Kinetics, molecular basis, and differentiation of L-lactate transport in spermatogenic cells

Round spermatid energy metabolism is closely dependent on the presence of L-lactate in the external medium. This L-lactate has been proposed to be supplied by Sertoli cells in the seminiferous tubules. L-Lactate, in conjunction with glucose, modulates intracellular Ca²⁺ concentration in round spermatids and pachytene spermatocytes. In spite of this central role of L-lactate in spermatogenic cell physiology, the mechanism of L-lactate transport, as well as possible differentiation during spermatogenesis, has not been studied in these cells. By measuring radioactive L-lactate transport and intracellular pH (pH_i) changes with pH_i fluorescent probes, we show that these cells transport L-lactate using monocarboxylate-H⁺ transport (MCT) systems. RT-PCR, in situ mRNA hybridization, and immunocyto- and immunohistochemistry data show that pachytene spermatocytes express mainly the MCT1 and MCT4 isoforms of the transporter (intermediate- and low-affinity transporters, respectively), while round spermatids, besides MCT1 and MCT4, also show expression of the MCT2 isoform (high-affinity transporter). These molecular data are consistent with the kinetic data of L-lactate transport in these cells demonstrating at least two transport components for L-lactate. These separate transport components reflect the ability of these cells to switch between the generation of glycolytic L-lactate in the presence of external glucose and the use of L-lactate when this substrate is available in the external environment. The supply of these substrates is regulated by the hormonal control of Sertoli cell glycolytic activity. cell differentiation; seminiferous tubules; spermatogenesis; testicle; meiosis     

Oxidation of milled wood lignin with laccase, tyrosinase and horseradish peroxidase

In this paper the oxidation of milled wood lignin (MWL), catalysed by three enzymes, i.e. laccase, tyrosinase and horseradish peroxidase (HRP) was studied. The oxidation was followed by measuring the consumption of O₂ during laccase and tyrosinase treatment and of H₂O₂ during HRP treatment. Both laccase and HRP were found to oxidise lignin effectively, whereas the effect of tyrosinase was negligible. The changes in MWL molecular-weight distributions caused in the reactions were analysed by gel permeation chromatography. Both laccase and HRP treatments were found to polymerise MWL. Peroxidase treatment was found to decrease the amount of phenolic hydroxyls in MWL, whereas no such effect could be detected in the laccase-treated sample. Both laccase and HRP treatments were, however, found to increase the amount of conjugated structures in MWL. The formation of phenoxy radicals during the treatments was studied by electron paramagnetic resonance spectroscopy. Phenoxy radicals were detected in both laccase and HRP-treated samples. The amount of the formed phenoxy radicals was found to be essentially constant during the detected time (i.e. 20–120 min after the addition of enzyme).     

Development of a novel compact sonicator for cell disruption

Ultrasound microbial cell disrupters operating at around 20 kHz are often physically large and, due to significant heating, can be unsuitable for small sample volumes where biochemical integrity of the extracted product is required. Development of a compact device based on a 63.5-mm diameter, 6.5-mm thick tubular transducer for rapid cell disruption in small-volume samples in a high-intensity acoustic cavitation field with minimal temperature rises is described here. Suspensions of Saccharomyces cerevisiae were exposed to cavitation for various times in the compact device and a 20-kHz probe sonicator. Cell disruption was assessed by protein release and by staining. Yeast cell disruption was greater in the novel 267-kHz sonicator than in the 20-kHz probe sonicator for the same exposure time. A 1-dimensional (1-D) transfer matrix model analysis for piezoelectric resonators was applied to an axial cross-section of the tubular sonicator to predict frequencies of mechanical resonance in the sample volume associated with maximum acoustic pressure. Admittance measurements identified frequencies of electrical resonance. Ultrasonic cavitation noise peaks were detected by a hydrophone at both the mechanical and electrical resonances. Cell breakage efficiency was twice as great in terms of protein released per dissipated watt at the mechanical resonance predicted by the model, compared to those at the electrical resonance frequencies. The results form a basis for rational design of an ultrasound cell disruption technique for small-volume samples.     

Strain development in Bacillus licheniformis: construction of biologically contained mutants deficient in sporulation and DNA repair

By introducing defined deletions in recA and an essential sporulation gene (spoIV), stable mutant strains of Bacillus licheniformis were obtained which are totally asporogenous and severely affected in DNA repair, and thus being UV-hypersensitive. Studies on growth in various liquid media as well as on amylase production revealed no differences of the mutants when compared to the wild type. Hence, such genes appear to be suitable disruption targets for achieving passive biological containment in this industrially exploited species.     

Structural insights into fusidic acid resistance and sensitivity in EF-G

Fusidic acid (FA) is a steroid antibiotic commonly used against Gram positive bacterial infections. It inhibits protein synthesis by stalling elongation factor G (EF-G) on the ribosome after translocation. A significant number of the mutations conferring strong FA resistance have been mapped at the interfaces between domains G, III and V of EF-G. However, direct information on how such mutations affect the structure has hitherto not been available. Here we present the crystal structures of two mutants of Thermus thermophilus EF-G, G16V and T84A, which exhibit FA hypersensitivity and resistance in vitro, respectively. These mutants also have higher and lower affinity for GTP respectively than wild-type EF-G. The mutations cause significant conformational changes in the switch II loop that have opposite effects on the position of a key residue, Phe90, which undergoes large conformational changes. This correlates with the importance of Phe90 in FA sensitivity reported in previous studies. These structures substantiate the importance of the domain G/domain III/domain V interfaces as a key component of the FA binding site. The mutations also cause subtle changes in the environment of the "P-loop lysine", Lys25. This led us to examine the conformation of the equivalent residue in all structures of translational GTPases, which revealed that EF-G and eEF2 form a group separate from the others and suggested that the role of Lys25 may be different in the two groups.     

Spatial models of virus-immune dynamics

To date, the majority of theoretical models describing the dynamics of infectious diseases in vivo are based on the assumption of well-mixed virus and cell populations. Because many infections take place in solid tissues, spatially structured models represent an important step forward in understanding what happens when the assumption of well-mixed populations is relaxed. Here, we explore models of virus and virus-immune dynamics where dispersal of virus and immune effector cells was constrained to occur locally. The stability properties of our spatial virus-immune dynamics models remained robust under almost all biologically plausible dispersal schemes, regardless of their complexity. The various spatial dynamics were compared to the basic non-spatial dynamics and important differences were identified: When space was assumed to be homogeneous, the dynamics generated by non-spatial and spatially structured models differed substantially at the peak of the infection. Thus, non-spatial models may lead to systematic errors in the estimates of parameters underlying acute infection dynamics. When space was assumed to be heterogeneous, spatial coupling not only changed the equilibrium properties of the uncoupled populations but also equalized the dynamics and thereby reduced the likelihood of dynamic elimination of the infection. In line with experimental and clinical observations, long-lasting oscillation periods were virtually absent. When source-sink dynamics were considered, the long-term outcome of the infection depended critically on the degree of spatial coupling. The infection collapsed when emigration from source sites became too large. Finally, we discuss the implications of spatially structured models on medical treatment of infectious diseases, and note that a huge gap exists in data accurately describing infection dynamics in solid tissues.     

Epitope down-modulation as a mechanism for the coexistence of competing T-cells

Efficient immune responses against pathogens are frequently characterized by the simultaneous targeting of multiple epitopes. However, it remains unclear how the targeting of multiple epitopes is maintained in the face of competition for antigenic stimulation. Here, we investigate this question by using mathematical models of the population dynamics of a viral pathogen, antigen presentation sites and T-cells. We first show that direct competition for access to antigen presenting sites and indirect competition through killing of the pathogen select for dominance of the T-cell response with the highest affinity for its epitope. We then incorporate in our model that epitopes can become down-modulated following interaction with epitope specific T-cells. We demonstrate that epitope down-modulation leads to differentiation of epitope presentation on antigen presenting sites. This differentiation promotes the coexistence of multiple epitope specific responses. Hence, we propose that the functional relevance of epitope down-modulation may be to enable the persistence of a broad immune response despite competition for antigenic stimulation.     

Toll-like receptor 3 is induced by and mediates antiviral activity against rhinovirus infection of human bronchial epithelial cells

Rhinoviruses (RV) are the major cause of the common cold and acute exacerbations of asthma and chronic obstructive pulmonary disease. Toll-like receptors (TLRs) are a conserved family of receptors that recognize and respond to a variety of pathogen-associated molecular patterns. TLR3 recognizes double-stranded RNA, an important intermediate of many viral life cycles (including RV). The importance of TLR3 in host responses to virus infection is not known. Using BEAS-2B (a human bronchial epithelial cell-line), we demonstrated that RV replication increased the expression of TLR3 mRNA and TLR3 protein on the cell surface. We observed that blocking TLR3 led to a decrease in interleukin-6, CXCL8, and CCL5 in response to poly(IC) but an increase following RV infection. Finally, we demonstrated that TLR3 mediated the antiviral response. This study demonstrates an important functional requirement for TLR3 in the host response against live virus infection and indicates that poly(IC) is not always a good model for studying the biology of live virus infection.     

Adaptive omnivory and species coexistence in tri-trophic food webs

The commonness of omnivory in natural communities is puzzling, because simple dynamic models of tri-trophic systems with omnivory are prone to species extinction. In particular, the intermediate consumer is frequently excluded by the omnivore at high levels of enrichment. It has been suggested that adaptive foraging by the omnivore may facilitate coexistence, because the intermediate consumer should persist more easily if it is occasionally dropped from the omnivore's diet. We explore theoretically how species permanence in tri-trophic systems is affected if the omnivore forages adaptively according to the "diet rule", i.e., feeds on the less profitable of its two prey species only if the more profitable one is sufficiently rare. We show that, compared to systems where omnivory is fixed, adaptive omnivory may indeed facilitate 3-species persistence. Counter to intuition, however, facilitation of 3-species coexistence requires that the intermediate consumer is a more profitable prey than the basal resource. Consequently, adaptive omnivory does not facilitate persistence of the intermediate consumer but enlarges the persistence region of the omnivore towards parameter space where a fixed omnivore would be excluded by the intermediate consumer. Overall, the positive effect of adaptive omnivory on 3-species persistence is, however, small. Generally, whether omnivory is fixed or adaptive, 3-species permanence is most likely when profitability (=conversion efficiency into omnivores) is low for basal resources and high for intermediate consumers.