Characteristics of β-adrenergic receptors in longitudinal muscle membranes of rat uterus: Changes in kinetic properties of the receptor during gestation

Binding characteristics of β-adrenergic receptors of longitudinal muscle membranes isolated from different stages of pregnant rat myometrium were investigated using [³H]dihydroalprenolol. Between Days 15 and 21 of gestation, the ratio of β₁- and β₂-adrenergic receptors of longitudinal membranes was constant. The membranes were found to be predominant in β₂-adrenergic receptors. The concentration of longitudinal muscle β-adrenergic receptors increased significantly during the last 7 days of gestation. Kinetic binding studies implied that the affinity of the membrane β-adrenergic receptors decreased through a slight decrease in the association rate and a large increase in the dissociation rate with progression of pregnancy. A Scatchard plot indicated that longitudinal muscle in β-adrenergic receptors on Days 15 and 18 constitute a single class of independent sites. By contrast, the dissociation kinetics, the convex downward curvature in a Scatchard plot and a Hill coefficient (h) of less than 1.00 of [³H] DHA binding to β-receptors of muscle on Day 21 suggested the existence of negatively cooperative multiple binding sites for β-adrenergic ligand. These results suggest that changes in the dynamics of uterus β-adrenergic receptors play an important role in the onset of labor.     

Crystal structure of meso-2,3-butanediol dehydrogenase in a complex with NAD+ and inhibitor mercaptoethanol at 1.7 A resolution for understanding of chiral substrate recognition mechanisms

The crystal structure of a ternary complex of meso-2,3-butanediol dehydrogenase with NAD+ and a competitive inhibitor, mercaptoethanol, has been determined at 1.7 A resolution by means of molecular replacement and refined to a final R-factor of 0.194. The overall structure is similar to those of the other short chain dehydrogenase/reductase enzymes. The NAD+ binding site, and the positions of catalytic residues Ser139, Tyr152, and Lys156 are also conserved. The crystal structure revealed that mercaptoethanol bound specifically to meso-2,3-butanediol dehydrogenase. Two residues around the active site, Gln140 and Gly183, forming hydrogen bonds with the inhibitor, are important but not sufficient for distinguishing stereoisomerism of a chiral substrate.     

Disturbed activation of endoplasmic reticulum stress transducers by familial Alzheimer's disease-linked presenilin-1 mutations

Recent studies have shown independently that presenilin-1 (PS1) null mutants and familial Alzheimer's disease (FAD)-linked mutants should both down-regulate signaling of the unfolded protein response (UPR). However, it is difficult to accept that both mutants possess the same effects on the UPR. Furthermore, contrary to these observations, neither loss of PS1 and PS2 function nor expression of FAD-linked PS1 mutants were reported to have a discernable impact on the UPR. Therefore, re-examination and detailed analyses are needed to clarify the relationship between PS1 function and UPR signaling. Here, we report that PS1/PS2 null and dominant negative PS1 mutants, which are mutated at aspartate residue 257 or 385, did not affect signaling of the UPR. In contrast, FAD-linked PS1 mutants were confirmed to disturb UPR signaling by inhibiting activation of both Ire1alpha and ATF6, both of which are endoplasmic reticulum (ER) stress transducers in the UPR. Furthermore, PS1 mutants also disturbed activation of PERK (PKR-like ER kinase), which plays a crucial role in inhibiting translation during ER stress. Taken together, these observations suggested that PS1 mutations could affect signaling pathways controlled by each of the respective ER-stress transducers, possibly through a gain-of-function.     

Scratch cytologic findings on surgically resected solitary fibrous tumors of the pleura

OBJECTIVE: To ascertain the cytologic characteristics of solitary fibrous tumors of the pleura (SFTPs) on smear preparations. STUDY DESIGN: Fine needle aspiration cytology (FNAC) was initially attempted preoperatively in five cases, but the specimens were inappropriate for interpretation because only a few tumor cells were obtained. Therefore, scratch smears made at the time of operation were used. Papanicolaou and immunocytochemical staining was performed in all 10 cases, 2 of which were malignant. RESULTS: As expected, cellular tumors yielded more cells. The cytologic appearance was variable, showing spindle/bipolar, dendritic/stellate and intermediate cells. Atypical cells reminiscent of sarcoma were also present in cellular, benign tumors. Highly atypical epithelioid cells were obtained in two malignant cases. Immunocytochemically, the tumor cells were positive for CD34 and vimentin and negative for cytokeratin, regardless of histologic differences and cell shape. CONCLUSION: It seems difficult to diagnose SFTPs with certainty by FNAC, partly because the cell morphology of SFTPs resembles a wide variety of heterogeneous groups of spindle cell tumors and partly because only a few tumor cells were available in the FNAC specimens in the present study. However, a cytologic diagnosis of SFTP is possible if cytologic preparations yield CD34-positive cells with spindle/bipolar or dendritic/stellate morphology.     

Different functional aspects of the group II subfamily (Types IIA and V) and type X secretory phospholipase A(2)s in regulating arachidonic acid release and prostaglandin generation. Implications of cyclooxygenase-2 induction and phospholipid scramblase-mediated cellular membrane perturbation.

We have recently reported that members of the heparin-binding group II subfamily of secretory PLA(2)s (sPLA(2)s) (types IIA and V), when transfected into 293 cells, released [(3)H]arachidonic acid (AA) preferentially in response to interleukin-1 (IL-1) and acted as "signaling" PLA(2)s that were functionally coupled with prostaglandin biosynthesis. Here we show that these group II subfamily sPLA(2)s and the type X sPLA(2) behave in a different manner, the former being more efficiently coupled with the prostaglandin-biosynthetic pathway than the latter, in 293 transfectants. Type X sPLA(2), which bound only minimally to cell surface proteoglycans, augmented the release of both [(3)H]AA and [(3)H]oleic acid in the presence of serum but not IL-1. Both types IIA and V sPLA(2), the AA released by which was efficiently converted to prostaglandin E(2), markedly augmented IL-1-induced expression of cyclooxygenase (COX)-2 in a heparin-sensitive fashion, whereas type X sPLA(2) lacked the ability to augment COX-2 expression, thereby exhibiting the poor prostaglandin E(2)-biosynthetic response unless either of the COX isozymes was forcibly introduced into type X sPLA(2)-expressing cells. Implication of phospholipid scramblase, an enzyme responsible for the perturbation of plasma membrane asymmetry, revealed that the scramblase-transfected cells became more sensitive to types IIA and V, but not X, sPLA(2), releasing both [(3)H]AA and [(3)H]oleic acid in an IL-1-independent manner. Thus, although phospholipid scramblase-mediated alteration in plasma membrane asymmetry actually led to the increased cellular susceptibility to the group II subfamily of sPLA(2)s, several lines of evidence suggest that it does not entirely mimic their actions on cells after IL-1 signaling. Interestingly, coexpression of type IIA or V, but not X, sPLA(2) and phospholipid scramblase resulted in a marked reduction in cell growth, revealing an unexplored antiproliferative aspect of particular classes of sPLA(2).     

Production of L-2,3-butanediol by a new pathway constructed in Escherichia coli

AIMS: A metabolic pathway for L-2,3-butanediol (BD) as the main product has not yet been found. To rectify this situation, we attempted to produce L-BD from diacetyl (DA) by producing simultaneous expression of diacetyl reductase (DAR) and L-2,3-butanediol dehydrogenase (BDH) using transgenic bacteria, Escherichia coli JM109/pBUD-comb. METHODS AND RESULTS: The meso-BDH of Klebsiella pneumoniae was used for its DAR activity to convert DA to L-acetoin (AC) and the L-BDH of Brevibacterium saccharolyticum was used to reduce L-AC to L-BD. The respective gene coding each enzyme was connected in tandem to the MCS of pFLAG-CTC (pBUD-comb). The divided addition of DA as a source, addition of 2% glucose, and the combination of static and shaking culture was effective for the production. CONCLUSIONS: L-BD (2200 mg l(-1)) was generated from 3000 mg l(-1) added of DA, which corresponded to a 73% conversion rate. Meso-BD as a by-product was mixed by 2% at most. SIGNIFICANCE AND IMPACT OF THE STUDY: An enzyme system for converting DA to L-BD was constructed with a view to using DA-producing bacteria in the future.