Cutting Edge: Regulatory T cells prevent efficient clearance of Mycobacterium tuberculosis

Mycobacterium tuberculosis remains one of the top microbial killers of humans causing approximately 2 million deaths annually. More than 90% of the 2 billion individuals infected never develop active disease, indicating that the immune system is able to generate mechanisms that control infection. However, the immune response generally fails to achieve sterile clearance of bacilli. Using adoptive cell transfer into C57BL/6J-Rag1(tm1Mom) mice (Rag1(-/-)), we show that regulatory T cells prevent eradication of tubercle bacilli by suppressing an otherwise efficient CD4+ T cell response. This protective CD4+ T cell response was not correlated with increased numbers of IFN-gamma- or TNF-alpha-expressing cells or general expression levels of IFN-gamma or inducible NO synthase in infected organs compared with wild-type C57BL/6 animals. Furthermore, suppression of protection by cotransferred regulatory T cells was neither accompanied by a general increase of IL-10 expression nor by higher numbers of IL-10-producing CD4+ T cells.     

Acetyl-<Emphasis Type="SmallCaps">l</Emphasis>-carnitine prevents carbon tetrachloride-induced oxidative stress in various tissues of Wistar rats

Acetyl-l-carnitine (ALCAR) has been shown to prevent experimental selenite cataractogenesis, a manifestation of oxidative stress, but little is known about its potential in other settings of oxidative stress. The present study was based on the hypothesis that ALCAR prevents carbon tetrachloride (CCl₄)-induced oxidative stress in vital tissues. Male albino Wistar rats were divided into three groups, each of six rats. Group I (control) rats received only vehicle (1 ml/kg b.w.) for 4 days; Group II (CCl₄-exposed, untreated) rats received CCl₄ (2 ml/kg b.w.) on the second and third days and vehicle on the first and fourth days; Group III (CCl₄-exposed, ALCAR-treated) rats received ALCAR (200 mg/kg b.w.) for 4 days and CCl₄ on the second and third days. All administrations were made intraperitoneally. After the experimental period, significantly (P < 0.05) elevated mean serum levels of aspartate transaminase, alanine transaminase, alkaline phosphatase, and lactate dehydrogenase were observed in Group II rats when compared to Group I and Group III rats. The mean levels of vitamin C, vitamin E, and reduced glutathione and the mean activities of superoxide dismutase, catalase, and glutathione peroxidase were significantly (P < 0.05) lower in samples of hemolysate and of liver, kidney, and brain tissues of Group II rats than those in Group I and Group III rats. The mean level of lipid peroxidation was significantly (P < 0.05) higher in Group II rats than that in Group I and Group III rats. Moreover, the CCl₄-induced upregulation of inducible nitric oxide synthase expression was prevented by ALCAR in the liver and brain tissues. These results suggest that ALCAR is able to prevent the CCl₄-induced oxidative stress.     

An integrative approach combining ion mobility mass spectrometry,X-ray crystallography,and nuclear magnetic resonance spectroscopy to study the conformational dynamics of α1-antitrypsin upon ligand binding

Native mass spectrometry (MS) methods permit the study of multiple protein species within solution equilibria, whereas ion mobility (IM)-MS can report on conformational behavior of specific states. We used IM-MS to study a conformationally labile protein (α₁-antitrypsin) that undergoes pathological polymerization in the context of point mutations. The folded, native state of the Z-variant remains highly polymerogenic in physiological conditions despite only minor thermodynamic destabilization relative to the wild-type variant. Various data implicate kinetic instability (conformational lability within a native state ensemble) as the basis of Z α₁-antitrypsin polymerogenicity. We show the ability of IM-MS to track such disease-relevant conformational behavior in detail by studying the effects of peptide binding on α₁-antitrypsin conformation and dynamics. IM-MS is, therefore, an ideal platform for the screening of compounds that result in therapeutically beneficial kinetic stabilization of native α₁-antitrypsin. Our findings are confirmed with high-resolution X-ray crystallographic and nuclear magnetic resonance spectroscopic studies of the same event, which together dissect structural changes from dynamic effects caused by peptide binding at a residue-specific level. IM-MS methods, therefore, have great potential for further study of biologically relevant thermodynamic and kinetic instability of proteins and provide rapid and multidimensional characterization of ligand interactions of therapeutic interest.PDB Code(s): 4PYW     

Visualisation and stereological assessment of blood and lymphatic vessels

The physiological processes involved in tissue development and regeneration also include the parallel formation of blood and lymphatic vessel circulations which involves their growth, maturation and remodelling. Both vascular systems are also frequently involved in the development and progression of pathological conditions in tissues and organs. The blood vascular system circulates oxygenated blood and nutrients at appropriate physiological levels for tissue survival, and efficiently removes all waste products including carbon dioxide. This continuous network consists of the heart, aorta, arteries, arterioles, capillaries, post-capillary venules, venules, veins and vena cava. This system exists in an interstitial environment together with the lymphatic vascular system, including lymph nodes, which aids maintenance of body fluid balance and immune surveillance. To understand the process of vascular development, vascular network stability, remodelling and/or regression in any research model under any experimental conditions, it is necessary to clearly and unequivocally identify and quantify all elements of the vascular network. By utilising stereological methods in combination with cellular markers for different vascular cell components, it is possible to estimate parameters such as surface density and surface area of blood vessels, length density and length of blood vessels as well as absolute vascular volume. This review examines the current strategies used to visualise blood vessels and lymphatic vessels in two- and three-dimensions and the basic principles of vascular stereology used to quantify vascular network parameters.     

Polarization of the C. elegans zygote proceeds via distinct establishment and maintenance phases

Polarization of the C. elegans zygote along the anterior-posterior axis depends on cortically enriched (PAR) and cytoplasmic (MEX-5/6) proteins, which function together to localize determinants (e.g. PIE-1) in response to a polarizing cue associated with the sperm asters. Using time-lapse microscopy and GFP fusions, we have analyzed the localization dynamics of PAR-2, PAR-6, MEX-5, MEX-6 and PIE-1 in wild-type and mutant embryos. These studies reveal that polarization involves two genetically and temporally distinct phases. During the first phase (establishment), the sperm asters at one end of the embryo exclude the PAR-3/PAR-6/PKC3 complex from the nearby cortex, allowing the ring finger protein PAR-2 to accumulate in an expanding 'posterior' domain. Onset of the establishment phase involves the non-muscle myosin NMY-2 and the 14-3-3 protein PAR-5. The kinase PAR-1 and the CCCH finger proteins MEX-5 and MEX-6 also function during the establishment phase in a feedback loop to regulate growth of the posterior domain. The second phase begins after pronuclear meeting, when the sperm asters begin to invade the anterior. During this phase (maintenance), PAR-2 maintains anterior-posterior polarity by excluding the PAR-3/PAR-6/PKC3 complex from the posterior. These findings provide a model for how PAR and MEX proteins convert a transient asymmetry into a stably polarized axis.     

Different thresholds for detection and discrimination of odors in the honey bee (Apis mellifera)

Naturally occurring odors used by animals for mate recognition, food identification and other purposes must be detected at concentrations that vary across several orders of magnitude. Olfactory systems must therefore have the capacity to represent odors over a large range of concentrations regardless of dramatic changes in the salience, or perceived intensity, of a stimulus. The stability of the representation of an odor relative to other odors across concentration has not been extensively evaluated. We tested the ability of honey bees to discriminate pure odorants across a range of concentrations at and above their detection threshold. Our study showed that pure odorant compounds became progressively easier for honey bees to discriminate with increasing concentration. Discrimination is, therefore, a function of odorant concentration. We hypothesize that the recruitment of sensory cell populations across a range of concentrations may be important for odor coding, perhaps by changing its perceptual qualities or by increasing its salience against background stimuli, and that this mechanism is a general property of olfactory systems.     

Serine Phosphorylation of the Insulin-like Growth Factor I (IGF-1) Receptor C-terminal Tail Restrains Kinase Activity and Cell Growth

Insulin-like growth factor I receptor (IGF-1R) signaling is essential for cell, organ, and animal growth. The C-terminal tail of the IGF-1R exhibits regulatory function, but the mechanism is unknown. Here, we show that mutation of Ser-1248 (S1248A) enhances IGF-1R in vitro kinase activity, autophosphorylation, Akt/mammalian target of rapamycin activity, and cell growth. Ser-1248 phosphorylation is mediated by GSK-3β in a mechanism that involves a priming phosphorylation on Ser-1252. GSK-3β knock-out cells exhibit reduced IGF-1R cell surface expression, enhanced IGF-1R kinase activity, and signaling. Examination of crystallographic structures of the IGF-1R kinase domain revealed that the (1248)SFYYS(1252) motif adopts a conformation tightly packed against the kinase C-lobe when Ser-1248 is in the unphosphorylated state that favors kinase activity. S1248A mutation is predicted to lock the motif in this position. In contrast, phosphorylation of Ser-1248 will drive profound structural transition of the sequence, critically affecting connection of the C terminus as well as exposing potential protein docking sites. Decreased kinase activity of a phosphomimetic S1248E mutant and enhanced kinase activity in mutants of its predicted target residue Lys-1081 support this auto-inhibitory model. Thus, the SFYYS motif controls the organization of the IGF-1R C terminus relative to the kinase domain. Its phosphorylation by GSK-3β restrains kinase activity and regulates receptor trafficking and signaling.