Succinate Dehydrogenase is the Regulator of Respiration in Mycobacterium tuberculosis

In chronic infection, Mycobacterium tuberculosis bacilli are thought to enter a metabolic program that provides sufficient energy for maintenance of the protonmotive force, but is insufficient to meet the demands of cellular growth. We sought to understand this metabolic downshift genetically by targeting succinate dehydrogenase, the enzyme which couples the growth processes controlled by the TCA cycle with the energy production resulting from the electron transport chain. M. tuberculosis contains two operons which are predicted to encode succinate dehydrogenase enzymes (sdh-1 and sdh-2); we found that deletion of Sdh1 contributes to an inability to survive long term stationary phase. Stable isotope labeling and mass spectrometry revealed that Sdh1 functions as a succinate dehydrogenase during aerobic growth, and that Sdh2 is dispensable for this catalysis, but partially overlapping activities ensure that the loss of one enzyme can incompletely compensate for loss of the other. Deletion of Sdh1 disturbs the rate of respiration via the mycobacterial electron transport chain, resulting in an increased proportion of reduced electron carrier (menaquinol) which leads to increased oxygen consumption. The loss of respiratory control leads to an inability to recover from stationary phase. We propose a model in which succinate dehydrogenase is a governor of cellular respiration in the adaptation to low oxygen environments.     

Efficacy of Brain-Derived Neurotrophic Factor and Neurotrophin-3 on Neurochemical and Behavioral Deficits Associated with Partial Nigrostriatal Dopamine Lesions

Abstract: Brain-derived neurotrophic factor (BDNF) promotes the survival of dopamine (DA) neurons, enhances expression of DA neuron characteristics, and protects these cells from 6-hydroxydopamine (6-OHDA) toxicity in vitro. We tested the ability of BDNF or neurotrophin-3 (NT-3) to exert similar protective effects in vivo during chronic delivery of 6-OHDA to the rat neostriatum. Chronic infusions of BDNF or NT-3 (12 µg/day) above the substantia nigra were started 6 days before and continued during an 8-day chronic intrastriatal infusion of 6-OHDA. In control and neurotrophin-treated animals, 6-OHDA treatment selectively depleted 50–60% of nigrostriatal DA nerve terminals but produced little if any loss of pars compacta DA cell bodies. This partial DA lesion resulted in three rotations per minute toward the lesioned hemisphere after treatment with the DA release-inducing drug d-amphetamine. Compared with supranigral infusions of vehicle, BDNF and NT-3 decreased the number of these ipsiversive rotations by 70 and 48% and increased by 20- and 10-fold, respectively, the number of contraversive rotations observed after amphetamine injection. When challenged with the DA receptor agonist apomorphine, BDNF- and NT-3-treated animals also exhibited a seven- and 3.5-fold increase in the number of contraversive rotations relative to the vehicle group, respectively. Compared with vehicle, BDNF increased striatal levels of homovanillic acid (HVA; 86%), 3,4-dihydroxyphenylacetic acid (DOPAC; 42%), and 5-hydroxyindoleacetic acid (5-HIAA; 32%) and the HVA/DA (43%) and 5-HIAA/serotonin (34%) ratios in the DA-denervated striatum. NT-3 augmented only striatal 5-HIAA levels (24%). Neither factor altered the 6-OHDA-induced decrease in striatal DA levels or high-affinity DA uptake and thus did not protect against the destruction of DA terminals and did not alter striatal D₁ or D₂ ligand binding. Choline, GABA, and glutamate uptake in the striatum were not altered by the lesion or neurotrophin treatment. Thus, BDNF and to a lesser extent NT-3 reverse rotational behavioral deficits and augment striatal DA and 5-HT metabolism in a partial DA lesion model.     

Drosophila Boi limits Hedgehog levels to suppress follicle stem cell proliferation

Stem cells depend on signals from cells within their microenvironment, or niche, as well as factors secreted by distant cells to regulate their maintenance and function. Here we show that Boi, a Hedgehog (Hh)-binding protein, is a novel suppressor of proliferation of follicle stem cells (FSCs) in the Drosophila ovary. Hh is expressed in apical cells, distant from the FSC niche, and diffuses to reach FSCs, where it promotes FSC proliferation. We show that Boi is expressed in apical cells and exerts its suppressive effect on FSC proliferation by binding to and sequestering Hh on the apical cell surface, thereby inhibiting Hh diffusion. Our studies demonstrate that cells distant from the local niche can regulate stem cell function through ligand sequestration, a mechanism that likely is conserved in other epithelial tissues.     

Multiplex Identification of Microbes

We have adapted molecular inversion probe technology to identify microbes in a highly multiplexed procedure. This procedure does not require growth of the microbes. Rather, the technology employs DNA homology twice: once for the molecular probe to hybridize to its homologous DNA and again for the 20-mer oligonucleotide barcode on the molecular probe to hybridize to a commercially available molecular barcode array. As proof of concept, we have designed, tested, and employed 192 molecular probes for 40 microbes. While these particular molecular probes are aimed at our interest in the microbes in the human vagina, this molecular probe method could be employed to identify the microbes in any ecological niche.The substantial majority of the earth''s bacteria have not been grown in culture and do not form colonies on agar plates (for examples, see references 19 and 21). These statements are particularly true of the bacteria living in or on human beings (for examples, see references 2, 6, and 7). The Human Microbiome Project (for examples, see references 2, 5, 24, and 27) is employing DNA sequencing and other genome-based technologies to reveal the plethora of microbes living in or on humans. Our goal was to develop a massively multiplex method employing currently commercially available reagents to identify those microbes at the species level.Several useful approaches to the identification of microbes that do not form colonies on agar plates have been published. Many scientists have employed dideoxy sequencing of the PCR-amplified 16S rRNA gene to identify microbes (for an example, see reference 22). Dideoxy sequencing is expensive, cumbersome, and slow. “Next-generation” sequencing reduces the cost of sequencing but produces much shorter read lengths. As examples, Tarnberg et al. (26), Jonasson et al. (11), and Sundquist et al. (25) employed pyrosequencing of small portions of the 16S rRNA gene to identify microbes. These scientists could not always identify the microbes down to the species level. “Checkerboard DNA-DNA hybridization” (CDH) is a technology that is more than a decade old (23). Nikolaitchouk et al. (14) have applied CDH to identify the microbes in the human female genital tract and achieved a 13-plex reaction. Dumonceaux et al. (4) coupled microbe-specific oligonucleotides to fluorescently labeled microspheres. Subsequently, the microbes were detected and counted by flow cytometry. Dumonceaux et al. (4) achieved a 9-plex reaction. DeSantis et al. (3) designed and employed a microarray containing 297,851 oligonucleotide probes derived from the 16S rRNA gene from 842 subfamilies of prokaryotes. DeSantis et al. (3) concluded that their microarray revealed greater prokaryotic diversity than dideoxy sequencing of a “typically sized clone library.”None of these ingenious methods meets the requirements for a robust, commercially available, highly multiplex technology. Therefore, we have adapted an independent method to identify microbes: molecular inversion probes (8) coupled with a commercial molecular barcode array. This method does not require growth of the microbes. Rather, molecular probe technology is a nucleic acid-based technology employing DNA homology twice: once for the molecular probe to hybridize to its homologous DNA and again for the 20-mer oligonucleotide barcode on the molecular probe to hybridize to a commercially available oligonucleotide barcode array. We present here data demonstrating proof of concept in which molecular probes were designed, tested, and employed to detect microbes in simulated clinical samples. Because of our ongoing interest in the bacteria that inhabit the adult human vagina (10), we focus on that ecological niche. However, this method is sufficiently general that it can be applied to detect the microbes in any ecological niche, e.g., soil and the ocean.