An integrated gene regulatory network controls stem cell proliferation in teeth

Epithelial stem cells reside in specific niches that regulate their self-renewal and differentiation, and are responsible for the continuous regeneration of tissues such as hair, skin, and gut. Although the regenerative potential of mammalian teeth is limited, mouse incisors grow continuously throughout life and contain stem cells at their proximal ends in the cervical loops. In the labial cervical loop, the epithelial stem cells proliferate and migrate along the labial surface, differentiating into enamel-forming ameloblasts. In contrast, the lingual cervical loop contains fewer proliferating stem cells, and the lingual incisor surface lacks ameloblasts and enamel. Here we have used a combination of mouse mutant analyses, organ culture experiments, and expression studies to identify the key signaling molecules that regulate stem cell proliferation in the rodent incisor stem cell niche, and to elucidate their role in the generation of the intrinsic asymmetry of the incisors. We show that epithelial stem cell proliferation in the cervical loops is controlled by an integrated gene regulatory network consisting of Activin, bone morphogenetic protein (BMP), fibroblast growth factor (FGF), and Follistatin within the incisor stem cell niche. Mesenchymal FGF3 stimulates epithelial stem cell proliferation, and BMP4 represses Fgf3 expression. In turn, Activin, which is strongly expressed in labial mesenchyme, inhibits the repressive effect of BMP4 and restricts Fgf3 expression to labial dental mesenchyme, resulting in increased stem cell proliferation and a large, labial stem cell niche. Follistatin limits the number of lingual stem cells, further contributing to the characteristic asymmetry of mouse incisors, and on the basis of our findings, we suggest a model in which Follistatin antagonizes the activity of Activin. These results show how the spatially restricted and balanced effects of specific components of a signaling network can regulate stem cell proliferation in the niche and account for asymmetric organogenesis. Subtle variations in this or related regulatory networks may explain the different regenerative capacities of various organs and animal species.     

MicroRNAs show a wide diversity of expression profiles in the developing and mature central nervous system

Background  

MicroRNA (miRNA) encoding genes are abundant in vertebrate genomes but very few have been studied in any detail. Bioinformatic tools allow prediction of miRNA targets and this information coupled with knowledge of miRNA expression profiles facilitates formulation of hypotheses of miRNA function. Although the central nervous system (CNS) is a prominent site of miRNA expression, virtually nothing is known about the spatial and temporal expression profiles of miRNAs in the brain. To provide an overview of the breadth of miRNA expression in the CNS, we performed a comprehensive analysis of the neuroanatomical expression profiles of 38 abundant conserved miRNAs in developing and adult zebrafish brain.     

Association of simian virus 40 vp1 with 70-kilodalton heat shock proteins and viral tumor antigens

Proper folding of newly synthesized viral proteins in the cytoplasm is a prerequisite for the formation of infectious virions. The major capsid protein Vp1 of simian virus 40 forms a series of disulfide-linked intermediates during folding and capsid formation. In addition, we report here that Vp1 is associated with cellular chaperones (HSP70) and a cochaperone (Hsp40) which can be coimmunoprecipitated with Vp1. Studies in vitro demonstrated the ATP-dependent interaction of Vp1 and cellular chaperones. Interestingly, viral cochaperones LT and ST were essential for stable interaction of HSP70 with the core Vp1 pentamer Vp1 (22-303). LT and ST also coimmunoprecipitated with Vp1 in vivo. In addition to these identified (co)chaperones, stable, covalently modified forms of Vp1 were identified for a folding-defective double mutant, C49A-C87A, and may represent a “trapped” assembly intermediate. By a truncation of the carboxyl arm of Vp1 to prevent the Vp1 folding from proceeding beyond pentamers, we detected several apparently modified Vp1 species, some of which were absent in cells transfected with the folding-defective mutant DNA. These results suggest that transient covalent interactions with known or unknown cellular and viral proteins are important in the assembly process.     

Coexpression and Heteromerization of Two Neuronal K-Cl Cotransporter Isoforms in Neonatal Brain

The neuron-specific K-Cl cotransporter KCC2 maintains the low intracellular chloride concentration required for the fast hyperpolarizing actions of inhibitory neurotransmitters. The KCC2 gene codes for two isoforms, KCC2a and KCC2b, which differ in their N termini. The relative expression and cellular distribution of the two KCC2 protein isoforms are unknown. Here, we characterize an antibody against the KCC2a isoform and show that a previously described antibody against KCC2 is specific for the KCC2b isoform (Hubner, C. A., Stein, V., Hermans-Borgmeyer, I., Meyer, T., Ballanyi, K., and Jentsch, T. J. (2001) Neuron 30, 515–524). Immunostaining of dissociated hippocampal cultures confirms that both KCC2 isoforms are neuron-specific. Immunoblot analysis indicates that KCC2b is the major KCC2 isoform in the adult brain, whereas in the neonatal mouse central nervous system, half of total KCC2 protein is KCC2a. At this stage, the two KCC2 isoforms are largely colocalized and show similar patterns of distribution in the brain. When coexpressed in HEK293 cells, KCC2a and KCC2b proteins form heteromeric complexes. Moreover, the two isoforms can be coimmunoprecipitated from the neonatal brain, suggesting the presence of endogenous KCC2a-KCC2b heteromers. Consistent with this, native gel analysis shows that a substantial part of endogenous KCC2 isoforms in the neonatal brain constitute dimers.The neuron-specific K⁺-Cl^- cotransporter KCC2 extrudes potassium and chloride ions from neurons, thus maintaining the low intracellular chloride concentration [Cl]_i necessary for the fast hyperpolarizing actions of inhibitory neurotransmitters γ-aminobutyric acid (GABA) and glycine (1). KCC2 is an ∼140-kDa plasma membrane protein that belongs to the cation chloride cotransporter (CCC)⁴ family (2, 3). CCCs, including KCC2, are thought to exist in the plasma membrane as functional oligomers, although the mechanisms whereby oligomerization affects their transport activity are unclear (4–8).We have recently shown that the KCC2 gene (alias Slc12a5) generates two mRNAs, KCC2a and KCC2b, by an alternative promoter and first exon usage (9). The difference between the KCC2a and KCC2b proteins lies in the most N-terminal part; the 40 unique amino acids in KCC2a include a putative binding sequence for the Ste20-related proline-alanine-rich kinase (SPAK). KCC2 null mutant mice deficient for both KCC2 isoforms show a disrupted breathing rhythm and die immediately after birth (10, 11), whereas selective KCC2b isoform knock-out mice exhibit spontaneous seizures but can survive up to 3 weeks after birth (9, 12). Thus, KCC2a obviously supports some vital functions of lower brain structures.In general, KCC2 expression in the CNS follows neuronal maturation; it is first detected in the postmitotic neurons of the spinal cord and brainstem and is then gradually increased in higher brain structures (13, 14). Our previous work has shown that during postnatal development, KCC2a mRNA expression remains relatively constant, whereas KCC2b mRNA is strongly up-regulated in the cortex during postnatal development (9). This indicates that KCC2b is responsible for the developmental shift from depolarizing to hyperpolarizing GABAergic responses.Here, we study the relative expression and cellular distribution of KCC2a and KCC2b proteins in the mouse brain. We characterize a new antibody specific for the KCC2a isoform and demonstrate that a previously described antibody against KCC2 (11) is specific for the KCC2b isoform. The relative expression of the KCC2 protein isoforms determined by immunoblot analysis correlates well with their mRNA levels (9). KCC2a and KCC2b proteins have a similar level and pattern of expression in the neonatal mouse brain and are colocalized in most neurons in non-cortical lower brain areas. Coimmunoprecipitation (coIP) experiments and coexpression followed by native gel analysis indicate that KCC2a-KCC2b heteromers can form in vitro and may exist in vivo.     

Molecular Basis of Catalytic Chamber-assisted Unfolding and Cleavage of Human Insulin by Human Insulin-degrading Enzyme

Insulin is a hormone vital for glucose homeostasis, and insulin-degrading enzyme (IDE) plays a key role in its clearance. IDE exhibits a remarkable specificity to degrade insulin without breaking the disulfide bonds that hold the insulin A and B chains together. Using Fourier transform ion cyclotron resonance (FTICR) mass spectrometry to obtain high mass accuracy, and electron capture dissociation (ECD) to selectively break the disulfide bonds in gas phase fragmentation, we determined the cleavage sites and composition of human insulin fragments generated by human IDE. Our time-dependent analysis of IDE-digested insulin fragments reveals that IDE is highly processive in its initial cleavage at the middle of both the insulin A and B chains. This ensures that IDE effectively splits insulin into inactive N- and C-terminal halves without breaking the disulfide bonds. To understand the molecular basis of the recognition and unfolding of insulin by IDE, we determined a 2.6-Å resolution insulin-bound IDE structure. Our structure reveals that IDE forms an enclosed catalytic chamber that completely engulfs and intimately interacts with a partially unfolded insulin molecule. This structure also highlights how the unique size, shape, charge distribution, and exosite of the IDE catalytic chamber contribute to its high affinity (∼100 nm) for insulin. In addition, this structure shows how IDE utilizes the interaction of its exosite with the N terminus of the insulin A chain as well as other properties of the catalytic chamber to guide the unfolding of insulin and allowing for the processive cleavages.IDE³ is an ∼110-kDa zinc metalloprotease that is evolutionarily conserved from bacteria to humans (1, 2). It was first discovered based on its high affinity to bind insulin (∼100 nm) and degrade it into pieces (3, 4). Insulin is a 5.8-kDa hormone that plays a central role in glucose homeostasis and the development of diabetes in humans. Consistent with the in vitro activity of IDE for insulin degradation, loss-of-function mutations of IDE in rodents result in elevated insulin levels and glucose intolerance (5). In addition, a nucleotide polymorphism of the human IDE gene is linked to type 2 diabetes (6). Later studies showed that IDE can also degrade amyloid-β (Aβ), a peptide vital to the progression of Alzheimer disease (7, 8). Accumulating evidence from rodent models and human genetic analyses also indicate the physiological role of IDE in the clearance of Aβ (5, 9–12).Despite nearly 60 years of studies on IDE, the molecular basis by which IDE binds, unfolds, and degrades insulin has only begun to be elucidated. Different from ATP-dependent proteases, IDE does not require the additional energy source such as ATP to unfold, bind, and cleave its substrates (4, 13). Insulin consists of the A and B chains that are held together by two inter- and one intra-chain disulfide bonds. Remarkably, IDE does not require disulfide bond isomerase activity to unfold and cleave insulin (4). Thus, IDE needs to overcome the stability created by the disulfide bonds of insulin. Structural analysis reveals that human IDE contains a catalytic chamber formed by the internal cavity of two roughly equally sized ∼55-kDa N- and C-terminal halves (IDE-N and IDE-C, respectively) (2). Within this chamber, only one catalytic center exists. However, IDE cleaves insulin at multiple sites on both the insulin A and B chains to completely inactivate this hormone. It remains unclear whether the cleavages of insulin by IDE proceed in a sequential or stochastic manner.IDE represents an emerging protease family that utilizes an enclosed catalytic chamber to selectively recognize and unfold the substrates for their degradation (1). The volume of the enclosed chamber of IDE (∼16,000 ų) allows the preferential exclusion of peptides that are greater than ∼75 amino acids long. This chamber also has unique electrostatic properties; the internal cavity of IDE-N is predominantly negative, whereas that of IDE-C is positive. Inside the catalytic chamber, IDE has an exosite that is an evolutionarily conserved substrate-binding site ∼30 Å away from the catalytic groove. This exosite is used to anchor the N-terminal end of IDE substrates. The unique size, electrostatic potential, and exosite of Ides'' catalytic chamber are postulated as key factors for the selective binding and unfolding of IDE substrates (1, 2, 14). In addition, one common feature among the known IDE substrates is their higher propensity to form amyloid fibers (8). Amyloidogenic peptides tend to unfold by themselves, which could facilitate their unfolding and subsequent cleavage by IDE. However, the molecular basis of how the catalytic chamber of IDE binds, unfolds, and cleaves insulin into pieces and how the flexibility of this substrate contributes to its cleavage by IDE remain elusive.IDE is known to cut insulin at multiple sites, and the resulting cleavage products are quite complex (4, 15–18). Here we took advantage of the high mass accuracy of Fourier transform ion cyclotron resonance (FTICR) mass spectrometry and the selective targeting of disulfide bonds by electron capture dissociation (ECD) in our mass spectrometry (MS) analysis to unambiguously identify IDE-degraded fragments of human insulin, as well as the time-dependent production of these fragments. We also present a 2.6- Å insulin-bound IDE structure, revealing extensive shape and charge complementarity of the partially unfolded insulin with the enclosed catalytic chamber and a potential path for the unfolding of insulin. Together, our data elucidate the molecular basis by which IDE engulfs, unfolds, and effectively cleaves insulin into pieces.     

Screening and Evaluation of Human Intestinal Lactobacilli for the Development of Novel Gastrointestinal Probiotics

The aim of this study was to screen intestinal lactobacilli strains for their advantageous properties to select those that could be used for the development of novel gastrointestinal probiotics. Ninety-three isolates were subjected to screening procedures. Fifty-nine percent of the examined lactobacilli showed the ability to auto-aggregate, 97% tolerated a high concentration of bile (2% w/v), 50% survived for 4 h at pH 3.0, and all strains were unaffected by a high concentration of pancreatin (0.5% w/v). One Lactobacillus buchneri strain was resistant to tetracycline. None of the tested strains caused lysis of human erythrocytes. Six potential probiotic strains were selected for safety evaluation in a mouse model. Five of 6 strains caused no translocation, and were considered safe. In conclusion, several strains belonging to different species and fermentation groups were found that have properties required for a potential probiotic strain. This study was the first phase of a multi-phase study aimed to develop a novel, safe and efficient prophylactic and therapeutic treatment system against gastrointestinal infections using genetically modified probiotic lactobacilli.     

The seed dispersal catapult of Cardamine parviflora (Brassicaceae) is efficient but unreliable

? Premise of the study: Seed dispersal performance is an essential component of plant fitness. Despite their significance in shaping performance, the mechanical processes that drive dispersal are poorly understood. We have quantified seed dispersal mechanics in Cardamine parviflora (Brassicaceae), a ballistic disperser that launches seeds with specialized catapult-like structures. To determine which aspects of catapult function dictate interspecific dispersal differences, we compared this disperser with other ballistic dispersers. Comparison with brassicas that lack ballistic dispersal may also provide insight into the evolution of this mechanism. ? Methods: Catapult performance was quantified using high-speed video analysis of dehiscence, ballistic modeling of seed trajectories, and measuring the mechanical energy storage capacity of the spring-like siliqua valve tissue that launched the seeds. ? Key results: The siliquae valves coiled rapidly outward, launching the seeds in 4.7 ± 1.3 ms (mean ± SD, N = 11). Coiling was likely driven by the bilayered valve structure. The catapult was 21.3 ± 10.3% efficient (mean ± SD, N = 11) at transferring stored elastic energy to the seeds as kinetic energy. The majority of seeds (71.4%) were not launched effectively. ? Conclusions: The efficiency of the C. parviflora catapult was high in comparison to that of a ballistic diplochore, a dispersal mode associated with poor ballistic performance, although the unreliability of the launch mechanism limited dispersal distance. Effective launching requires temporary seed-valve adhesion. The adhesion mechanism may be the source of the unreliability. Valve curvature is likely driven by the bilayered valve structure, a feature absent in nonballistic brassicas.     

Overoxidation of 2-Cys Peroxiredoxin in Prokaryotes: CYANOBACTERIAL 2-CYS PEROXIREDOXINS SENSITIVE TO OXIDATIVE STRESS*?

In eukaryotic organisms, hydrogen peroxide has a dual effect; it is potentially toxic for the cell but also has an important signaling activity. According to the previously proposed floodgate hypothesis, the signaling activity of hydrogen peroxide in eukaryotes requires a transient increase in its concentration, which is due to the inactivation by overoxidation of 2-Cys peroxiredoxin (2-Cys Prx). Sensitivity to overoxidation depends on the structural GGLG and YF motifs present in eukaryotic 2-Cys Prxs and is believed to be absent from prokaryotic enzymes, thus representing a paradoxical gain of function exclusive to eukaryotic organisms. Here we show that 2-Cys Prxs from several prokaryotic organisms, including cyanobacteria, contain the GG(L/V/I)G and YF motifs characteristic of sensitive enzymes. In search of the existence of overoxidation-sensitive 2-Cys Prxs in prokaryotes, we have analyzed the sensitivity to overoxidation of 2-Cys Prxs from two cyanobacterial strains, Anabaena sp. PCC7120 and Synechocystis sp. PCC6803. In vitro analysis of wild type and mutant variants of the Anabaena 2-Cys Prx showed that this enzyme is overoxidized at the peroxidatic cysteine residue, thus constituting an exception among prokaryotes. Moreover, the 2-Cys Prx from Anabaena is readily and reversibly overoxidized in vivo in response to high light and hydrogen peroxide, showing higher sensitivity to overoxidation than the Synechocystis enzyme. These cyanobacterial strains have different strategies to cope with hydrogen peroxide. While Synechocystis has low content of less sensitive 2-Cys Prx and high catalase activity, Anabaena contains abundant and sensitive 2-Cys Prx, but low catalase activity, which is remarkably similar to the chloroplast system.     

Selecting thioredoxins for disulphide proteomics: target proteomes of three thioredoxins from the cyanobacterium Synechocystis sp. PCC 6803

Searching for enzymes and other proteins which can be redox-regulated by dithiol/disulphide exchange is a rapidly expanding area of functional proteomics. Recently, several experimental approaches using thioredoxins have been developed for this purpose. Thioredoxins comprise a large family of redox-active enzymes capable of reducing protein disulphides to cysteines and of participating in a variety of processes, such as enzyme modulation, donation of reducing equivalents and signal transduction. In this study we screened the target proteomes of three different thioredoxins from the unicellular cyanobacterium Synechocystis sp. PCC 6803, using site-directed active-site cysteine-to-serine mutants of its m-, x- and y-type thioredoxins. The properties of a thioredoxin that determine the outcome of such analyses were found to be target-binding capacity, solubility and the presence of non-active-site cysteines. Thus, we explored how the choice of thioredoxin affects the target proteomes and we conclude that the m-type thioredoxin, TrxA, is by far the most useful for screening of disulphide proteomes. Furthermore, we improved the resolution of target proteins on non-reducing/reducing 2-DE, leading to the identification of 14 new potentially redox-regulated proteins in this organism. The presence of glycogen phosphorylase among the newly identified targets suggests that glycogen breakdown is redox-regulated in addition to glycogen synthesis.