Loss of HR6B ubiquitin-conjugating activity results in damaged synaptonemal complex structure and increased crossing-over frequency during the male meiotic prophase

The ubiquitin-conjugating enzymes HR6A and HR6B are the two mammalian homologs of Saccharomyces cerevisiae RAD6. In yeast, RAD6 plays an important role in postreplication DNA repair and in sporulation. HR6B knockout mice are viable, but spermatogenesis is markedly affected during postmeiotic steps, leading to male infertility. In the present study, increased apoptosis of HR6B knockout primary spermatocytes was detected during the first wave of spermatogenesis, indicating that HR6B performs a primary role during the meiotic prophase. Detailed analysis of HR6B knockout pachytene nuclei showed major changes in the synaptonemal complexes. These complexes were found to be longer. In addition, we often found depletion of synaptonemal complex proteins from near telomeric regions in the HR6B knockout pachytene nuclei. Finally, we detected an increased number of foci containing the mismatch DNA repair protein MLH1 in these nuclei, reflecting a remarkable and consistent increase (20 to 25%) in crossing-over frequency. The present findings reveal a specific requirement for the ubiquitin-conjugating activity of HR6B in relation to dynamic aspects of the synaptonemal complex and meiotic recombination in spermatocytes.     

Progesterone, but not progesterone-independent activation of progestin receptors by a mating stimulus, rapidly decreases progestin receptor immunoreactivity in female rat brain

Recent studies suggest that progestin receptors may be activated in vivo by neurotransmitters in the absence of ligand. More specifically, vaginal-cervical stimulation (VCS) can influence sexual behavior by activating progestin receptors in the absence of progesterone. Another way to test if progestin receptors are influenced by particular stimuli is to examine progestin receptor immunostaining. We report that progestin receptor immunoreactivity is decreased in the forebrain of estradiol-primed ovariectomized (OVX) rats within 1 h after a subcutaneous injection of progesterone, a time by which rapid down-regulation of progestin receptors does not seem to have occurred. In estradiol-primed OVX rats, VCS also decreased progestin receptor immunoreactivity within 1 h in the medial preoptic area, but not in any other area examined. To determine if the decrease in immunoreactivity by VCS was due to adrenal secretions or by ligand-independent activation of progestin receptors, we repeated the experiment in estradiol-primed OVX/adrenalectomized rats. Prior removal of the adrenal glands blocked the rapid decrease in progestin receptor immunoreactivity, even though data from other experiments suggest that progestin receptors are activated by VCS at this time. These studies suggest the possibility that progestin receptors may be affected differentially by progesterone-dependent or by progesterone-independent pathways. This raises the possibility that activation of progestin receptors by these two distinct pathways may lead to different neuronal consequences.     

13C and 1H Nuclear Magnetic Resonance Study of Glycogen Futile Cycling in Strains of the Genus Fibrobacter

We investigated the carbon metabolism of three strains of Fibrobacter succinogenes and one strain of Fibrobacter intestinalis. The four strains produced the same amounts of the metabolites succinate, acetate, and formate in approximately the same ratio (3.7/1/0.3). The four strains similarly stored glycogen during all growth phases, and the glycogen-to-protein ratio was close to 0.6 during the exponential growth phase. ¹³C nuclear magnetic resonance (NMR) analysis of [1-¹³C]glucose utilization by resting cells of the four strains revealed a reversal of glycolysis at the triose phosphate level and the same metabolic pathways. Glycogen futile cycling was demonstrated by ¹³C NMR by following the simultaneous metabolism of labeled [¹³C]glycogen and exogenous unlabeled glucose. The isotopic dilutions of the CH₂ of succinate and the CH₃ of acetate when the resting cells were metabolizing [1-¹³C]glucose and unlabeled glycogen were precisely quantified by using ¹³C-filtered spin-echo difference ¹H NMR spectroscopy. The measured isotopic dilutions were not the same for succinate and acetate; in the case of succinate, the dilutions reflected only the contribution of glycogen futile cycling, while in the case of acetate, another mechanism was also involved. Results obtained in complementary experiments are consistent with reversal of the succinate synthesis pathway. Our results indicated that for all of the strains, from 12 to 16% of the glucose entering the metabolic pathway originated from prestored glycogen. Although genetically diverse, the four Fibrobacter strains studied had very similar carbon metabolism characteristics.     

The Capsule and S-Layer: Two Independent and Yet Compatible Macromolecular Structures in Bacillus anthracis

Bacillus anthracis, the etiological agent of anthrax, is a gram-positive spore-forming bacterium. Fully virulent bacilli are toxinogenic and capsulated. Two abundant surface proteins, including the major antigen, are components of the B. anthracis surface layer (S-layer). The B. anthracis paracrystalline S-layer has previously only been found in noncapsulated vegetative cells. Here we report that the S-layer proteins are also synthesized under conditions where the poly-γ-d-glutamic acid capsule is present. Structural and immunological analyses show that the capsule is exterior to and completely covers the S-layer proteins. Nevertheless, analysis of single and double S-layer protein mutants shows that the presence of these proteins is not required for normal capsulation of the bacilli. Similarly, the S-layer proteins assemble as a two-dimensional crystal, even in the presence of the capsule. Thus, both structures are compatible, and yet neither is required for the correct formation of the other.

Bacillus anthracis, a gram-positive spore-forming bacterium, is the causative agent of anthrax. This disease, to which many animals, including humans, are susceptible, involves toxemia and septicemia. In the mammalian host, B. anthracis bacilli synthesize two toxins (lethal and edema toxins) (31) and a capsule (18) encoded by two large plasmids, pXO1 and pXO2, respectively (12, 21). The capsule is composed of poly-γ-d-glutamic acid and has antiphagocytic properties (13, 31, 37). Although unusual, a similar capsule is also found on Bacillus licheniformis bacilli (9). In the absence of pXO2 or the inducer bicarbonate, the cell does not produce a capsule and the cell wall appears layered. These layers are composed of fragments displaying a highly patterned ultrastructure (10, 16). This type of cell surface is now referred to as the surface layer (S-layer).S-layers are present on the surfaces of many archaea and bacteria (for reviews, see references 29 and 30). Most are formed by noncovalent, entropy-driven assembly of a single (glyco)protein protomer on the bacterial surface, giving rise to proteinaceous paracrystalline layers. Generally, a single S-layer is present, constituting 5 to 10% of total cell protein. Its synthesis is thus presumably energy consuming for the bacterium. Numerous bacteria have S-layers, suggesting that they play important roles in the interaction between the cell and its environment. Various functions have been proposed for S-layers, including shape maintenance and molecular sieving, and they can serve in phage fixation. The S-layer may be a virulence factor, protecting pathogenic bacteria against complement killing, facilitating binding of bacteria to host molecules, or enhancing their ability to associate with macrophages (for reviews, see references 27 and 29).Some bacteria, such as cyanobacteria or Azotobacter spp., possess both a capsule and an S-layer; however, to our knowledge, their structural relationships have not been analyzed through simultaneous genetic and cytologic studies. Both of these features have been independently described for the surface of the pathogenic bacterium B. anthracis. The components of the B. anthracis S-layer are two abundant surface proteins, EA1 and Sap (6, 20). Previous analyses of the B. anthracis S-layer used plasmid-cured strains; consequently, the interaction, if any, between the capsule and the S-layer could not be studied. Temporal or environmental regulation could be such that only one or the other structure is ever present at the cell surface. However, we show that S-layer proteins are synthesized under conditions where the bacilli are capsulated. We determined the localizations of capsule and S-layer components and analyzed whether the S-layer is necessary for proper capsulation. Finally, the assembly of the S-layer proteins in a two-dimensional crystal was examined in the presence of the capsule.     

Protection of DNA by salts against thermodegradation at temperatures typical for hyperthermophiles

The effect of physiological concentrations of KCl and MgCl₂ on the chemical stability of double-stranded and single-stranded DNA has been studied at temperatures typical for hyperthermophiles. These two salts protect both double and single-stranded DNA against heat-induced cleavage by inhibiting depurination. High KCl concentrations also protect DNA cleavage at apurinic sites, while high MgCl₂ concentrations stimulate this cleavage. It has been previously proposed that salt protects double-stranded DNA against depurination by stabilizing the double helix. However, the inhibition of the depurination of single-stranded DNA by KCl and MgCl₂ indicates that this effect is more probably due to a direct interaction of salts with purine nucleotides. These results suggest that the number and nature of heat-induced DNA lesions which have to be repaired might be quite different from one hyperthermophile to another, depending on their intracellular salt concentration. High salt concentrations might be also useful to protect DNA in long polymerase chain reaction (PCR) experiments and for long-term preservation. Received: October 12, 1997 / Accepted: January 29, 1998     

Airway Response to Methacholine following Eucapnic Voluntary Hyperpnea in Athletes

Aim

To evaluate the changes in airway responsiveness to methacholine inhalation test (MIT) when performed after an eucapnic voluntary hyperpnea challenge (EVH) in athletes.

Methods

Two MIT preceded (visit 1) or not (visit 2) by an EVH, were performed in 28 athletes and 24 non-athletes. Twelve athletes and 13 non-athletes had airway hyperresponsiveness (AHR) to methacholine, and 11 athletes and 11 non-athletes had AHR to EVH (EVH+).

Results

The MIT PC₂₀ post-EVH was significantly lower compared to baseline MIT PC₂₀ by 1.3±0.7 doubling-concentrations in EVH+ athletes only (p<0.0001). No significant change was observed in EVH- athletes and EVH+/EVH- non-athletes. A significant correlation between the change in MIT PC₂₀ post-EVH and EVH+/EVH- status and athlete/nonathlete status was found (Adjusted R²=0.26 and p<0.001). Three (11%) athletes and one (4%) non-athlete had a change in the diagnosis of AHR when MIT was performed consecutively to EVH.

Conclusion

The responsiveness to methacholine was increased by a previous indirect challenge in EVH+ athletes only. The mechanisms for such increase remain to be determined. MIT and EVH should ideally be performed on separate occasions as there is a small but possible risk to obtain a false-positive response to methacholine when performed immediately after the EVH.

Trial Registration

ClinicalTrials.gov NCT00686491     

Activation of Type I and III Interferon Response by Mitochondrial and Peroxisomal MAVS and Inhibition by Hepatitis C Virus

Sensing viruses by pattern recognition receptors (PRR) triggers the innate immune system of the host cell and activates immune signaling cascades such as the RIG-I/IRF3 pathway. Mitochondrial antiviral-signaling protein (MAVS, also known as IPS-1, Cardif, and VISA) is the crucial adaptor protein of this pathway localized on mitochondria, peroxisomes and mitochondria-associated membranes of the endoplasmic reticulum. Activation of MAVS leads to the production of type I and type III interferons (IFN) as well as IFN stimulated genes (ISGs). To refine the role of MAVS subcellular localization for the induction of type I and III IFN responses in hepatocytes and its counteraction by the hepatitis C virus (HCV), we generated various functional and genetic knock-out cell systems that were reconstituted to express mitochondrial (mito) or peroxisomal (pex) MAVS, exclusively. Upon infection with diverse RNA viruses we found that cells exclusively expressing pexMAVS mounted sustained expression of type I and III IFNs to levels comparable to cells exclusively expressing mitoMAVS. To determine whether viral counteraction of MAVS is affected by its subcellular localization we employed infection of cells with HCV, a major causative agent of chronic liver disease with a high propensity to establish persistence. This virus efficiently cleaves MAVS via a viral protease residing in its nonstructural protein 3 (NS3) and this strategy is thought to contribute to the high persistence of this virus. We found that both mito- and pexMAVS were efficiently cleaved by NS3 and this cleavage was required to suppress activation of the IFN response. Taken together, our findings indicate comparable activation of the IFN response by pex- and mitoMAVS in hepatocytes and efficient counteraction of both MAVS species by the HCV NS3 protease.     

CTXφ Replication Depends on the Histone-Like HU Protein and the UvrD Helicase

The Vibrio cholerae bacterium is the agent of cholera. The capacity to produce the cholera toxin, which is responsible for the deadly diarrhea associated with cholera epidemics, is encoded in the genome of a filamentous phage, CTXφ. Rolling-circle replication (RCR) is central to the life cycle of CTXφ because amplification of the phage genome permits its efficient integration into the genome and its packaging into new viral particles. A single phage-encoded HUH endonuclease initiates RCR of the proto-typical filamentous phages of enterobacteriaceae by introducing a nick at a specific position of the double stranded DNA form of the phage genome. The rest of the process is driven by host factors that are either essential or crucial for the replication of the host genome, such as the Rep SF1 helicase. In contrast, we show here that the histone-like HU protein of V. cholerae is necessary for the introduction of a nick by the HUH endonuclease of CTXφ. We further show that CTXφ RCR depends on a SF1 helicase normally implicated in DNA repair, UvrD, rather than Rep. In addition to CTXφ, we show that VGJφ, a representative member of a second family of vibrio integrative filamentous phages, requires UvrD and HU for RCR while TLCφ, a satellite phage, depends on Rep and is independent from HU.     

Response of the Cholesterol Metabolism to a Negative Energy Balance in Dairy Cows Depends on the Lactational Stage

The response of cholesterol metabolism to a negative energy balance (NEB) induced by feed restriction for 3 weeks starting at 100 days in milk (DIM) compared to the physiologically occurring NEB in week 1 postpartum (p.p.) was investigated in 50 dairy cows (25 control (CON) and 25 feed-restricted (RES)). Blood samples, liver biopsies and milk samples were taken in week 1 p.p., and in weeks 0 and 3 of feed restriction. Plasma concentrations of total cholesterol (C), phospholipids (PL), triglycerides (TAG), very low density lipoprotein-cholesterol (VLDL-C) and low density lipoprotein-cholesterol (LDL-C) increased in RES cows from week 0 to 3 during feed restriction and were higher in week 3 compared to CON cows. In contrast, during the physiologically occurring NEB in week 1 p.p., C, PL, TAG and lipoprotein concentrations were at a minimum. Plasma phospholipid transfer protein (PLTP) and lecithin:cholesterol acyltransferase (LCAT) activities did not differ between week 0 and 3 for both groups, whereas during NEB in week 1 p.p. PLTP activity was increased and LCAT activity was decreased. Milk C concentration was not affected by feed restriction in both groups, whereas milk C mass was decreased in week 3 for RES cows. In comparison, C concentration and mass in milk were elevated in week 1 p.p. Hepatic mRNA abundance of sterol regulatory element-binding factor-2 (SREBF-2), 3-hydroxy-3-methylglutaryl-coenzyme A synthase 1 (HMGCS1), 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR), and ATP-binding cassette transporter (ABCA1) were similar in CON and RES cows during feed restriction, but were upregulated during NEB in week 1 p.p. compared to the non-lactating stage without a NEB. In conclusion, cholesterol metabolism in dairy cows is affected by nutrient and energy deficiency depending on the stage of lactation.     

In-Depth Characterization of Live Vaccines Used in Europe for Oral Rabies Vaccination of Wildlife

Although rabies incidence has fallen sharply over the past decades in Europe, the disease is still present in Eastern Europe. Oral rabies immunization of wild animal rabies has been shown to be the most effective method for the control and elimination of rabies. All rabies vaccines used in Europe are modified live virus vaccines based on the Street Alabama Dufferin (SAD) strain isolated from a naturally-infected dog in 1935. Because of the potential safety risk of a live virus which could revert to virulence, the genetic composition of three commercial attenuated live rabies vaccines was investigated in two independent laboratories using next genome sequencing. This study is the first one reporting on the diversity of variants in oral rabies vaccines as well as the presence of a mix of at least two different variants in all tested batches. The results demonstrate the need for vaccine producers to use new robust methodologies in the context of their routine vaccine quality controls prior to market release.