spermatogenic output (CREM-positive round spermatids) in all age groups studied. In men beyond their mid-40s (group 2), we detected increased nuclear and nucleolar size in Sertoli cells, indirectly indicating an elevated protein turnover. From the 7th decade (group 3) of life onwards, testes showed increased proliferation of undifferentiated spermatogonia, decreased spermatogenic efficiency and elevated numbers of proliferating A-dark spermatogonia. DISCUSSION AND CONCLUSION: Maintaining normal sperm output seems to be an intrinsic determinant of spermatogenesis. Ageing appears to affect this output and might provoke compensatory proliferation increase in A spermatogonia which, in turn, might hamper germ cell integrity.

Ab initio determination of the crystal structure of cytochrome c6 and comparison with plastocyanin.

BACKGROUND: Electron transfer between cytochrome f and photosystem I (PSI) can be accomplished by the heme-containing protein cytochrome c6 or by the copper-containing protein plastocyanin. Higher plants use plastocyanin as the only electron donor to PSI, whereas most green algae and cyanobacteria can use either, with similar kinetics, depending on the copper concentration in the culture medium. RESULTS: We report here the determination of the structure of cytochrome c6 from the green alga Monoraphidium braunii. Synchrotron X-ray data with an effective resolution of 1.2 A and the presence of one iron and three sulfur atoms enabled, possibly for the first time, the determination of an unknown protein structure by ab initio methods. Anisotropic refinement was accompanied by a decrease in the 'free' R value of over 7% the anisotropic motion is concentrated at the termini and between residues 38 and 53. The heme geometry is in very good agreement with a new set of heme distances derived from the structures of small molecules. This is probably the most precise structure of a heme protein to date. CONCLUSIONS: On the basis of this cytochrome c6 structure, we have calculated potential electron transfer pathways and made comparisons with similar analyses for plastocyanin. Electron transfer between the copper redox center of plastocyanin to PSI and from cytochrome f is believed to involve two sites on the protein. In contrast, cytochrome c6 may well use just one electron transfer site, close to the heme unit, in its corresponding reactions with the same two redox partners.

An alternative interpretation of cellular 'selfish spermatogonial selection'-clusters in the human testis indicates the need for 3-D-analyses.

The 'selfish spermatogonial selection'- model was proposed to explain the paternal age effect (PAE) of some congenital disorders associated with point mutations in male germ cells. According to this, spermatogonia carrying pathogenic mutations gain a selection advantage over non-mutated spermatogonia which leads to an increased number of mutated spermatogonia and consequently spermatozoa over time. Recently, an immunohistochemical approach using the premeiotic marker melanoma antigen family A4 (MAGE A4) was undertaken by the Wilkie group to confirm the presence of microclones of putatively mutated spermatogonia in testes of elderly men. The objective of our study was the age-dependent assessment of testes from men with normal spermatogenesis using MAGE A4 immunohistochemistry to identify and corroborate cellular clusters indicative for 'selfish spermatogonial selection' in our cohort. We analyzed testicular tissues obtained from men with normal spermatogenesis assigned to three age groups [(1) 28.8 ± 2.7 years; (2) 48.1 ± 1 years; (3) 71.9 ± 6.8 years, n/group = 8]. We could detect very similar distribution patterns of MAGE A4-positive cells and the presence of several types of microclusters as reported previously. However, these cellular clusters, indicative for clonal expansion, were not only present in testes from elderly men but also in those from age group 1 and 2. Using graphical three-dimensional modelling, we identified that cross-section directions e.g. longitudinal sections might provoke misleading interpretation of spermatogonial clusters, in particular when the tissue processing is limited. Thus, appropriate fixation and embedding is needed for reliable analysis of testicular sections. We therefore propose a more careful interpretation of such spermatogonial clusters and recommend a 3-D analysis to unequivocally determine 'selfish spermatogonial selection'-manifestations.

Permeation of phloretin across bilayer lipid membranes monitored by dipole potential and microelectrode measurements.

The transmembrane diffusion of phloretin across planar bilayer lipid membranes is studied under steady-state conditions. Diffusion restrictions and adsorption related effects are measured independently. The adsorption of aligned phloretin dipoles generates a change in the intrinsic dipole potential difference between the inner and outer leaflets of the lipid bilayer. It is monitored by capacitive current measurements carried out with a direct current (dc) bias. The variation of the intramembrane electric field indicates a saturation of the binding sites at the membrane interface. In contrast, pH profile measurements undertaken in the immediate membrane vicinity show a constant membrane permeability. If phloretin binding and transmembrane diffusion are treated as two competitive events rather than subsequent steps in the transport queue the contradictory results become explainable. A mathematical model is developed where it is assumed that diffusing phloretin molecules are randomly oriented, i.e., that they do not contribute to the intrinsic membrane potential. Only the dipoles adsorbing onto the membrane are oriented. Based on these theory the membrane permeability is calculated from the capacitive current data. It is found to agree very well with the permeability deduced from the microelectrode measurements.

Changes of the membrane potential profile induced by verapamil and propranolol.

The effects of the organic calcium channel blocker verapamil and the beta-receptor blocker propranolol on dipole (phi(d)) and surface (phi(s)) potentials of bilayer lipid membranes were studied. The boundary potentials (phi(b)= phi(d) + phi(s)) of black lipid membranes, monitored by conductance measurements in the presence of nonactin and by capacitive current measurements were compared with phi(s) calculated from the electrophoretic mobility of lipid vesicles. It was shown that the increase of boundary potential, induced by the adsorption of the positively charged propranolol, was caused solely by an increase in surface potential. Although phi(s) also increases due to the adsorption of verapamil, phi(b) diminishes. A sharp decrease of the dipole potential was shown to be responsible for this effect. From Langmuir adsorption isotherm the dissociation constant Kd of verapamil was estimated. The uncharged form of verapamil (Kd=(0.061+/-0.01) mM at pH 10.5) has a tenfold higher affinity to a neutral bilayer membrane than the positively charged form. The alteration of membrane dipole potential due to verapamil adsorption may have important implications for both membrane translocation and partitioning of small or hydrophobic ions and charged groups of membrane proteins.

Membrane fusion mediated by ricin and viscumin.

The ribosome inactivating plant proteins (RIPs) ricin and viscumin but not Ricinus communis agglutinin are able induce vesicle-vesicle fusion. A model is suggested in which the toxicity of the RIPs is partially determined by their fusogenicity. Herein, fusion is hypothesized to allow the RIPs to leak across endocytic vesicles to approve their access to cytoplasmic ribosomes.

Crystal structure of a cobalt-activated diphtheria toxin repressor-DNA complex reveals a metal-binding SH3-like domain.

The diphtheria toxin repressor (DtxR) is the prototype of a family of iron-dependent regulator (IdeR) proteins, which are activated by divalent iron and bind DNA to prevent the transcription of downstream genes. In Corynebacterium diphtheriae, DtxR regulates not only the expression of diphtheria toxin encoded by a corynebacteriophage, but also of components of the siderophore-mediated iron-transport system. Here we report the crystal structure of wild-type DtxR, a 226 residue three-domain dimeric protein, activated by cobalt and bound to a 21 bp DNA duplex based on the consensus operator sequence. Two DtxR dimers surround the DNA duplex which is distorted compared to canonical B -DNA. The SH3-like third domain interacts with the metal at site 1 via the side-chains of Glu170 and Gln173, revealing for the first time a metal-binding function for this class of domains. The SH3-like domain is also in contact with the DNA-binding first domain and with the second, or dimerization, domain. The DNA-binding helices in the first domain are shifted by 3 to 5 A when compared to the apo-repressor, and fit into the major groove of the duplex bound. These shifts are due to a hinge-binding motion of the DNA-binding domain with respect to the dimerization domains of DtxR. The third domain might play a role in regulating this hinge motion.

Crystal structure of the iron-dependent regulator (IdeR) from Mycobacterium tuberculosis shows both metal binding sites fully occupied.

Iron-dependent regulators are a family of metal-activated DNA binding proteins found in several Gram-positive bacteria. These proteins are negative regulators of virulence factors and of proteins of bacterial iron-uptake systems. In this study we present the crystal structure of the iron-dependent regulator (IdeR) from Mycobacterium tuberculosis, the causative agent of tuberculosis. The protein crystallizes in the hexagonal space group P62 with unit cell dimensions a=b=92.6 A, c=63.2 A. The current model comprises the N-terminal DNA-binding domain (residues 1-73) and the dimerization domain (residues 74-140), while the third domain (residues 141-230) is too disordered to be included. The molecule lies on a crystallographic 2-fold axis that generates the functional dimer. The overall structure of the monomer shares many features with the homologous regulator, diphtheria toxin repressor (DtxR) from Corynebacterium diphtheriae. The IdeR structure in complex with Zinc reported here is, however, the first wild-type repressor structure with both metal binding sites fully occupied. This crystal structure reveals that both Met10 and most probably the Sgamma of Cys102 are ligands of the second metal binding site. In addition, there are important changes in the tertiary structure between apo-DtxR and holo-IdeR bringing the putative DNA binding helices closer together in the holo repressor. The mechanism by which metal binding may cause these structural changes between apo and holo wild-type repressor is discussed.

The importance of open emergency surgery in the treatment of acute mesenteric ischemia.

OBJECTIVE: Acute mesenteric ischemia (AMI) is a complex disease with a high mortality rate. A patient's chance of survival depends on early diagnosis and rapid revascularization to prevent progression of intestinal gangrene. We reviewed our experience with open surgery treatment in 54 cases of AMI. METHODS: A monocentric retrospective study was conducted between 01/01/2001 and 04/30/2014; 54 AMI patients with a mean age of 56.6 years underwent surgery (26 women and 28 men). Retrospectively, the risk factors, management until diagnosis, vascular therapy and follow-up were evaluated. RESULTS: The symptom upon admission was an acute abdominal pain event. The delay time from admission to surgery was, on average, 13.9 h (n = 34). The therapeutic procedures were open surgical operations. The complication rate was (53.7 %) (n = 29). The 30-day mortality was 29.6 % (n = 16). The late mortality rate was 24.1 % (n = 13), and the cumulative survival risk was 44.6 %. Survival was, on average, 60.54 months; however, in the over 70-year-old patient subgroup, the survival rate was 9.5 months (p = 0.035). The mortality rate was 27 % (n = 22) in the <12 h delay group, 20 % (n = 5) in the 12-24 h delay group, and 50 % (n = 7) in the > 24 h delay group. CONCLUSIONS: The form of therapy depends on the intraoperative findings and the type of occlusion. Although the mortality rate has decreased in the last decade, in patients over 70 years of age, a significantly worse prognosis was seen.

Comparison of high-resolution structures of the diphtheria toxin repressor in complex with cobalt and zinc at the cation-anion binding site.

The diphtheria toxin repressor (DtxR) from Corynebacterium diphtheriae is a divalent-metal activated repressor of chromosomal genes responsible for siderophore-mediated iron-uptake and of a gene on several corynebacteriophages that encodes diphtheria toxin. Even though DtxR is the best characterized iron-dependent repressor to date, numerous key properties of the protein still remain to be explained. One is the role of the cation-anion pair discovered in its first metal-binding site. A second is the reason why zinc exhibits its activating effect only at a concentration 100-fold higher than other divalent cations. In the presently reported 1.85 A resolution Co-DtxR structure at 100K, the sulfate anion in the cation-anion-binding site interacts with three side chains that are all conserved in the entire DtxR family, which points to a possible physiological role of the anion. A comparison of the 1.85 A Cobalt-DtxR structure at 100K and the 2.4 A Zinc-DtxR structure at room temperature revealed no significant differences. Hence, the difference in efficiency of Co2+ and Zn2+ to activate DtxR remains a mystery and might be hidden in the properties of the intriguing second metal-binding site. Our studies do, however, provide a high resolution view of the cationanion-binding site that has most likely evolved to interact not only with a cation but also with the anion in a very precise manner.

The role of structural domains in RIP II toxin model membrane binding.

The interaction of plant toxin ricin and MLI binding subunits to liposomes containing monosialoganglioside (GM1), bearing a terminal galactose residue, has been examined as a possible receptor model. For the first time we demonstrate that ricin B-chain but not ricin provokes liposome aggregation at 10 M% GM1 concentration, whereas in the presence of either ricin A-chain or galactose the aggregation is inhibited. The B-subunit of plant toxin MLI from Viscum album has similar lectin specificity and activity but cannot aggregate GM1 liposomes. The ability of the B-chain to aggregate liposomes adds a new crucial step in the toxin transmembrane penetration mechanism. We demonstrate here possible ricin B-chain interactions with membranes proceeding via two sites, namely (a) a galactose-binding domain and (b) a hydrophobic interchain domain. In close contact with two phospholipid bilayers, ricin B-chain may determine the geometry of the fusion site. These events can provoke A-chain translocation which follows membrane fusion.

The TB structural genomics consortium: a resource for Mycobacterium tuberculosis biology.

The TB Structural Genomics Consortium is an organization devoted to encouraging, coordinating, and facilitating the determination and analysis of structures of proteins from Mycobacterium tuberculosis. The Consortium members hope to work together with other M. tuberculosis researchers to identify M. tuberculosis proteins for which structural information could provide important biological information, to analyze and interpret structures of M. tuberculosis proteins, and to work collaboratively to test ideas about M. tuberculosis protein function that are suggested by structure or related to structural information. This review describes the TB Structural Genomics Consortium and some of the proteins for which the Consortium is in the progress of determining three-dimensional structures.

Effects of ultrasound on agglutination and aggregation of human erythrocytes in vitro.

A new experimental approach has shown that human erythrocytes of different blood groups were induced to form more agglutinates at a sound pressure of 70-240 kPa in vitro than the control erythrocytes. Similar effects were observed for alcian blue and dextran stimulated aggregation and for spontaneous aggregation. The increase of agglutination or aggregation was reversible. Heating and acoustic cavitation were shown not to be responsible for this effect. Bulk fluid movement produced by ultrasound irradiation appeared to cause the described phenomenon. Possible underlying mechanisms connecting the acoustic streaming and agglutination or aggregation behaviour of the cells are proposed.

Absolute configurations of Emycin D, E and F; mimicry of centrosymmetric space groups by mixtures of chiral stereoisomers.

The crystal structures of Emycin E (1), di-o-bromobenzoyl-Emycin F (2) and o-bromobenzoyl-Emycin D (3) have been determined by X-ray analysis at low temperature. Emycin E and o-bromobenzoyl-Emycin D both crystallize with two molecules in a triclinic unit cell. These two structures can be solved and refined either in the centrosymmetric space group P1;, with apparent disorder localized at or around the expected chiral centre, or in the non-centrosymmetric space group P1 as mixtures of two diastereomers without disorder. Only the latter interpretation is consistent with the chemical and spectroscopic evidence. Refinements in the centrosymmetric and non-centrosymmetric space groups are compared in this paper and are shown to favour the chemically correct interpretation, more decisively so in the case of the bromo derivative as a result of the anomalous dispersion of bromine. Structures (1) and (3) provide a dramatic warning of the dangers inherent in the conventional wisdom that if a structure can be refined satisfactorarily in both centrosymmetric and non-centrosymmetric space groups, the former should always be chosen. In these two cases, despite apparently acceptable intensity statistics and R factors (5.87 and 3.55%), the choice of the centrosymmetric space group leads to the serious chemical error that the triclinic unit cell contains a racemate rather than two chiral diastereomers! The weakest reflections are shown to be most sensitive to the correct choice of space group, underlining the importance of refining against all data rather than against intensities greater than a specified threshold. The use of similar-distance restraints is shown to be beneficial in both P1 refinements. Di-o-bromobenzoyl-Emycin F crystallizes in the monoclinic space group P2(1) with one molecule in the asymmetric unit and so does not give rise to these problems of interpretation. The absolute configuration of the two bromo derivatives, and hence the Emycins in general, was determined unambiguously as S at the chiral centre C3.

The main inconsequences in the present radiological protection concept for the general population.

The present concept of radiation protection emphasizes the protection of man against ionizing radiation from manmade sources. But the overwhelming radiation exposure in a given population results from the natural radiation environment (NRE) and the use of radiation in medicine, while less than 2% of the total dose is due to occupational exposure, fallout, releases from nuclear industries, etc. Nevertheless, ICRP recommendations do not apply to, or include, "normal" NRE exposure, nor do they deal with medical exposure of patients by applying risk/benefit considerations. The increasing problems associated with various types of NRE exposures (recycling of waste materials, energy-efficient dwellings) and the continuously increasing use of ionizing radiation in medical practices (x-ray and nuclear diagnostic examinations) require a revision of the present radiation protection concept. In this paper suggestions are made concerning the applicability of a unified concept for controlling all sources of ionizing radiation.

Radiation exposure of the respiratory tract and associated carcinogenic risk due to inhaled radon daughters.

The atmospheric content of radon and its decay products contributes significantly to the radiation exposure of man even in the normal environment. In this study, the risk for lung cancer induction associated with normal exposure to natural radionuclides is assessed in Salzburg, Austria. Altogether more than a thousand rooms have been investigated by using combined radon grab-sampling methods together with continuous measurements of radon and daughters at different control stations. Dose calculations were carried out for 729 demoscopically selected test persons, considering individual differences in age, sex and life-style and atmospheric nuclide concentrations at different sites. Using a specially developed age-dependent lung model, the dose frequency distribution of the absorbed dose to the basal cells of the bronchial epithelium could be evaluated for the population of Salzburg. Dose modifications caused by anatomical and physiological variabilities as well as microdosimetric considerations of the stochastic events during energy deposition result in a significantly increased dispersion of the dose histogram. Induction of lung cancer due to inhalation of radon and decay products can be correlated with cumulative exposure based on experience with lung cancer incidence amongst uranium miners. Using the recommended range of lifetime risk values of 20-45 X 10(-5)/WLM (UN77), it is shown for Salzburg that about 15% of the observed number of lung cancer cases may be induced by natural radionuclides.