The Mycobacterium tuberculosis Secreted Protein Rv0203 Transfers Heme to Membrane Proteins MmpL3 and MmpL11

Mycobacterium tuberculosis is the causative agent of tuberculosis, which is becoming an increasingly global public health problem due to the rise of drug-resistant strains. While residing in the human host, M. tuberculosis needs to acquire iron for its survival. M. tuberculosis has two iron uptake mechanisms, one that utilizes non-heme iron and another that taps into the vast host heme-iron pool. To date, proteins known to be involved in mycobacterial heme uptake are Rv0203, MmpL3, and MmpL11. Whereas Rv0203 transports heme across the bacterial periplasm or scavenges heme from host heme proteins, MmpL3 and MmpL11 are thought to transport heme across the membrane. In this work, we characterize the heme-binding properties of the predicted extracellular soluble E1 domains of both MmpL3 and MmpL11 utilizing absorption, electron paramagnetic resonance, and magnetic circular dichroism spectroscopic methods. Furthermore, we demonstrate that Rv0203 transfers heme to both MmpL3-E1 and MmpL11-E1 domains at a rate faster than passive heme dissociation from Rv0203. This work elucidates a key step in the mycobacterial uptake of heme, and it may be useful in the development of anti-tuberculosis drugs targeting this pathway.     

A Mathematical and Numerical Investigation of the Hemodynamical Origins of Oscillations in Microvascular Networks

Evidence is presented to show that self-sustained oscillations of purely hemodynamical origin are possible in some arcade-type microvascular networks supplied with steady boundary conditions, but that in others the oscillations disappear with sufficient reduction of the time step Δt, showing them to be numerical artefacts. In an attempt to elucidate the mechanisms involved in the onset of fluctuations, we proceed to perform a linear stability analysis for the convective model of Kiani et al. (Microvasc. Res. 45:219–232, 1993; Am. J. Physiol. 266(35):H1822–H1828, 1994), and show that this leads via a system of delay differential equations to a nonlinear eigenvalue problem. This result generalises the characteristic equation obtained by Carr et al. (Ann. Biomed. Eng. 33:764–771, 2005) and Geddes et al. (SIAM J. Appl. Dyn. Syst. 6(4):694–727, 2007) who solved a special case in a two node network. An implicit numerical method is proposed for the computation of blood flows in networks using the convective model. In a moderate size subnetwork of one of the networks chosen by Kiani et al. (Am. J. Physiol. 266(35):H1822–H1828, 1994), the topology, vessel lengths, and diameters of which were based on microvascular networks in the rat mesentery, we compare results generated using the original explicit numerical method of Kiani et al. (Am. J. Physiol. 266(35):H1822–H1828, 1994) with those from our implicit scheme. From the linear stability theory, a critical value D _RBC,crit of a red blood cell diameter parameter D _RBC in the plasma skimming model of Fenton et al. (Pflügers Arch. 403:396–401, 1985b) is identified for the onset of oscillations about steady state and both the explicit and implicit methods are used to calculate the inflow hematocrit solutions in all vessels of the subnetwork at the critical parameter value, subject to perturbed initial conditions. The results of the implicit method are demonstrated to be in excellent and superior agreement with the predictions of the linear analysis in this case. For values of D _RBC slightly larger than D _RBC,crit the bifurcating periodic solutions calculated using either the explicit or implicit schemes are characteristic of those of a supercritical Hopf bifurcation and the graphs of D _RBC vs. oscillation amplitude would seem to converge as Δt→0.     

Secretions from the female gametophyte and their role in spermatozoid induction in Cycas revoluta

In cycads, spermatozoids are released from pollen tubes and swim in fluid toward the archegonia. The source of this fluid was examined using Cycas revoluta Thunb. ovules placed in culture. Dissected female gametophytes just before fertilization produced copious fluid on their upper surface. The fluid first appeared around the archegonial chamber and then on the inside of the archegonial chamber. When this fluid was applied to dry turgid pollen tubes, they discharged spermatozoids 12 h later. The archegonial neck appeared as two semi-spherical swellings, whereas the four neck cells later became visible and they separated in a schizogenous manner. Many globose particles appear on the top of the archegonial neck cells when the fluid is present. The contents of pollen tubes, spermatozoids and surrounding liquid intermingle with the secreted fluid. The female gametophyte differs in ultrastructure during the stages before and after fluid secretion, the latter showing changes suggestive of fluid secretion from the female gametophyte.     

The Touch Dome Defines an Epidermal Niche Specialized for Mechanosensory Signaling

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Inhibition of Dopamine Transporter Activity by G Protein βγ Subunits

Uptake through the Dopamine Transporter (DAT) is the primary mechanism of terminating dopamine signaling within the brain, thus playing an essential role in neuronal homeostasis. Deregulation of DAT function has been linked to several neurological and psychiatric disorders including ADHD, schizophrenia, Parkinson’s disease, and drug addiction. Over the last 15 years, several studies have revealed a plethora of mechanisms influencing the activity and cellular distribution of DAT; suggesting that fine-tuning of dopamine homeostasis occurs via an elaborate interplay of multiple pathways. Here, we show for the first time that the βγ subunits of G proteins regulate DAT activity. In heterologous cells and brain tissue, a physical association between Gβγ subunits and DAT was demonstrated by co-immunoprecipitation. Furthermore, in vitro pull-down assays using purified proteins established that this association occurs via a direct interaction between the intracellular carboxy-terminus of DAT and Gβγ. Functional assays performed in the presence of the non-hydrolyzable GTP analog GTP-γ-S, Gβγ subunit overexpression, or the Gβγ activator mSIRK all resulted in rapid inhibition of DAT activity in heterologous systems. Gβγ activation by mSIRK also inhibited dopamine uptake in brain synaptosomes and dopamine clearance from mouse striatum as measured by high-speed chronoamperometry in vivo. Gβγ subunits are intracellular signaling molecules that regulate a multitude of physiological processes through interactions with enzymes and ion channels. Our findings add neurotransmitter transporters to the growing list of molecules regulated by G-proteins and suggest a novel role for Gβγ signaling in the control of dopamine homeostasis.     

Extent and Degree of Shoreline Oiling: Deepwater Horizon Oil Spill,Gulf of Mexico,USA

The oil from the 2010 Deepwater Horizon spill in the Gulf of Mexico was documented by shoreline assessment teams as stranding on 1,773 km of shoreline. Beaches comprised 50.8%, marshes 44.9%, and other shoreline types 4.3% of the oiled shoreline. Shoreline cleanup activities were authorized on 660 km, or 73.3% of oiled beaches and up to 71 km, or 8.9% of oiled marshes and associated habitats. One year after the spill began, oil remained on 847 km; two years later, oil remained on 687 km, though at much lesser degrees of oiling. For example, shorelines characterized as heavily oiled went from a maximum of 360 km, to 22.4 km one year later, and to 6.4 km two years later. Shoreline cleanup has been conducted to meet habitat-specific cleanup endpoints and will continue until all oiled shoreline segments meet endpoints. The entire shoreline cleanup program has been managed under the Shoreline Cleanup Assessment Technique (SCAT) Program, which is a systematic, objective, and inclusive process to collect data on shoreline oiling conditions and support decision making on appropriate cleanup methods and endpoints. It was a particularly valuable and effective process during such a complex spill.     

Proteomic analysis of Aspergillus fumigatus – clinical implications

Introduction: Aspergillus fumigatus is a ubiquitous saprophytic fungus capable of producing small airborne spores, which are frequently inhaled by humans. In healthy individuals, the fungus is rapidly cleared by innate mechanisms, including immune cells. However, in individuals with impaired lung function or immunosuppression the spores can germinate and prompt severe allergic responses, and disease with limited or extensive invasiveness.

Areas covered: The traits that make A. fumigatus a successful colonizer and pathogen of humans are multi-factorial. Thus, a global investigative approach is required to elucidate the mechanisms utilized by the fungus to cause disease.

Expert commentary: In doing so, a better understanding of disease pathology can be achieved with improved therapeutic/diagnostic solutions, thereby improving patient outcome. Proteomic analysis permits such investigations and recent work has yielded insight into these mechanisms.     

A Rep recognition sequence is necessary but not sufficient for nicking of DNA by adeno-associated virus type-2 Rep proteins

The strand-specific, site-specific endonuclease (nicking) activity of the Rep68 and Rep78 (Rep68/78) proteins of adeno-associated virus type 2 (AAV) is involved in AAV replication, and appears to be involved in AAV site-specific integration. Rep68/78 cuts within the inverted terminal repeats (ITRs) of the AAV genome and in the AAV preferred integration locus on human chromosome 19 (AAVS1). The known endonuclease cut sites are 11-16 bases away from the primary binding sites, known as Rep recognition sequences (RRSs). A linear, double-stranded segment of DNA, containing an RRS and a cut site, has previously been shown to function as a substrate for the Rep68/78 endonuclease activity. We show here that mutation of the Rep recognition sequence, within such a DNA segment derived from the AAV ITRs, eliminates the ability of this substrate to be cleaved detectably by Rep78. Rep78 nicks the RRS-containing site from AAVS1 about half as well as the linear ITR sequence. Eighteen other RRS-containing sequences found in the human genome, but outside AAVS1, are not cleaved by Rep78. These results may help to explain the specificity of AAV integration.