Preparation and evaluation of 99mTc(V)-DMSA complex: studies in medullary carcinoma of thyroid

Consequent to the promising results reported with 99mTc(V)-DMSA for imaging certain types of soft tissue tumors, we have developed methods to prepare this radiopharmaceutical in three ways: from freshly prepared reagents, through the use of a two component kit and use of the standard renal DMSA kit by a modified recipe. The 99mTc(V)-DMSA complex has been subjected to paper electrophoretic and chromatographic procedures and also biodistribution studies. The distinctly different behaviour of this new product compared to that of the well known renal DMSA complex has been clearly established. Scintiimaging in a preliminary clinical trial in patients with medullary carcinoma of the thyroid has been encouraging.     

Horizontal transfer of domains in ssrA gene among Enterobacteriaceae

The tmRNA (transfer messenger RNA), encoded by ssrA gene, is involved in rescuing of stalled ribosomes by a process called trans-translation. Additionally, regions of the ssrA gene (coding for tmRNA) were reported to serve as integration sites for various bacteriophages. Though variations in ssrA genes were reported, their functional relevance is less studied. In this study, we investigated the horizontal gene transfer (HGT) of ssrA among the members of Enterobacteriaceae. This was done by predicting recombination signals in ssrA gene (belonging to Enterobacteriaceae) using RDP5 (Recombination Detection Program 5). Our results revealed 7 recombination signals in ssrA gene belonging to different species. We further showed that the recombination signals were more in the domains present in the 3′ end than 5′ end of tmRNA. Of note, the mRNA region was reported in many recombination signals. Further, members belonging to genera Yersinia, Erwinia, Dickeya and Enterobacter were highly represented in the recombination signals. Sequence analysis revealed the presence of integration sites for different class of bacteriophages in ssrA gene. The locations of phage recognition sites are comparable with recombination signals. Taken together, our results revealed a diverse nature of HGT and recombination which possibly due to transduction mediated by phages.     

Structural basis for p50RhoGAP BCH domain–mediated regulation of Rho inactivation

Spatiotemporal regulation of signaling cascades is crucial for various biological pathways, under the control of a range of scaffolding proteins. The BNIP-2 and Cdc42GAP Homology (BCH) domain is a highly conserved module that targets small GTPases and their regulators. Proteins bearing BCH domains are key for driving cell elongation, retraction, membrane protrusion, and other aspects of active morphogenesis during cell migration, myoblast differentiation, and neuritogenesis. We previously showed that the BCH domain of p50RhoGAP (ARHGAP1) sequesters RhoA from inactivation by its adjacent GAP domain; however, the underlying molecular mechanism for RhoA inactivation by p50RhoGAP remains unknown. Here, we report the crystal structure of the BCH domain of p50RhoGAP Schizosaccharomyces pombe and model the human p50RhoGAP BCH domain to understand its regulatory function using in vitro and cell line studies. We show that the BCH domain adopts an intertwined dimeric structure with asymmetric monomers and harbors a unique RhoA-binding loop and a lipid-binding pocket that anchors prenylated RhoA. Interestingly, the β5-strand of the BCH domain is involved in an intermolecular β-sheet, which is crucial for inhibition of the adjacent GAP domain. A destabilizing mutation in the β5-strand triggers the release of the GAP domain from autoinhibition. This renders p50RhoGAP active, thereby leading to RhoA inactivation and increased self-association of p50RhoGAP molecules via their BCH domains. Our results offer key insight into the concerted spatiotemporal regulation of Rho activity by BCH domain–containing proteins.

Small GTPases are molecular switches that cycle between an active GTP-bound state and an inactive GDP-bound state and are primarily involved in cytoskeletal reorganization during cell motility, morphogenesis, and cytokinesis (1, 2). These small GTPases are tightly controlled by activators and inactivators, such as guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs), respectively (3, 4), which are multidomain proteins that are themselves regulated through their interactions with other proteins, lipids, secondary messengers, and/or by posttranslational modifications (5–7). Despite our understanding of the mechanisms of action of GTPases, GAPs, and GEFs, little is known about how they are further regulated by other cellular proteins in tightly controlled local environments.The BNIP-2 and Cdc42GAP Homology (BCH) domain has emerged as a highly conserved and versatile scaffold protein domain that targets small GTPases, their GEFs, and GAPs to carry out various cellular processes in a spatial, temporal, and kinetic manner (8–15). BCH domain–containing proteins are classified into a distinct functional subclass of the CRAL_TRIO/Sec14 superfamily, with ∼175 BCH domain–containing proteins (in which 14 of them are in human) present across a range of eukaryotic species (16). Some well-studied BCH domain–containing proteins include BNIP-2, BNIP-H (CAYTAXIN), BNIP-XL, BNIP-Sα, p50RhoGAP (ARHGAP1), and BPGAP1 (ARHGAP8), with evidence to show their involvement in cell elongation, retraction, membrane protrusion, and other aspects of active morphogenesis during cell migration, growth activation and suppression, myoblast differentiation, and neuritogenesis (17–21). Aside from interacting with small GTPases and their regulators, some of these proteins can also associate with other signaling proteins, such as fibroblast growth factor receptor tyrosine kinases, myogenic Cdo receptor, p38-MAP kinase, Mek2/MP1, and metabolic enzymes, such as glutaminase and ATP-citrate lyase (17–26). Despite the functional diversity and versatility of BCH domain–containing proteins, the structure of the BCH domain and its various modes of interaction remain unknown. The BCH domain resembles the Sec14 domain (from the CRAL-TRIO family) (16, 27, 28), a domain with lipid-binding characteristics, which may suggest that the BCH domain could have a similar binding strategy. However, to date, the binding and the role of lipids in BCH domain function remain inconclusive.Of the BCH domain–containing proteins, we have focused on the structure and function of p50RhoGAP. p50RhoGAP comprises an N-terminal BCH domain and a C-terminal GAP domain separated by a proline-rich region. We found that p50RhoGAP contains a noncanonical RhoA-binding motif in its BCH domain and is associated with GAP-mediated cell rounding (13). Further, we showed previously that deletion of the BCH domain dramatically enhanced the activity of the adjacent GAP domain (13); however, the full dynamics of this interaction is unclear. Previously, it has been reported that the BCH and other domains regulate GAP activity in an autoinhibited manner (18, 21, 29, 30) involving the interactions of both the BCH and GAP domains, albeit the mechanism remains to be investigated. It has also been shown that a lipid moiety on Rac1 (a Rho GTPase) is necessary for its inactivation by p50RhoGAP (29, 31), which may imply a role in lipid binding. An understanding of how the BCH domain coordinates with the GAP domain to affect the local activity of RhoA and other GTPases would offer a previously unknown insight into the multifaceted regulation of Rho GTPase inactivation.To understand the BCH domain–mediated regulation of p50RhoGAP and RhoA activities, we have determined the crystal structure of a homologous p50RhoGAP BCH domain from S. pombe for functional interrogation. We show that the BCH domain adopts an intertwined dimeric structure with asymmetric monomers and harbors a unique RhoA-interacting loop and a lipid-binding pocket. Our results show that the lipid-binding region of the BCH domain helps to anchor the prenylation tail of RhoA while the loop interacts directly with RhoA. Moreover, we show that a mutation in the β5-strand releases the autoinhibition of the GAP domain by the BCH domain. This renders the GAP domain active, leading to RhoA inactivation and the associated phenotypic effects in yeast and HeLa cells. The released BCH domain also contributes to enhanced p50RhoGAP–p50RhoGAP interaction. Our findings offer crucial insights into the regulation of Rho signaling by BCH domain–containing proteins.     

Correlation between NT proBNP and left ventricular ejection fraction in elderly patients presenting to emergency department with dyspnoea

ObjectiveShortness of breath is a common complaint for which the elderly seek medical attention in the emergency department (ED). Differentiating cardiac from respiratory causes of dyspnoea in this population is quite a challenge. N Terminal pro brain-natriuretic-peptide (NT proBNP) has been studied extensively as a biomarker of left ventricular (LV) failure.MethodsThe NT proBNP was measured in 100 patients above 60 years of age who presented to the ED with shortness of breath. The level was compared with echocardiographic findings to assess correlation with ejection fraction (EF).ResultsThe NT proBNP values increased significantly as the functional severity of heart failure (HF) increased (P < 0.001). The mean NT proBNP level was 1503.33 pg/mL. Patients with respiratory causes of dyspnoea had a mean NT proBNP level of 309.28 pg/mL with normal LV function.ConclusionThe NT proBNP levels had a good correlation with worsening LVEF.