Nerve-induced responses of mouse vaginal smooth muscle.

Neural and agonist-induced contractions of proximal (i.e. upper half adjacent to the cervix) and distal mouse vaginal smooth muscle strips were investigated. We hypothesised that nerve-mediated vaginal contractions arise through activity of cholinergic nerves. Nerve activation by bursts of electrical field stimulation (EFS) caused a primary transient contraction often accompanied by a secondary transient contraction, both larger in proximal than distal tissues (i.e. primary: 7-fold larger; secondary: 3-fold larger). Our hypothesis was supported as we found that cholinergic nerves mediated the primary transient contraction in both proximal and distal vaginal strips, as EFS responses were enhanced by neostigmine an anticholinesterase, massively inhibited by the competitive muscarinic receptor antagonist atropine and not affected by the non-selective α-adrenergic receptor antagonist phentolamine. Primary transient contractions were halved in amplitude by the L-type Ca2+ channel blocker nifedipine and markedly inhibited by the sarco-endoplasmic reticulum calcium ATPase (SERCA) inhibitor cyclopiazonic acid (CPA). Resultant secondary transient contractions were abolished by nifedipine. Notably, the selective α1-adrenergic receptor agonist phenylephrine caused tonic contracture in distal but not proximal strips. Low-frequency EFS often initiated recurrent transient contractions similar to those elicited by CCh. Immunohistochemical studies demonstrated innervation of the smooth muscle. Findings of enhanced proximal cholinergic nerve-induced transient contractions, evidence that maintained nerve stimulation could cause recurrent contractions and the finding of distal phenylephrine-mediated tonic contraction have implications on insemination.

Assembly of synthetic locked phycocyanobilin derivatives with phytochrome in vitro and in vivo in Ceratodon purpureus and Arabidopsis.

Phytochromes are photoreceptors with a bilin chromophore in which light triggers the conversion between the red light-absorbing form, Pr, and the far-red-light-absorbing form, Pfr. Here we performed in vitro and in vivo studies using locked phycocyanobilin derivatives, termed 15 Z anti phycocyanobilin (15ZaPCB) and 15 E anti PCB (15EaPCB). Recombinant bacterial and plant phytochromes incorporated either chromophore in a noncovalent or covalent manner. All adducts were photoinactive. The absorption spectra of the 15ZaPCB and 15EaPCB adducts were comparable with those of the Pr and Pfr form, respectively. Feeding of 15EaPCB, but not 15ZaPCB, to protonemal filaments of the moss Ceratodon purpureus resulted in increased chlorophyll accumulation, modulation of gravitropism, and induction of side branches in darkness. The effect of locked chromophores on phytochrome responses, such as induction of seed germination, inhibition of hypocotyl elongation, induction of cotyledon opening, randomization of gravitropism, and gene regulation, were investigated in wild-type Arabidopsis thaliana and the phytochrome-chromophore-deficient long hypocotyl mutant hy1. All phytochrome responses were induced in darkness by 15EaPCB, not only in the mutant but also in the wild type. These studies show that the 15Ea stereochemistry of the chromophore results in the formation of active Pfr-like phytochrome in the cell. Locked chromophores might be used to investigate phytochrome responses in many other organisms without the need to isolate mutants. The induction of phytochrome responses in the hy1 mutant by 15EaPCB were however less efficient than by red light irradiation given to biliverdin-rescued seeds or seedlings.

Diagnosis of a SMARCA4-deficient undifferentiated tumor using multigene panel testing: A case report.

Key Clinical Message: SMARCA4-deficient thoracic carcinoma is a malignant tumor that may present as cancer of unknown primary. This tumor is refractory and requires a novel approach. In addition to identifying therapeutic targets, multigene panel testing can reveal novel genetic mutations, leading to more pathologically relevant diagnoses and appropriate tumor care. Abstract: SMARCA4-deficient undifferentiated tumors are characterized by SMARCA4 inactivation. We present a case of a 74-year-old man with an undifferentiated tumor and a novel SMARCA4 mutation detected using multigene panel testing. The tumor was multiagent and refractory to three chemotherapy lines. The test results helped guide appropriate medical management.

Serological response 5 months after the BNT162b2 COVID-19 vaccination in patients with various hematological disorders in Japan.

Purpose: Patients with hematological malignancies are at an increased risk of severe infection with coronavirus disease 2019 (COVID-19). However, developing an adequate immune response after vaccination is difficult, especially in patients with lymphoid neoplasms. Since the long-term effects of the BNT162b2 vaccine are unclear, the humoral immune response 5 months after the two vaccinations in patients with hematological disorders was analyzed. Materials and Methods: Samples were collected from 96 patients vaccinated twice with BNT162b2 and treated with at least one line of an antitumor or immunosuppressive drug in our hospital from November 2021 to February 2022. Serum anti-severe acute respiratory syndrome coronavirus 2 (anti-SARS-CoV-2) spike (S) antibody titers were analyzed. Patients were age- and sex-matched using propensity matching and compared with a healthy control group. Patients with serum anti-SARS-CoV-2 S antibodies were defined as 'responder' if >50 U/mL. The patients had B-cell non-Hodgkin lymphoma (B-NHL), multiple myeloma, chronic myeloid leukemia, etc. Results: Patients had significantly low antibody levels (median, 55.3 U/mL vs. 809.8 U/mL; p<0.001) and a significantly low response rate (p<0.001). Multivariate analysis showed that patients with B-NHL, aged >72 years, were associated with a low response to vaccination. There were no significant differences between patients with chronic myeloid leukemia and healthy controls. Conclusion: Our study shows that patients with hematological disorders are at risk of developing severe COVID-19 infections because of low responsiveness to vaccination. Moreover, the rate of antibody positivity differed between the disease groups. Further studies are warranted to determine an appropriate preventive method for these patients, especially those with B-NHL.

The cyanobacteriochrome, TePixJ, isomerizes its own chromophore by converting phycocyanobilin to phycoviolobilin.

The cyanobacterial phototaxis regulator protein, TePixJ, is a member of the subfamily of cyanobacteriochromes that binds phycoviolobilin (PVB) as a chromophore and exhibits reversible photoconversion between blue light-absorbing (Pb) and green light-absorbing (Pg) forms. We reconstituted the PVB-binding photoactive holocomplex in vivo and in vitro. Coexpression of the apoprotein and phycocyanobilin (PCB) in Escherichia coli (in vivo reconstitution) produced a mixture of the PCB-bound and PVB-bound holoproteins. Reconstitution in vitro of the apoprotein and synthetic PCB quickly generated a photoactive complex, which covalently bound PCB and exhibited partially reversible photoconversion between two species by UV-vis spectroscopy (with a λ(max) values of 430 and 545 nm). Further incubation produced slow isomerization of PCB to PVB with concomitant improvement of photoreactivity. Site-directed mutagenesis confirmed that Cys522, and a second conserved Cys (Cys494), are both essential for the assembly of the photoactive complex. Fourier transform infrared (FTIR) spectroscopy revealed green light-induced cross-linking, and blue light-induced release, of a thiol group, possibly that of Cys494. These results suggest that the Pb/Pg-type cyanobacteriochrome TePixJ is assembled in at least three steps: (i) rapid and stable chromophorylation of PCB, (ii) additional photoreversible chromophorylation, and (iii) subsequent slow isomerization of PCB to PVB. In addition to its known autolyase activity with Cys522 and photoreversible isomerase activity (of the Z and E isomers at C15 and C16 of PCB), the GAF domain of TePixJ therefore appears to have other roles: as an isomerase (converting PCB to PVB) and as a photoreversible autolyase with a second conserved Cys residue.

Interaction of anti-aggregation agent dimethylethylammonium propane sulfonate with acidic fibroblast growth factor.

Prevention of aggregation is critical for analyzing protein structure. Non-detergent sulfobetaines (NDSBs) are known to prevent protein aggregation, but the molecular mechanisms of their anti-aggregation effect are poorly understood. To elucidate the underlying mechanisms, we analyzed the effects of dimethylethylammonium propane sulfonate (NDSB-195) on acidic fibroblast growth factor (aFGF). NDSB-195 (0.5M) increased both aggregation and denaturation temperatures of aFGF by 4 degrees C. Chemical shift perturbation analyses indicated that many affected residues were located at the junction between a beta-strand (or 3(10)-helix) and a loop, irrespective of the chemical properties of the residue. The apparent dissociation constants of the interaction ranged from 0.04 to 3M, indicating weak interactions between NDSB and protein molecules.