The
American Chemical Society (ACS) Green Chemistry Institute (GCI)
Pharmaceutical Roundtable conducted a study to elucidate the value
of continuous processing, which had been defined as a key research
area for green engineering. In the course of defining the business
case for continuous processing, individual cases were collected and
evaluated to determine specific drivers to implement continuous processing
and to find key success factors. The magnitude and timing of effects
and the relation to the principles of green chemistry were investigated.
Glutamate is the main excitatory transmitter in the brain, while ATP represents the most important energy currency in any living cell. Yet, these chemicals play an important role in both processes, enabling them with dual-acting functions in metabolic and intercellular signaling pathways. Glutamate can fuel ATP production, while ATP can act as a transmitter in intercellular signaling. We discuss the interface between glutamate and ATP in signaling and metabolism of astrocytes. Not only do glutamate and ATP cross each other's paths in physiology of the brain, but they also do so in its pathology. We present the fabric of this process in (patho)physiology through the discussion of synthesis and metabolism of ATP and glutamate in astrocytes as well as by providing a general description of astroglial receptors for these molecules along with the downstream signaling pathways that may be activated. It is astroglial receptors for these dual-acting molecules that could hold a key for medical intervention in pathological conditions. We focus on two examples disclosing the role of activation of astroglial ATP and glutamate receptors in pathology of two kinds of brain tissue, gray matter and white matter, respectively. Interventions at the interface of metabolism and signaling show promise for translational medicine.
The expression of the leukotoxin (ltx) operon varies significantly among Actinobacillus actinomycetemcomitans strains. The dual promoters driving ltx expression in the highly toxic strain JP2 have been previously characterized (
A robust, green, and sustainable
manufacturing process has been
developed for the synthesis of gefapixant citrate, a P2X3 receptor
antagonist that is under investigation for the treatment of refractory
and unexplained chronic cough. The newly developed commercial process
features low process mass intensity (PMI), short synthetic sequence,
high overall yield, minimal environmental impact, and significantly
reduced API costs. The key innovations are the implementation of a
highly efficient two-step methoxyphenol synthesis, an innovative pyrimidine
synthesis in flow, a simplified sulfonamide synthesis, and a novel
salt metathesis approach to consistently deliver the correct active
pharmaceutical ingredient (API) salt form in high purity.
Streptococcus gordonii M5 expresses a lectin on its surface (SSP-5) which binds to human salivary agglutinin (SAG). This interaction requires sialic acid residues of SAG and divalent cations and may mediate the colonization of oral tissues by this organism. In this report, we show that the binding of SAG to SSP-5 requires calcium and that SSP-5 is a high-affinity calcium-binding protein. SAG-mediated aggregation of S. gordonii M5 was inhibited by 1 mM EDTA, and the restoration of aggregation occurred only upon the readdition of calcium. To ascertain the level at which calcium exerts its effects, the calcium-binding properties of SSP-5 were evaluated by using a 45Ca binding assay. In addition, a kinetic analysis of calcium binding was carried out by using fura2, a fluorescent calcium-binding dye. These analyses showed that SSP-5 is a high-affinity calcium-binding protein that binds 1 mol of calcium per mol of protein and has a dissociation constant of 0.45 + 0.2 ,uM. The calcium-binding capacity of SSP-5 was also calculated independently to be 1.0 + 0.2 mol of Ca per mol of SSP-5 by column chromatography on Sephadex G-25 equilibrated with 10 ,uM 45Ca. To localize the calcium binding site of SSP-5, a series of C-terminal deletion mutants were expressed in Escherichia coli and evaluated for calcium-binding activity. Deletion of the 250 C-terminal residues of SSP-5 had little effect on calcium binding. However, deletion of residues 1168 to 1250 resulted in the loss of calcium-binding activity, suggesting that this region is important for calcium binding by SSP-5.
BackgroundHeterotrimeric guanine nucleotide binding proteins of the G12/13 subfamily, which includes the α-subunits Gα12 and Gα13, stimulate the monomeric G protein RhoA through interaction with a distinct subset of Rho-specific guanine nucleotide exchange factors (RhoGEFs). The structural features that mediate interaction between Gα13 and RhoGEFs have been examined in crystallographic studies of the purified complex, whereas a Gα12:RhoGEF complex has not been reported. Several signaling responses and effector interactions appear unique to Gα12 or Gα13, despite their similarity in amino acid sequence.MethodsTo comprehensively examine Gα12 for regions involved in RhoGEF interaction, we screened a panel of Gα12 cassette substitution mutants for binding to leukemia-associated RhoGEF (LARG) and for activation of serum response element mediated transcription.ResultsWe identified several cassette substitutions that disrupt Gα12 binding to LARG and the related p115RhoGEF. These Gα12 mutants also were impaired in activating serum response element mediated signaling, a Rho-dependent response. Most of these mutants matched corresponding regions of Gα13 reported to contact p115RhoGEF, but unexpectedly, several RhoGEF-uncoupling mutations were found within the N- and C-terminal regions of Gα12. Trypsin protection assays revealed several mutants in these regions as retaining conformational activation. In addition, charge substitutions near the Gα12 N-terminus selectively disrupted binding to LARG but not p115RhoGEF.ConclusionsSeveral structural aspects of the Gα12:RhoGEF interface differ from the reported Gα13:RhoGEF complex, particularly determinants within the C-terminal α5 helix and structurally uncharacterized N-terminus of Gα12. Furthermore, key residues at the Gα12 N-terminus may confer selectivity for LARG as a downstream effector.
Summary
In the adult ventricular-subventricular zone (V-SVZ), neural stem cells (NSCs) give rise to transit-amplifying progenitor (TAP) cells. These progenitors reside in different subniche locations, implying that cell movement must accompany lineage progression, but the dynamic behaviors of adult NSCs and TAPs remain largely unexplored. Here, we performed live time-lapse imaging with computer-based image analysis of young and aged 3D V-SVZ wholemounts from transgenic mice with fluorescently distinguished NSCs and TAP cells. Young V-SVZ progenitors are highly dynamic, with regular process outgrowth and retraction and cell migration. However, these activities dramatically declined with age. An examination of single-cell RNA sequencing (RNA-seq) data revealed age-associated changes in the
Rho-Rock
pathway that are important for cell motility. Applying a small molecule to inhibit ROCK transformed young into old V-SVZ progenitor cell dynamic behaviors. Hence RHO-ROCK signaling is critical for normal adult NSC and TAP movement and interactions, which are compromised with age, concomitant with the loss of regenerative ability.
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