Human Semaphorin 3 Variants Link Melanocortin Circuit Development and Energy Balance

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Insect cell expression and purification of recombinant SARS‐COV‐2 spike proteins that demonstrate ACE2 binding

The COVID‐19 pandemic caused by SARS‐CoV‐2 infection has led to socio‐economic shutdowns and the loss of over 5 million lives worldwide. There is a need for the identification of therapeutic targets to treat COVID‐19. SARS‐CoV‐2 spike is a target of interest for the development of therapeutic targets. We developed a robust SARS‐CoV‐2 S spike expression and purification protocol from insect cells and studied four recombinant SARS‐CoV‐2 spike protein constructs based on the original SARS‐CoV‐2 sequence using a baculovirus expression system: a spike protein receptor‐binding domain that includes the SD1 domain (RBD) coupled to a fluorescent tag (S‐RBD‐eGFP), spike ectodomain coupled to a fluorescent tag (S‐Ecto‐eGFP), spike ectodomain with six proline mutations and a foldon domain (S‐Ecto‐HexaPro(+F)), and spike ectodomain with six proline mutations without the foldon domain (S‐Ecto‐HexaPro(‐F)). We tested the yield of purified protein expressed from the insect cell lines Spodoptera frugiperda (Sf9) and Trichoplusia ni (Tni) and compared it to previous research using mammalian cell lines to determine changes in protein yield. We demonstrated quick and inexpensive production of functional glycosylated spike protein of high purity capable of recognizing and binding to the angiotensin converting enzyme 2 (ACE2) receptor. To further confirm functionality, we demonstrate binding of eGFP fused construct of the spike ectodomain (S‐Ecto‐eGFP) to surface ACE2 receptors on lung epithelial cells by flow cytometry analysis and show that it can be decreased by means of receptor manipulation (blockade or downregulation).     

A convenient preparation of aldonohydroxamic acids in water and crystal structure of L-erythronohydroxamic acid

Hydroxamic acids derived from aldonic acids, namely aldonohydroxamic acids, have become an increasingly important class of inhibitors of enzymes involved in the metabolism of carbohydrates. We now report the straightforward preparation of various types of aldonohydroxamic acids by a new methodology involving the use of commercial 50% aqueous hydroxylamine as the source of the free base hydroxylamine that reacts directly with the corresponding aldonolactone dissolved in water. The reaction proceeds almost instantaneously in water at room temperature, yielding generally pure products in quantitative yield. To date, this methodology is probably the most facile and efficient way to synthesize aldonohydroxamic acids. We also determined by X-ray diffraction analysis the first crystal structure of a free aldonohydroxamic acid reported to date. Crystals of L-erythronohydroxamic acid belonged to the monoclinic system, space group P2(1), a=5.511(3), b=7.556(1), c=8.071(3) A, beta=109.10 degrees, and Z=2.     

Tissue factor is not involved in the mitogenic activity of factor VIIa

The present study was undertaken to evaluate in vitro the importance of tissue factor in the mitogenic effect of factor VIIa for embryonic fibroblasts. For that purpose, embryonic fibroblasts were isolated from either wild-type or transgenic mice showing a single inactivation of the tissue factor gene or expressing a truncated form (lacking the cytosolic domain) of this protein. Factor VIIa stimulated in a dose-dependent manner the growth of the 3 types of fibroblasts, thus showing that TF is not involved in the mitogenic activity of factor VIIa. The mitogenic activity of factor VIIa disappeared in serum immunopurified in factor X and was almost totally inhibited by DX9065, a selective factor Xa inhibitor, showing that this effect of factor VIIa occurred via factor Xa generated during the incubation period. Hirudin did not show any significant effect on factor VIIa-induced fibroblast proliferation, thus showing that the effect observed for factor VIIa was selectively mediated by factor Xa and was not due to thrombin formation. Our results therefore represent the first evidence for the possible importance of factor Xa in the mitogenic effect of factor VIIa and show the negligible role of tissue factor in this process.     

Studies of mitochondrial respiration in muscle cells in situ: Use and misuse of experimental evidence in mathematical modelling

Applications of permeabilized cell and skinned fiber techniques in combination with methods of mathematical modelling for studies of mitochondrial function in the cell are critically evaluated. Mathematical models may be useful tools for explaining biological phenomena, but only if they are selected by fitting the computing results with real experimental data. Confocal microscopy has been used in experiments with permeabilized cardiomyocytes and myocardial fibers to determine the maximal diffusion distance from medium to the core of cells, which is shown not to exceed 8-10 microm. This is a principal index for correctly explaining high apparent Km for exogenous ADP (200-300 microM) in regulation of mitochondrial respiration in oxidative muscle cells in situ. The best fitting of the results of in silico studies may be achieved by using of the compartmentalized energy transfer model. From these results, it may be concluded that in cardiac muscle cells the mitochondria and ATPases are organized into intracellular energetic units (ICEUs) separated from the bulk phase of cytoplasm by some barriers which limit the diffusion of adenine nucleotides. In contrast, alternative models based on the concept of the cell as homogenous system do not explain the observed experimental phenomena and have led to misleading conclusions. The various sources of experimental and conceptual errors are analyzed.     

Effects of Acute Lorazepam Administration on Aminergic Activity in Normal Elderly Subjects: Relationship to Performance Effects and Apolipoprotein Genotype

The effects of acute lorazepam challenges on plasma (p) HVA, MHPG, and 5-HIAA, and their relationship to drug-induced cognitive and motor deficits and the apolipoprotein (APOE)-epsilon4 allele were examined. Eighteen healthy elderly (8 epsilon4 carriers) received placebo or acute oral lorazepam doses (0.5 mg or 1 mg) in random sequence, 1-week apart. Cognitive assessment and plasma levels of pHVA, pMHPG, and p5-HIAA were determined at baseline and at 1, 2.5, and 5 h postchallenge. There was no drug-to-placebo difference in monoamine levels and no consistent relationship between changes in monoamine levels and cognitive performance, regardless of epsilon4 status. However, the 1.0 mg dose increased p5-HIAA in epsilon4 carriers, whereas it caused a reduction in noncarriers. Higher baseline pMHPG and p5-HIAA levels were associated with better baseline memory. The epsilon4 allele may modulate the effect of lorazepam on p5-HIAA, but further studies are needed to confirm this finding and elucidate its possible significance.     

Analysis of the soluble matrix of vaterite otoliths of juvenile herring (Clupea harengus): do crystalline otoliths have less protein?

Otoliths are calcium carbonate concretions laid down in the inner ear of fish and used in fish age estimation. Otoliths precipitate in the form of aragonite but aberrant precipitation may result in vaterite formation instead of aragonite. Vaterite otoliths are more translucent than aragonite. The quantity of HCl-soluble proteins (SP) was measured in the vaterite otoliths and their aragonite pairs of one year old reared herring Clupea harengus to assess the changes induced by the precipitation of vaterite in the amount of soluble proteins in the otolith. Results showed that vaterite otoliths had as much soluble proteins as their aragonite pairs (p>0.05). Due to the lower density of the vaterite, vaterite otoliths were lighter than their aragonite pairs (p<0.05) which explained that protein concentrations were significantly higher (p<0.05) than in aragonite otoliths. These results indicate that the precipitation of vaterite in otoliths did not affect the inclusion of soluble proteins. Furthermore, they suggest that soluble proteins do not affect the translucent or opaque appearance of otoliths. Differences in translucency may instead be caused by the amounts of insoluble proteins or by differences in the physical properties of proteins. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) of the otolith proteins revealed two bands at 50 and 62 kDa in both aragonite and vaterite otoliths suggesting that the precipitation of vaterite in the otolith is not controlled by either of these two proteins present in the otolith.     

Increased zinc content in transplastomic tobacco plants expressing a polyhistidine-tagged Rubisco large subunit

Rubisco is a hexadecameric enzyme composed of two subunits: a small subunit (SSU) encoded by a nuclear gene (rbcS), and a large subunit (LSU) encoded by a plastid gene (rbcL). Due to its high abundance, Rubisco represents an interesting target to express peptides or small proteins as fusion products at high levels. In an attempt to modify the plant metal content, a polyhistidine sequence was fused to Rubisco, the most abundant protein of plants. Plastid transformation was used to express a polyhistidine (6x) fused to the C-terminal extremity of the tobacco LSU. Transplastomic tobacco plants were generated by cotransformation of polyethylene glycol-treated protoplasts using two vectors: one containing the 16SrDNA marker gene, conferring spectinomycin resistance, and the other the polyhistidine-tagged rbcL gene. Homoplasmic plants containing L8-(His)6S8 as a single enzyme species were obtained. These plants contained normal Rubisco amounts and activity and displayed normal photosynthetic properties and growth. Interestingly, transplastomic plants accumulated higher zinc amounts than the wild-type when grown on zinc-enriched media. The highest zinc increase observed exceeded the estimated chelating ability of the polyhistidine sequence, indicating a perturbation in intracellular zinc homeostasis. We discuss the possibility of using Rubisco to express foreign peptides as fusion products and to confer new properties to higher plants.