Termination and activation of store‐operated cyclic AMP production

Diverse pathophysiological processes (e.g. obesity, lifespan determination, addiction and male fertility) have been linked to the expression of specific isoforms of the adenylyl cyclases (AC1‐AC10), the enzymes that generate cyclic AMP (cAMP). Our laboratory recently discovered a new mode of cAMP production, prominent in certain cell types, that is stimulated by any manoeuvre causing reduction of free [Ca²⁺] within the lumen of the endoplasmic reticulum (ER) calcium store. Activation of this ‘store‐operated’ pathway requires the ER Ca²⁺ sensor, STIM1, but the identity of the enzymes responsible for cAMP production and how this process is regulated is unknown. Here, we used sensitive FRET‐based sensors for cAMP in single cells combined with silencing and overexpression approaches to show that store‐operated cAMP production occurred preferentially via the isoform AC3 in NCM460 colonic epithelial cells. Ca²⁺ entry via the plasma membrane Ca²⁺ channel, Orai1, suppressed cAMP production, independent of store refilling. These findings are an important first step towards defining the functional significance and to identify the protein composition of this novel Ca²⁺/cAMP crosstalk system.     

Proteomics reveals selective regulation of proteins in response to memory-related serotonin stimulation in Aplysia californica ganglia

The marine mollusk Aplysia californica (Aplysia) is a powerful model for learning and memory due to its minimalistic nervous system. Key proteins, identified to be regulated by the neurotransmitter serotonin in Aplysia, have been successfully translated to mammalian models of learning and memory. Based upon a recently published large‐scale analysis of Aplysia proteomic data, the current study investigated the regulation of protein levels 24 and 48 h after treatment with serotonin in Aplysia ganglia using a 2‐D gel electrophoresis approach. Protein spots were quantified and protein‐level changes of selected proteins were verified by Western blotting. Among those were Rab GDP dissociation inhibitor alpha (RabGDIα), synaptotagmin‐1 and deleted in azoospermia‐associated protein (DAZAP‐1) in cerebral ganglia, calreticulin, RabGDIα, DAZAP‐1, heterogeneous nuclear ribonucleoprotein F (hnRNPF), RACK‐1 and actin‐depolymerizing factor (ADF) in pleural ganglia and DAZAP‐1, hnRNPF and ADF in pedal ganglia. Protein identity of the majority of spots was confirmed by a gel‐based mass spectrometrical method (FT‐MS). Taken together, protein‐level changes induced by the learning‐related neurotransmitter serotonin in Aplysia ganglia are described and a role for the abovementioned proteins in synaptic plasticity is proposed.     

CpG drives human transitional B cells to terminal differentiation and production of natural antibodies

The receptor TLR9, recognizing unmethylated bacterial DNA (CpG), is expressed by B cells and plays a role in the maintenance of serological memory. Little is known about the response of B cells stimulated with CpG alone, without additional cytokines. In this study, we show for the first time the phenotypic modification, changes in gene expression, and functional events downstream to TLR9 stimulation in human B cell subsets. In addition, we demonstrate that upon CpG stimulation, IgM memory B cells differentiate into plasma cells producing IgM Abs directed against the capsular polysaccharides of Streptococcus pneumoniae. This novel finding proves that IgM memory is the B cell compartment responsible for the defense against encapsulated bacteria. We also show that cord blood transitional B cells, corresponding to new bone marrow emigrants, respond to CpG. Upon TLR9 engagement, they de novo express AID and Blimp-1, genes necessary for hypersomatic mutation, class-switch recombination, and plasma cell differentiation and produce Abs with anti-pneumococcal specificity. Transitional B cells, isolated from cord blood, have not been exposed to pneumococcus in vivo. In addition, it is known that Ag binding through the BCR causes apoptotic cell death at this stage of development. Therefore, the ability of transitional B cells to sense bacterial DNA through TLR9 represents a tool to rapidly build up the repertoire of natural Abs necessary for our first-line defense at birth.     

Phosphite-derivatized ruthenium-carbohydrate complexes in the catalytic hydration of nitriles. short communication

The synthesis of metal complexes with bioligands is one option to introduce chirally defined ligands to catalysts. Herein, the hydration of nitriles to the corresponding carboxamides by use of ruthenium(II) complexes is described, which were obtained by attaching derivatives of the 3,5,6-bicyclophosphite-alpha-D-glucofuranoside ligand.     

Functional Characterization of a Newly Identified Group B Streptococcus Pullulanase Eliciting Antibodies Able to Prevent Alpha-Glucans Degradation

Streptococcal pullulanases have been recently proposed as key components of the metabolic machinery involved in bacterial adaptation to host niches. By sequence analysis of the Group B Streptococcus (GBS) genome we found a novel putative surface exposed protein with pullulanase activity. We named such a protein SAP. The sap gene is highly conserved among GBS strains and homologous genes, such as PulA and SpuA, have been described in other pathogenic streptococci. The SAP protein contains two N-terminal carbohydrate-binding motifs, followed by a catalytic domain and a C-terminal LPXTG cell wall-anchoring domain. In vitro analysis revealed that the recombinant form of SAP is able to degrade α-glucan polysaccharides, such as pullulan, glycogen and starch. Moreover, NMR analysis showed that SAP acts as a type I pullulanase. Studies performed on whole bacteria indicated that the presence of α-glucan polysaccharides in culture medium up-regulated the expression of SAP on bacterial surface as confirmed by FACS analysis and confocal imaging. Deletion of the sap gene resulted in a reduced capacity of bacteria to grow in medium containing pullulan or glycogen, but not glucose or maltose, confirming the pivotal role of SAP in GBS metabolism of α-glucans. As reported for other streptococcal pullulanases, we found specific anti-SAP antibodies in human sera from healthy volunteers. Investigation of the functional role of anti-SAP antibodies revealed that incubation of GBS in the presence of sera from animals immunized with SAP reduced the capacity of the bacterium to degrade pullulan. Of interest, anti-SAP sera, although to a lower extent, also inhibited Group A Streptococcus pullulanase activity. These data open new perspectives on the possibility to use SAP as a potential vaccine component inducing functional cross-reacting antibodies interfering with streptococcal infections.     

Lakeside cemeteries in the Sahara: 5000 years of holocene population and environmental change

Background

Approximately two hundred human burials were discovered on the edge of a paleolake in Niger that provide a uniquely preserved record of human occupation in the Sahara during the Holocene (∼8000 B.C.E. to the present). Called Gobero, this suite of closely spaced sites chronicles the rapid pace of biosocial change in the southern Sahara in response to severe climatic fluctuation.

Methodology/Principal Findings

Two main occupational phases are identified that correspond with humid intervals in the early and mid-Holocene, based on 78 direct AMS radiocarbon dates on human remains, fauna and artifacts, as well as 9 OSL dates on paleodune sand. The older occupants have craniofacial dimensions that demonstrate similarities with mid-Holocene occupants of the southern Sahara and Late Pleistocene to early Holocene inhabitants of the Maghreb. Their hyperflexed burials compose the earliest cemetery in the Sahara dating to ∼7500 B.C.E. These early occupants abandon the area under arid conditions and, when humid conditions return ∼4600 B.C.E., are replaced by a more gracile people with elaborated grave goods including animal bone and ivory ornaments.

Conclusions/Significance

The principal significance of Gobero lies in its extraordinary human, faunal, and archaeological record, from which we conclude the following:

1. The early Holocene occupants at Gobero (7700–6200 B.C.E.) were largely sedentary hunter-fisher-gatherers with lakeside funerary sites that include the earliest recorded cemetery in the Sahara.
2. Principal components analysis of craniometric variables closely allies the early Holocene occupants at Gobero with a skeletally robust, trans-Saharan assemblage of Late Pleistocene to mid-Holocene human populations from the Maghreb and southern Sahara.
3. Gobero was abandoned during a period of severe aridification possibly as long as one millennium (6200–5200 B.C.E).
4. More gracile humans arrived in the mid-Holocene (5200–2500 B.C.E.) employing a diversified subsistence economy based on clams, fish, and savanna vertebrates as well as some cattle husbandry.
5. Population replacement after a harsh arid hiatus is the most likely explanation for the occupational sequence at Gobero.
6. We are just beginning to understand the anatomical and cultural diversity that existed within the Sahara during the Holocene.

     

Dynamic coupling of the putative coiled-coil domain of ORAI1 with STIM1 mediates ORAI1 channel activation

STIM1 and ORAI1 (also termed CRACM1) are essential components of the classical calcium release-activated calcium current; however, the mechanism of the transmission of information of STIM1 to the calcium release-activated calcium/ORAI1 channel is as yet unknown. Here we demonstrate by F?rster resonance energy transfer microscopy a dynamic coupling of STIM1 and ORAI1 that culminates in the activation of Ca(2+) entry. F?rster resonance energy transfer imaging of living cells provided insight into the time dependence of crucial events of this signaling pathway comprising Ca(2+) store depletion, STIM1 multimerization, and STIM1-ORAI1 interaction. Accelerated store depletion allowed resolving a significant time lag between STIM1-STIM1 and STIM1-ORAI1 interactions. Store refilling reversed both STIM1 multimerization and STIM1-ORAI1 interaction. The cytosolic STIM1 C terminus itself was able, in vitro as well as in vivo, to associate with ORAI1 and to stimulate channel function, yet without ORAI1-STIM1 cluster formation. The dynamic interaction occurred via the C terminus of ORAI1 that includes a putative coiled-coil domain structure. An ORAI1 C terminus deletion mutant as well as a mutant (L273S) with impeded coiled-coil domain formation lacked both interaction as well as functional communication with STIM1 and failed to generate Ca(2+) inward currents. An N-terminal deletion mutant of ORAI1 as well as the ORAI1 R91W mutant linked to severe combined immune deficiency syndrome was similarly impaired in terms of current activation despite being able to interact with STIM1. Hence, the C-terminal coiled-coil motif of ORAI1 represents a key domain for dynamic coupling to STIM1.