Baculovirus-directed expression of the human insulin receptor and an insulin-binding ectodomain

In this report we describe the use of the baculovirus expression system to overproduce the human insulin holoreceptor (HIR) and a truncated, secretory version of the HIR cDNA (HIRsec) consisting of the alpha subunit and the extracellular portion of the beta subunit (beta'). Sf9 cells infected with the full-length HIR viruses synthesize recombinant HIR (rHIR) with an insulin-binding alpha subunit of apparent Mr = 110,000 and a beta subunit of apparent Mr = 80,000. Uncleaved alpha beta proreceptor accumulates in infected cells. Both of these forms assemble into higher order disulfide-linked dimers or heterotetramers of apparent Mr greater than 350,000. Insulin-binding activity in cells infected with rHIR viruses is present predominantly on the extracellular aspect of the plasma membrane (greater than 80%). Insulin binding to the full-length rHIR occurs with typical complex kinetics with Kd1 = 0.5-1 x 10(-9) M and Kd2 = 10(-7) M and receptors are present in large amounts in infected cells (1 x 10(6) receptors/cell; 1-2 mg HIR/10(9) cells). The full-length rHIR undergoes insulin-dependent autophosphorylation; half-maximal activation of beta subunit autophosphorylation occurs at 1-2 x 10(-8) M. The alpha beta proreceptor also becomes phosphorylated in vitro. Analysis of tryptic phosphopeptides derived from in vitro autophosphorylated beta subunit and alpha beta proreceptor reveals a pattern of phosphorylation that is indistinguishable from that of authentic placental HIR. Sf9 cells infected with rHIRsec viruses synthesize and secrete an (alpha beta')2 heterotetrameric complex having an insulin-binding alpha subunit of apparent Mr = 110,000 and a truncated beta' subunit of apparent Mr = 45,000 that lacks kinase activity. The rHIRsec complex purified from the conditioned medium of infected cells binds insulin with high affinity (Kd = 10(-9) M).     

Discovery and functional interrogation of SARS-CoV-2 RNA-host protein interactions

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Inhibition of dopamine uptake by N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, a cause of parkinsonism

N-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine has been reported to cause parkinsonism in man and monkeys, producing behavioral effects within 5 min of administration. The compound reversibly and competively inhibited (IC50 = 2 microM) dopamine uptake into PC12, a clonal line of rat pheochromocytoma cells that store and secrete dopamine and acetylcholine. Uptake of choline and 2-deoxyglucose was not affected. Prolonged exposure to the compound was lethal to PC12; survivors of this treatment lost the ability to store dopamine and acetylcholine and to extend neurites upon incubation with nerve growth factor.     

Rhinophyma: treatment with electrocautery and silver-impregnated porcine xenograft

A typical case of advanced rhinophyma is reported. Partial excision reduced the size of the nose by two-thirds, leaving a thin layer of dermis. This was covered with silver-impregnated porcine xenograft, which allowed efficient drainage and remained adherent in its initial application for 2 weeks. A total of four applications of silver-impregnated porcine xenografts--the last two involving only very small areas--were required for complete epithelialization at 5 weeks after surgery. We believe that this xenograft is an ideal dressing for wounds resulting from partial excision of rhinophymatous tissue, providing the beneficial effects of biological dressings, including the hemostatic effects of collagen with the added benefit of silver's potent antibacterial properties. Silver-impregnated porcine xenograft reduces patient discomfort and results in rapid healing with excellent cosmetic results.     

Demography of an endangered,long-lived fish: Informing management options in the face of cyclic and stochastic climate variation

June sucker (Chasmistes liorus) is a long-lived, endangered fish endemic to Utah Lake, Utah. For several decades June sucker have failed to recruit sufficient numbers to the adult size classes such that the current wild population consists of a small number of old adults and it continues to decline. Vital rates of June sucker are influenced by climate-driven variation in lake level and inflow from the Provo River. We used population projection matrix modeling to assess effects of cyclic and stochastic environmental variation on population growth trajectories of June sucker in Utah Lake. The stable stage distribution is dominated by stage 1 individuals (93% of the total population) in contrast to the current situation where old age classes are the most abundant. Total population size is highly influenced by the stochastic component of climate variation; whereas, the adult population of June sucker closely tracks the systematic drought cycle. If changes in survival of larvae and juveniles can be coordinated such that positive changes in both parameters can occur somewhat simultaneously, then each parameter would only have to be increased by a factor of about 8.8 to achieve sustainable population growth (compared to a 77-fold increase for each parameter separately). Stochastic climatic variation has relatively little long-term effect on population growth. However, the multidecadal cyclic pattern of lake level and river discharge imposes a similar pattern on population growth rates of the June sucker, such that during some periods, populations decline even when the long-term trend is positive.     

A new phylogenetic test for comparing multiple high‐dimensional evolutionary rates suggests interplay of evolutionary rates and modularity in lanternfishes (Myctophiformes; Myctophidae)

The interplay between evolutionary rates and modularity influences the evolution of organismal body plans by both promoting and constraining the magnitude and direction of trait response to ecological conditions. However, few studies have examined whether the best‐fit hypothesis of modularity is the same as the shape subset with the greatest difference in evolutionary rate. Here, we develop a new phylogenetic comparative method for comparing evolutionary rates among high‐dimensional traits, and apply this method to analyze body shape evolution in bioluminescent lanternfishes. We frame the study of evolutionary rates and modularity through analysis of three hypotheses derived from the literature on fish development, biomechanics, and bioluminescent communication. We show that a development‐informed partitioning of shape exhibits the greatest evolutionary rate differences among modules, but that a hydrodynamically informed partitioning is the best‐fit modularity hypothesis. Furthermore, we show that bioluminescent lateral photophores evolve at a similar rate as, and are strongly integrated with, body shape in lanternfishes. These results suggest that overlapping life‐history constraints on development and movement define axes of body shape evolution in lanternfishes, and that the positions of their lateral photophore complexes are likely a passive outcome of the interaction of these ecological pressures.     

Computational and Functional Analyses of a Small-Molecule Binding Site in ROMK

The renal outer medullary potassium channel (ROMK, or Kir1.1, encoded by KCNJ1) critically regulates renal tubule electrolyte and water transport and hence blood volume and pressure. The discovery of loss-of-function mutations in KCNJ1 underlying renal salt and water wasting and lower blood pressure has sparked interest in developing new classes of antihypertensive diuretics targeting ROMK. The recent development of nanomolar-affinity small-molecule inhibitors of ROMK creates opportunities for exploring the chemical and physical basis of ligand-channel interactions required for selective ROMK inhibition. We previously reported that the bis-nitro-phenyl ROMK inhibitor VU591 exhibits voltage-dependent knock-off at hyperpolarizing potentials, suggesting that the binding site is located within the ion-conduction pore. In this study, comparative molecular modeling and in silico ligand docking were used to interrogate the full-length ROMK pore for energetically favorable VU591 binding sites. Cluster analysis of 2498 low-energy poses resulting from 9900 Monte Carlo docking trajectories on each of 10 conformationally distinct ROMK comparative homology models identified two putative binding sites in the transmembrane pore that were subsequently tested for a role in VU591-dependent inhibition using site-directed mutagenesis and patch-clamp electrophysiology. Introduction of mutations into the lower site had no effect on the sensitivity of the channel to VU591. In contrast, mutations of Val¹⁶⁸ or Asn¹⁷¹ in the upper site, which are unique to ROMK within the Kir channel family, led to a dramatic reduction in VU591 sensitivity. This study highlights the utility of computational modeling for defining ligand-ROMK interactions and proposes a mechanism for inhibition of ROMK.