Characterization of Antibodies against Receptor Activity-Modifying Protein 1 (RAMP1): A Cautionary Tale

Calcitonin gene-related peptide (CGRP) is a key component of migraine pathophysiology, yielding effective migraine therapeutics. CGRP receptors contain a core accessory protein subunit: receptor activity-modifying protein 1 (RAMP1). Understanding of RAMP1 expression is incomplete, partly due to the challenges in identifying specific and validated antibody tools. We profiled antibodies for immunodetection of RAMP1 using Western blotting, immunocytochemistry and immunohistochemistry, including using RAMP1 knockout mouse tissue. Most antibodies could detect RAMP1 in Western blotting and immunocytochemistry using transfected cells. Two antibodies (844, ab256575) could detect a RAMP1-like band in Western blots of rodent brain but not RAMP1 knockout mice. However, cross-reactivity with other proteins was evident for all antibodies. This cross-reactivity prevented clear conclusions about RAMP1 anatomical localization, as each antibody detected a distinct pattern of immunoreactivity in rodent brain. We cannot confidently attribute immunoreactivity produced by RAMP1 antibodies (including 844) to the presence of RAMP1 protein in immunohistochemical applications in brain tissue. RAMP1 expression in brain and other tissues therefore needs to be revisited using RAMP1 antibodies that have been comprehensively validated using multiple strategies to establish multiple lines of convincing evidence. As RAMP1 is important for other GPCR/ligand pairings, our results have broader significance beyond the CGRP field

Internal Carotid Artery Aneurysm Mimicking a Pituitary Adenoma: A Case Report and Review of the Literature

Background/Objective: A thrombosed internal carotid artery (ICA) aneurysm mimicking a pituitary adenoma can be catastrophic if unrecognized. We report a unique case of the rare presentation of ICA aneurysms masquerading as pituitary adenomas, which can preserve pituitary function when treated early. Case Report: A 54-year-old man with type 2 diabetes, aortic valve replacement, and stroke presented with sudden onset severe headache and left eye pain. Left third nerve palsy was noted. Laboratory studies showed low thyroid-stimulating hormone, follicle-stimulating hormone, luteinizing hormone, testosterone, and insulin-like growth factor 1 levels and baseline, post–30-minute, and post–60-minute cortisol levels of 16, 17, and 14 μg/dL, respectively, after adrenocorticotropic hormone stimulation. Magnetic resonance imaging of the pituitary revealed a heterogeneously enhancing 2.0 × 2.1 × 2.1-cm sellar/suprasellar mass with peripheral enhancement abutting the left cavernous sinus. Given the acute third nerve palsy without visual defects and magnetic resonance imaging findings, other sources of sellar occupying etiology were suspected. Therefore, carotid cerebral angiography was performed and revealed a mostly thrombosed left ICA aneurysm projected into the sellar/suprasellar region. The patient underwent successful endovascular treatment with a resolution of the cranial nerve palsy and hormonal abnormalities at 3-month follow-up. Discussion: Our case demonstrates the importance of swift recognition of ICA aneurysms masquerading as pituitary adenomas. Early recognition and treatment may lead to the complete resolution of presenting symptoms and hormonal deficiencies. Conclusion: Clinicians should have a high index of suspicion for ICA aneurysm in the differential diagnosis for a sellar mass. Careful evaluation is essential because misdiagnosis may lead to catastrophic consequences

Association of the circadian rhythmic expression of GmCRY1a with a latitudinal cline in photoperiodic flowering of soybean

Photoperiodic control of flowering time is believed to affect latitudinal distribution of plants. The blue light receptor CRY2 regulates photoperiodic flowering in the experimental model plant Arabidopsis thaliana. However, it is unclear whether genetic variations affecting cryptochrome activity or expression is broadly associated with latitudinal distribution of plants. We report here an investigation of the function and expression of two cryptochromes in soybean, GmCRY1a and GmCRY2a. Soybean is a short-day (SD) crop commonly cultivated according to the photoperiodic sensitivity of cultivars. Both cultivated soybean (Glycine max) and its wild relative (G. soja) exhibit a strong latitudinal cline in photoperiodic flowering. Similar to their Arabidopsis counterparts, both GmCRY1a and GmCRY2a affected blue light inhibition of cell elongation, but only GmCRY2a underwent blue light- and 26S proteasome-dependent degradation. However, in contrast to Arabidopsis cryptochromes, soybean GmCRY1a, but not GmCRY2a, exhibited a strong activity promoting floral initiation, and the level of protein expression of GmCRY1a, but not GmCRY2a, oscillated with a circadian rhythm that has different phase characteristics in different photoperiods. Consistent with the hypothesis that GmCRY1a is a major regulator of photoperiodic flowering in soybean, the photoperiod-dependent circadian rhythmic expression of the GmCRY1a protein correlates with photoperiodic flowering and latitudinal distribution of soybean cultivars. We propose that genes affecting protein expression of the GmCRY1a protein play an important role in determining latitudinal distribution of soybeans