Morphologic and hematologic characteristics of storage pool deficiency in beige rats (Chédiak-Higashi syndrome of rats)

Characterization of beige rats as having a platelet storage pool deficiency (SPD) was undertaken. Platelets from beige rats, an animal model of Chédiak-Higashi syndrome (CHS), completely lacked the ability to aggregate when stimulated with high dosages of collagen (50 micrograms/ml), and lacked secondary aggregation induced by adenosine diphosphate (ADP). Concentrations of ADP, ATP, and serotonin in the platelets of beige rats were significantly lower than those of control rats. However, platelet count remained within normal values. Electron microscopy revealed that platelets had fewer dense granules, whereas other organelles had normal structure. This morphologic and functional evidence confirms that platelets of beige rats have the typical characteristics of SPD.     

Potent and orally active ET(A) selective antagonists with 5,7-diarylcyclopenteno[1,2-b]pyridine-6-carboxylic acid structures

The synthesis and structure-activity relationships of a series of 5,7-diarylcyclopenteno[1,2-b]pyridine-6-carboxylic acids are described. Our efforts have been focused on modification of the aryl ring at the 5-position and the alkyl substituent at the 2-position of the bottom 4-methoxyphenyl ring in an effort to develop orally available ET(A) selective antagonists with safer profiles in terms of the P-450 enzyme inhibitory activity. Incorporation of a hydroxymethyl group as an alkyl substituent in methylenedioxyphenyl and 6-dihydrobenzofuran derivatives led to the identification of orally bioavailable ET(A) selective antagonists 1f and 7f. These compounds also showed not only excellent binding affinity (IC(50) < 0.10nM, more than 800-fold selectivity for the ET(A) receptor over the ET(B) receptor) but also sufficient oral bioavailability, 48% and 56%, respectively, in rats. Furthermore, these compounds did not exhibit either competitive or mechanism-based inhibition of human cytochrome P450 enzymes.     

Phenylalanine production by metabolically engineered Corynebacterium glutamicum with the pheA gene of Escherichia coli

The bifunctional enzyme chorismate mutase (CM)-prephenate dehydratase (PD), which is encoded by the pheA gene of Escherichia coli, catalyses the two consecutive key steps in phenylalanine biosynthesis. To utilize the enzyme for metabolic engineering of phenylalanine-producing Corynebacterium glutamicum KY10694, the intact gene was cloned on a multicopy vector to yield pEA11. C. glutamicum cells transformed with pEA11 exhibited a more than tenfold increase in CM and PD activities relative to the host cells. Moreover, the level of pheA expression was further elevated a fewfold when cells were starved of phenylalanine, suggesting that the attenuation regulation of pheA expression functions in heterogeneous C. glutanicum. Plasmid pEA11 encoding the wild-type enzyme was mutated to yield pEA22, which specified CM-PD exhibiting almost complete resistance to end-product inhibition. When pEA22 was introduced into KY10694, both the activities of CM and PD were highly maintained throughout the cultivation, thus leading to a 35% increased production (23 g/l) of phenylalanine.     

The exceptionally tight affinity of DnaA for ATP/ADP requires a unique aspartic acid residue in the AAA+ sensor 1 motif

Escherichia coli DnaA, an AAA+ superfamily protein, initiates chromosomal replication in an ATP-binding-dependent manner. Although DnaA has conserved Walker A/B motifs, it binds adenine nucleotides 10- to 100-fold more tightly than do many other AAA+ proteins. This study shows that the DnaA Asp-269 residue, located in the sensor 1 motif, plays a specific role in supporting high-affinity ATP/ADP binding. The affinity of the DnaA D269A mutant for ATP/ADP is at least 10- to 100-fold reduced compared with that of the wild-type and DnaA R270A proteins. In contrast, the abilities of DnaA D269A to bind a typical DnaA box, unwind oriC duplex in the presence of elevated concentrations of ATP, load DnaB onto DNA and support minichromosomal replication in a reconstituted system are retained. Whereas the acidic Asp residue is highly conserved among eubacterial DnaA homologues, the corresponding residue in many other AAA+ proteins is Asn/Thr and in some AAA+ proteins these neutral residues are essential for ATP hydrolysis but not ATP binding. As the intrinsic ATPase activity of DnaA is extremely weak, this study reveals a novel and specific function for the sensor 1 motif in tight ATP/ADP binding, one that depends on the alternate key residue Asp.     

Docking protein Gab2 positively regulates glycoprotein VI-mediated platelet activation

Gab2, a recently identified docking protein, contains a pleckstrin homology domain and potential binding sites for SH2 and SH3 domain-containing proteins. Gab2 has been shown to support growth, differentiation, and function in a number of haematopoietic cells, although its role in platelets remains to be determined. Here we report that cross-linking of the collagen receptor GPVI by the snake venom toxin convulxin stimulates tyrosine phosphorylation of Gab2. Furthermore, platelet aggregation induced by submaximal concentrations of convulxin is attenuated in the absence of Gab2, although recovery is seen with higher concentrations of the toxin. Consistent with this, tyrosine phosphorylation of Fc receptor gamma-chain, Syk, Btk, and phospholipase Cgamma2 by convulxin is reduced in the absence of Gab2. In comparison, the G protein-coupled receptor agonist, thrombin, does not induce phosphorylation of Gab2 and aggregation is unaltered in the absence of the toxin. These findings provide evidence for a functional role of Gab2 in supporting platelet activation by GPVI.     

Evaluation of platelet calcium ion mobilization by the use of various divalent ions.

Divalent ion mobilization in human platelets was evaluated with Fura-2 fluorescence changes induced by Ca2+, Sr2+, Ba2+ and Mn2+. Extracellular Ca2+, Sr2+ and Ba2+ all entered thrombin-stimulated platelets. These divalent ions were also able to refill the intracellular Ca2+ storage sites which had been depleted of Ca2+ by ionomycin treatment, and were released from the storage sites upon thrombin stimulation. However, only the refilling of the storage sites with Ca2+ and Sr2+, but not with Ba2+, were capable of suppressing the opening state of Ca2+ channels assessed with Mn2+ influx. Efflux of intracellularly accumulated divalent ions was observed with Ca2+ and Sr2+ but not with Ba2+. These findings indicate that there are subtle differences in the Ca(2+)-binding domains of the various systems involved in Ca2+ mobilization in platelets, some of which discriminate Ba2+ while accepting Sr2+.     

l-threonine production by l-aspartate- and l-homoserine-resistant mutant of Escherichia coli

Summary Growth and l-threonine productivity of l-threonine producer Escherichia coli H-4290 were inhibited by precursor amino acids, l-homoserine and l-aspartate. l-Threonine hyper-producers were isolated among the mutants resistant to l-homoserine and l-aspartate. Mutants H-4351 (Hom^r) and H-4578 (Hom^r, Asp^r) accumulated 22.2 g/l and 24.3 g/l of l-threonine in test tube cultures, while the parental strain H-4290 accumulated 18.2 g/l. The enzyme level of aspartokinase I (first enzyme of the threonine operon) was enhanced 2.3 times (H-4351) and 3 times (H-4578) that of H-4290. Mutant H-4578 accumulated 76 g/l of l-threonine in a 2-l jar fermentor after 75 h cultivation.Abbreviations DAP diaminopimeric acid - Met ^l poor growth in methionine-free medium - AHV -amino--hydroxyvaleric acid - Thr-N^- lack of ability to utilize l-threonine as a nitrogen source - Rif rifampicin - Lys+Met^r resistant to l-lysine and dl-methionine