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Coordinated carbon and nitrogen metabolism is crucial for bacteria living in the fluctuating environments. Intracellular carbon and nitrogen homeostasis is maintained by a sophisticated network, in which the widespread signaling protein PII acts as a major regulatory hub. In cyanobacteria, PII was proposed to regulate the nitrate uptake by an ABC (ATP-binding cassette)-type nitrate transporter NrtABCD, in which the nucleotide-binding domain of NrtC is fused with a C-terminal regulatory domain (CRD). Here, we solved three cryoelectron microscopy structures of NrtBCD, bound to nitrate, ATP, and PII, respectively. Structural and biochemical analyses enable us to identify the key residues that form a hydrophobic and a hydrophilic cavity along the substrate translocation channel. The core structure of PII, but not the canonical T-loop, binds to NrtC and stabilizes the CRD, making it visible in the complex structure, narrows the substrate translocation channel in NrtB, and ultimately locks NrtBCD at an inhibited inward-facing conformation. Based on these results and previous reports, we propose a putative transport cycle driven by NrtABCD, which is allosterically inhibited by PII in response to the cellular level of 2-oxoglutarate. Our findings provide a distinct regulatory mechanism of ABC transporter via asymmetrically binding to a signaling protein. [ABSTRACT FROM AUTHOR]

The myophage possesses a contractile tail that penetrates its host cell envelope. Except for investigations on the bacteriophage T4 with a rather complicated structure, the assembly pattern and tail contraction mechanism of myophage remain largely unknown. Here, we present the fine structure of a freshwater Myoviridae cyanophage Pam3, which has an icosahedral capsid of ~680 Å in diameter, connected via a three-section neck to an 840-Å-long contractile tail, ending with a three-module baseplate composed of only six protein components. This simplified baseplate consists of a central hub-spike surrounded by six wedge heterotriplexes, to which twelve tail fibers are covalently attached via disulfide bonds in alternating upward and downward configurations. In vitro reduction assays revealed a putative redox-dependent mechanism of baseplate assembly and tail sheath contraction. These findings establish a minimal myophage that might become a user-friendly chassis phage in synthetic biology. [ABSTRACT FROM AUTHOR]

Carboxysomes are membrane-free organelles for carbon assimilation in cyanobacteria. The carboxysome consists of a proteinaceous shell that structurally resembles virus capsids and internal enzymes including ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco), the primary carbon-fixing enzyme in photosynthesis. The formation of carboxysomes requires hierarchical self-assembly of thousands of protein subunits, initiated from Rubisco assembly and packaging to shell encapsulation. Here we study the role of Rubisco assembly factor 1 (Raf1) in Rubisco assembly and carboxysome formation in a model cyanobacterium, Synechococcus elongatus PCC7942 (Syn7942). Cryo-electron microscopy reveals that Raf1 facilitates Rubisco assembly by mediating RbcL dimer formation and dimer-dimer interactions. Syn7942 cells lacking Raf1 are unable to form canonical intact carboxysomes but generate a large number of intermediate assemblies comprising Rubisco, CcaA, CcmM, and CcmN without shell encapsulation and a low abundance of carboxysome-like structures with reduced dimensions and irregular shell shapes and internal organization. As a consequence, the Raf1-depleted cells exhibit reduced Rubisco content, CO2-fixing activity, and cell growth. Our results provide mechanistic insight into the chaperone-assisted Rubisco assembly and biogenesis of carboxysomes. Advanced understanding of the biogenesis and stepwise formation process of the biogeochemically important organelle may inform strategies for heterologous engineering of functional CO2-fixing modules to improve photosynthesis. [ABSTRACT FROM AUTHOR]

The tubulin-like GTPase protein FtsZ, which forms a discontinuous cytokinetic ring at mid-cell, is a central player to recruit the division machinery to orchestrate cell division. To guarantee the production of two identical daughter cells, the assembly of FtsZ, namely Z-ring, and its precise positioning should be finely regulated. In Streptococcus pneumoniae, the positioning of Z-ring at the division site is mediated by a bitopic membrane protein MapZ (mid-cell-anchored protein Z) through direct interactions between the intracellular domain (termed MapZ-N (the intracellular domain of MapZ)) and FtsZ. Using nuclear magnetic resonance titration experiments, we clearly assigned the key residues involved in the interactions. In the presence of MapZ-N, FtsZ gains a shortened activation delay, a lower critical concentration for polymerization and a higher cooperativity towards GTP hydrolysis. On the other hand, MapZ-N antagonizes the lateral interactions of singlestranded filaments of FtsZ, thus slows down the formation of highly bundled FtsZ polymers and eventually maintains FtsZ at a dynamic state. Altogether, we conclude that MapZ is not only an accelerator to trigger the polymerization of FtsZ, but also a brake to tune the velocity to form the end-product, FtsZ bundles. These findings suggest that MapZ is a multi-functional regulator towards FtsZ that controls both the precise positioning and proper timing of FtsZ polymerization. [ABSTRACT FROM AUTHOR]

The spr0693-spr0694-spr0695 operon of Streptococcus pneumoniae encodes a putative ATPbinding cassette (ABC)-type efflux pump involved in the resistance of antibiotics and antimicrobial peptides. Here we report the crystal structures of Spr0694-0695 at 3.3 Å and Spr0693 at 3.0 Å resolution, revealing a MacAB-like efflux pump. The dimeric Spr0694-0695 adopts a non-canonical fold of ABC transporter, the transmembrane domain of which consists of eight tightly packed transmembrane helices with an insertion of extracellular domain between the first and second helices, whereas Spr0693 forms a nanotube channel docked onto the ABC transporter. Structural analyses combined with ATPase activity and antimicrobial susceptibility assays, enable us to propose a putative substrate-entrance tunnel with a lateral access controlled by a guard helix. Altogether, our findings provide structural insights and putative transport mechanism of a MacAB-like efflux pump in Grampositive bacteria. [ABSTRACT FROM AUTHOR]

The coordination of carbon and nitrogen metabolism is essential for bacteria to adapt to nutritional variations in the environment, but the underlying mechanism remains poorly understood. In autotrophic cyanobacteria, high CO2 levels favor the carboxylase activity of ribulose 1,5 bisphosphate carboxylase/oxygenase (RuBisCO) to produce 3-phosphoglycerate, whereas low CO2 levels promote the oxygenase activity of RuBisCO, leading to 2-phosphoglycolate (2-PG) production. Thus, the 2-PG level is reversely correlated with that of 2-oxoglutarate (2-OG), which accumulates under a high carbon/nitrogen ratio and acts as a nitrogen-starvation signal. The LysR-type transcriptional repressor NAD(P)H dehydrogenase regulator (NdhR) controls the expression of genes related to carbon metabolism. Based on genetic and biochemical studies, we report here that 2-PG is an inducer of NdhR, while 2-OG is a corepressor, as found previously. Furthermore, structural analyses indicate that binding of 2-OG at the interface between the two regulatory domains (RD) allows the NdhR tetramer to adopt a repressor conformation, whereas 2-PG binding to an intradomain cleft of each RD triggers drastic conformational changes leading to the dissociation of NdhR from its target DNA. We further confirmed the effect of 2-PG or 2-OG levels on the transcription of the NdhR regulon. Together with previous findings, we propose that NdhR can sense 2-OG from the Krebs cycle and 2-PG from photorespiration, two key metabolites that function together as indicators of intracellular carbon/nitrogen status, thus representing a fine sensor for the coordination of carbon and nitrogen metabolism in cyanobacteria. [ABSTRACT FROM AUTHOR]

The pain-depression dyad is becoming widespread in the clinic and is attracting increasing attention. A previous study by our group found that 100-Hz electro-acupuncture (EA), but not 2-, 50- and 2/100-Hz EA, was effective against the reserpine-induced pain-depression dyad. This finding is in contrast to the fact that low-frequency EA is commonly used to treat supraspinal-originating diseases. The present study aimed to investigate the mechanism underlying the effects of 100-Hz EA on the pain-depression dyad. Repeated reserpine injection was found to induce allodynia and depressive behaviors in rats. It decreased 5-hydroxytryptamine (5-HT) levels and immunoreactive expressions in the dorsal raphe nucleus (DRN). 100-Hz EA alleviated the pain-depression dyad and upregulated 5-HT in the DRN of reserpine-injected rats. Intracerebroventricular injection of para-chlorophenylalanine, an inhibitor of 5-HT resynthesis, suppressed the upregulation of 5-HT in the DRN by 100-Hz EA and partially counteracted the analgesic and anti-depressive effects of 100-Hz EA. The present study was the first to demonstrate that 5-HT in the DRN is involved in mediating the analgesic and anti-depressive effects of 100-Hz EA on the pain-depression dyad. This finding provided a scientific basis for high-frequency EA as a potential treatment for the pain-depression dyad. [ABSTRACT FROM AUTHOR]

Exogenous and endogenous opioids have been shown to modulate the immune system. Morphine-induced immunosuppression has been investigated extensively. However, the immune-regulating function of endogenous opioid peptides is unclear. The present study aimed to evaluate the difference in effects on cellular immune function between recombinant rat β-endorphin (β-EP; 50 μg/kg) and plant source morphine (10 mg/kg) via intraperitoneal injection treatment in a rat model of bone cancer pain. Walker 256 cells were injected into a tibial cavity injection to establish the bone cancer pain model. The paw withdrawal thresholds and body weights were measured prior to surgery, at 6 days after surgery, and following 1, 3,6 and 8 treatments. The spleen cells were harvested for detection of T cell proliferation, natural killer (NK) cell cytotoxicity, and the relative quantities of T cell subtypes (CD3+, CD4+ and CD8+ cells). Plasma levels of interleukin-2 (IL-2) were also determined. It was found that single or multiple treatments with β-EP (a homogenous opioid peptide) and morphine (a heterogenous opioid) had good analgesic effects on bone cancer pain, while the analgesia provided by morphine was stronger than that of β-EP. Treatment with β-EP 3, 6 and 8 times increased the body weight gain in the rat model of bone cancer pain, while morphine treatment had on effect on it. With regard to immunomodulatory functions, β-EP treatment increased T cell proliferation and NK cell cytotoxicity, and increased the relative quantities of T cell subtypes, but no effect on T cell secretion. However, morphine treatment decreased T cell proliferation and the levels of T cell subtypes. These data indicate that opioids from different sources have different effects on cellular immune function in vivo. A small dose of homogenous opioid peptide exhibited positive effects (analgesia and immune enhancement) on cancer pain. These results provide experimental evidence supporting the exploitation of human opioids for the treatment of cancer pain. [ABSTRACT FROM AUTHOR]

Formation of the heterocyst envelope polysaccharide (HEP) is a key process for cyanobacterial heterocyst differentiation. The maturation of HEP in Anabaena sp. strain PCC 7120 is controlled by a gene cluster termed HEP island in addition to an operon alr3698-alr3699, which encodes two putative proteins termed Alr3698/HepD and Alr3699/HepE. Here we report the crystal structures of HepE in the apo-form and three complex forms that bind to UDP-glucose (UDPG), UDP&glucose, and UDP, respectively. The overall structure of HepE displays a typical GT-B fold of glycosyltransferases, comprising two separate β/α/β Rossmann-fold domains that form an inter-domain substrate- binding crevice. Structural analyses combined with enzymatic assays indicate that HepE is a glucosyltransferase using UDPG as a sugar donor. Further site-directed mutageneses enable us to assign the key residues that stabilize the sugar donor and putative acceptor. Based on the comparative structural analyses, we propose a putative catalytic cycle of HepE, which undergoes "openclosed- open" conformational changes upon binding to the substrates and release of products. These findings provide structural and catalytic insights into the first enzyme involved in the HEP biosynthesis pathway. [ABSTRACT FROM AUTHOR]

Previous studies have focused on strategies for pain relief based on the peripheral opioid system. However, little is known with regard to the profile of the peripheral opioid system in long-lasting inflammatory pain. In the current study, the intrinsic changes of the peripheral opioids were investigated in long-lasting inflammatory pain. A rat model of complete Freund's adjuvant (CFA)-induced inflammatory pain was established. Paw swelling and thermal hyperalgesia (paw withdrawal latency, PWL) were analyzed until day 18 after the CFA injection. The levels of peripheral opioids and their upstream inducers, corticotrophin-releasing factor (CRF) and interleukin (IL)-1β, were measured, and validation experiments were performed using opioid receptor antagonists. Long-lasting inflammatory pain was successfully induced in the rats, as shown by the significantly increased paw swelling and decreased PWLs. On day 18 after the CFA injection, the IL-1β levels were significantly elevated, while CRF remained at a normal level in the paw inflammatory tissue. In addition, met-enkephalin (Met-ENK) and dynorphin A (DYN A) levels were significantly increased, while the β-endorphin level remained normal. Local intraplantar administration of δ- and κ-opioid receptor antagonists resulted in more substantial pain, but did not significantly affect the PWLs of the normal control rats. Therefore, the results indicated that the increased levels of local Met-ENK and DYN A in CFA-induced long-lasting inflammatory pain may be involved in peripheral intrinsic analgesia. [ABSTRACT FROM AUTHOR]

Epidemic investigations reveal an intimate interrelationship between pain and depression. The effect of electroacupuncture (EA) on pain or depression has been demonstrated individually, but its effect on pain-depression dyad is unknown. Our study aimed to screen a dominant EA frequency on pain-depression dyad and determine the validity of acupoint selection based on meridian theory. The pain-depression dyad rat model was induced by reserpine and treated using EA with different frequencies at identical acupoints to extract a dominant frequency and then administrated dominant-frequency EA at different acupoints in the above models. Paw withdrawal latency (PWL), emotional behavior of elevated zero maze (EZM) test, and open field (OF) test were conducted. We found that 100 Hz EA at Zusanli (ST 36) and Sanyinjiao (SP 6) (classical acupoints for spleen-deficiency syndrome) were the most effective in improving PWL, travelling distance in the EZM, and maximum velocity in OF compared to EA with other frequencies; ST 36 and SP 6 were proved more effective than other acupoints beyond the meridian theory and nonacupoints under the same administration of EA. Therefore, we concluded that 100 Hz is the dominant frequency for treating the pain-depression dyad with EA, and acupoints on spleen and stomach meridians are preferable choices. [ABSTRACT FROM AUTHOR]

Neuropathic pain is an intractable problem in clinical practice. Accumulating evidence shows that electroacupuncture (EA) with low frequency can effectively relieve neuropathic pain. Transient receptor potential vanilloid type 1 (TRPV1) plays a key role in neuropathic pain. The study aimed to investigate whether neuropathic pain relieved by EA administration correlates with TRPV1 inhibition. Neuropathic pain was induced by right L5 spinal nerve ligation (SNL) in rats. 2 HzEA stimulation was administered. SNL induced mechanical allodynia in ipsilateral hind paw. SNL caused a significant reduction of TRPV1 expression in ipsilateral L5 dorsal root ganglia (DRG), but a significant up-regulation in ipsilateral L4 and L6 DRGs. Calcitonin gene-related peptide (CGRP) change was consistent with that of TRPV1. EA alleviated mechanical allodynia, and inhibited TRPV1 and CGRP overexpressions in ipsilateral L4 and L6 DRGs. SNL did not decrease pain threshold of contralateral hind paw, and TRPV1 expression was not changed in contralateral L5 DRG. 0.001, 0.01 mg/kg TRPV1 agonist 6ʹ-IRTX fully blocked EA analgesia in ipsilateral hind paw. 0.01 mg/kg 6' -IRTX also significantly decreased pain threshold of contralateral paw. These results indicated that inhibition of TRPV1 upregulation in ipsilateral adjacent undamaged DRGs contributed to low frequency EA analgesia for mechanical allodynia induced by spinal nerve ligation. [ABSTRACT FROM AUTHOR]

Although the beneficial effects of acupuncture in asthma treatment have been well documented, little is known regarding the biological basis of this treatment. Changes in the lung proteome of acupuncture-treated rats with asthma onset were comparatively analyzed using a two-dimensional gel electrophoresis (2DE) and mass-spectrometry- ( MS-) based proteomic approach. Acupuncture on specific acupuncture points appeared to improve respiratory function and reduce the total number of leukocytes and eosinophils in bronchoalveolar lavage fluid in OVA-induced asthma onset. Image analysis of 2DE gels revealed 32 differentially expressed acupuncture-specific protein spots in asthma onset; 30 of which were successfully identified as 28 unique proteins using LC- MS/ MS. Bioinformatic analyses indicated that these altered proteins are most likely involved in inflammation-related biological functions, and the functional associations of these proteins result in an inflammation signaling pathway. Acupuncture regulates the pathway at different levels by regulating several key nodal proteins, including downregulating of proinflammatory proteins (e.g., S100A8, RAGE, and S100A11) and upregulating of anti-inflammatory proteins (e.g., CC10, ANXA5, and sRAGE). These deregulated inflammation-related proteins may mediate, at least in part, the antiasthmatic effect of acupuncture. Further functional investigation of these acupuncture-specific effector proteins could identify new drug candidates for the prophylaxis and treatment of asthma. [ABSTRACT FROM AUTHOR]

2-oxogluatarate (2-OG), a metabolite of the highly conserved Krebs cycle, not only plays a critical role in metabolism, but also constitutes a signaling molecule in a variety of organisms ranging from bacteria to plants and animals. In cyanobacteria, the accumulation of 2-OG constitutes the signal of nitrogen starvation and NtcA, a global transcription factor, has been proposed as a putative receptor for 2-OG. Here we present three crystal structures of NtcA from the cyanobacterium Anabaena: the apoform, and two ligand-bound forms in complex with either 2-OG or its analogue 2,2-difluoropentanedioic acid. All structures assemble as homodimers, with each subunit composed of an N-terminal effector-binding domain and a C-terminal DNA-binding domain connected by a long helix (C-helix). The 2-OG binds to the effector-binding domain at a pocket similar to that used by cAMP in catabolite activator protein, but with a different pattern. Comparative structural analysis reveals a putative signal transmission route upon 2-OG binding. A tighter coiled-coil conformation of the two C-helices induced by 2-OG is crucial to maintain the proper distance between the two F-helices for DNA recognition. Whereas catabolite activator protein adopts a transition from off-to-on state upon CAMP binding, our structural analysis explains well why NtcA can bind to DNA even in its apoform, and how 2-OG just enhances the DNA-binding activity of NtcA. These.findings provided the structural insights into the function of a global transcription factor regulated by 2-0G. a metabolite standing at a crossroad between carbon and nitrogen metabolisms. [ABSTRACT FROM AUTHOR]

Hypoxia-inducible factor (HIF)-α subunits (HIF-1α, HIF-2α and HIF-3α), which play a pivotal role during the development of hypoxia-induced pulmonary hypertension (HPH), are regulated through post-translational hydroxylation by their three prolyl hydroxylase domain-containing proteins (PHD1, PHD2 and PHD3). PHDs could also be regulated by HIF. But differential and reciprocal regulation between HIF-α and PHDs during the development of HPH remains unclear. To investigate this problem, a rat HPH model was established. Mean pulmonary arterial pressure increased significantly after 7 d of hypoxia. Pulmonary artery remodeling index and right ventricular hypertrophy became evident after 14 d of hypoxia. HIF-1? and HIF-2? mRNA increased slightly after 7 d of hypoxia, but HIF-3? increased significantly after 3 d of hypoxia. The protein expression levels of all three HIF-α were markedly upregulated after exposure to hypoxia. PHD2 mRNA and protein expression levels were upregulated after 3 d of hypoxia; PHD1 protein declined after 14 d of hypoxia without significant mRNA changes. PHD3 mRNA and protein were markedly upregulated after 3 d of hypoxia, then the mRNA remained at a high level, but the protein declined after 14 d of hypoxia. In hypoxic animals, HIF-1α proteins negatively correlated with PHD2 proteins, whereas HIF-2α and HIF-3α proteins showed negative correlations with PHD3 and PHD1 proteins, respectively. All three HIF-α proteins were positively correlated with PHD2 and PHD3 mRNA. In the present study, HIF-α subunits and PHDs showed differential and reciprocal regulation, and this might play a key pathogenesis role in hypoxia-induced pulmonary hypertension. Edited by Faton H. AGANI [ABSTRACT FROM AUTHOR]

The muscularization of non-muscular pulmonary arterioles is an important pathological feature of hypoxic pulmonary vascular remodeling. However, the origin of the cells involved in this process is still not well understood. The present study was undertaken to test the hypothesis that transforming growth factor-β1 (TGF-β1) can induce transdifferentiation of fibroblasts into myofibroblasts, which might play a key role in the muscularization of non-muscular pulmonary arterioles. It was found that mean pulmonary arterial pressure increased significantly after 7 d of hypoxia. Pulmonary artery remodeling index and right ventricular hypertrophy became evident after 14 d of hypoxia. The distribution of nonmuscular, partially muscular, and muscular vessels was significantly different after 7 d of hypoxia. Immunocytochemistry results demonstrated that the expression of β-smooth muscle actin was increased in intra-acinar pulmonary arteries with increasing hypoxic time. TGF-β1 mRNA expression in pulmonary arterial walls was increased significantly after 14 d of hypoxia, but showed no obvious changes after 3 or 7 d of hypoxia. In pulmonary tunica adventitia and tunica media, TGF-β1 protein staining was poorly positive in control rats, but was markedly enhanced after 3 d of hypoxia, reaching its peak after 7 d of hypoxia. The myofibroblast phenotype was confirmed by electron microscopy, which revealed microfilaments and a well-developed rough endoplasmic reticulum. Taken together, our results suggested that TGF-β1 induces transdifferentiation of fibroblasts into myofibroblasts, which is important in hypoxic pulmonary vascular remodeling. Edited by Ming-Hua XU [ABSTRACT FROM AUTHOR]