The efficient detection of membrane protein with immunoblotting: lessons from cold-temperature denaturation

　Transmembrane proteins play essential roles in cell signaling, transport of membrane-impermeable molecules, cell-cell communication, and cell adhesion. Our recent work demonstrated that reactive oxygen species-generating NADPH oxidase 4 (Nox4), a protein with multiple transmembrane domains, is involved in cell migration by stabilizing vascular endothelial growth factor receptor 2 (VEGFR-2), a single-span transmembrane protein. During this study and with further verification, we developed a simple method to prepare protein samples without aggregating these membrane proteins for SDS-PAGE, immunoblotting, and deglycosylation assay. We found that heating was unnecessary for protein denaturation for SDS-PAGE and deglycosylation assay. Also, the detectable amounts of VEGFR-2 and Nox4 were increased in the sample treated at 4℃ compared with the sample treated at 98℃ . Moreover, the N-glycan of VEGFR-2 was digested by glycosidase at reaction temperature 4℃

Retrospective Evaluation of an Oral Propranolol Delivery Strategy in 25 Cases of Infantile Hemangioma

Objectives: The majority of infantile hemangiomas shrink on their own, but when there is a chance of functional impairment, ulceration, or aesthetic issues, an active therapeutic intervention is needed. Propranolol is effective in treating infantile hemangiomas in recent years, although little information is known regarding the consequences of long-term oral administration of propranolol for treating infantile hemangioma or regarding when to stop propranolol treatment. We looked back at the therapeutic effectiveness, sequelae, and side effects of propranolol in actual clinical settings for infantile hemangiomas. Methods: The study comprised 25 patients under the age of 6 months who had propranolol treatment for infantile hemangiomas in our department between April 2011 and May 2021. Results: At one month and 6 months following the commencement of treatment, propranolol treatment was successful in 72% and 96% of patients, respectively. In 84% of patients, there were cutaneous sequelae. Only one patient with sequelae of hair loss required early surgery because of a cosmetic problem. There were substantial side effects in one case, but these were not related to the propranolol dose. Conclusions: This study found that the effect of propranolol did not differ depending on the kind of infantile hemangiomas, although propranolol was expected to be extremely helpful for head lesions regardless of site. A degree of sequelae remained in many cases following propranolol therapy, whereas the subcutaneous type was less likely to develop sequelae with propranolol treatment

The α(1D)-adrenergic receptor directly regulates arterial blood pressure via vasoconstriction

To investigate the physiological role of the α(1D)-adrenergic receptor (α(1D)-AR) subtype, we created mice lacking the α(1D)-AR (α(1D)(–/–)) by gene targeting and characterized their cardiovascular function. In α(1D)(–/–) mice, the RT-PCR did not detect any transcript of the α(1D)-AR in any tissue examined, and there was no apparent upregulation of other α(1)-AR subtypes. Radioligand binding studies showed that α(1)-AR binding capacity in the aorta was lost, while that in the heart was unaltered in α(1D)(–/–) mice. Non-anesthetized α(1D)(–/–) mice maintained significantly lower basal systolic and mean arterial blood pressure conditions, relative to wild-type mice, and they showed no significant change in heart rate or in cardiac function, as assessed by echocardiogram. Besides hypotension, the pressor responses to phenylephrine and norepinephrine were decreased by 30–40% in α(1D)(–/–) mice. Furthermore, the contractile response of the aorta and the pressor response of isolated perfused mesenteric arterial beds to α(1)-AR stimulation were markedly reduced in α(1D)(–/–) mice. We conclude that the α(1D)-AR participates directly in sympathetic regulation of systemic blood pressure by vasoconstriction

Characterisation and Traceability of Calcium Carbonate from the Seaweed Lithothamnium calcareum

Calcium carbonate (CaCO3) from the seaweed Lithothamnium calcareum is a suitable dietary supplement for the prevention of osteoporosis, due to its chemical composition. This study compared CaCO3 from L. calcareum to CaCO3 from oyster shell and inorganic minerals that are already used in the pharmaceutical industry. The Rietveld refinement of the XRD showed that the mineral fraction of L. calcareum is composed of aragonite (50.3 wt%), magnesian calcite (45.3 wt%), calcite (4.4 wt%), comin contrast to oyster shell and inorganic minerals, which contain only calcite. The morphology of L. calcareum carbonate particles is granular xenomorphic, which is distinct from the scalenohedral form of inorganic calcite and the fibrous and scale-like fragments of oyster shell. The crystal structures of aragonite and magnesian calcite, present in L. calcareum, have higher contents of oligoelements than the pure calcite in other materials. The isotopic composition (stable isotopes of carbon and oxygen) is heavy in the CaCO3 from L. calcareum (δ13C = 1.1‰; δ18O = −0.1‰) and oyster shell (δ13C = −4‰; δ18O = −2.8‰) in marked contrast to the much lighter isotopic composition of inorganic mineral CaCO3 (δ13C = −19.2‰; δ18O = −26.3‰). The differences indicated above were determined through principal component analysis, where the first and second principal components are sufficient for the clear distinction and traceability of CaCO3 sources