Probing the rotor subunit interface of the ATP synthase from Ilyobacter tartaricus

The interaction between the c(11)ring and the gammaepsilon complex, forming the rotor of the Ilyobacter tartaricus ATP synthase, was probed by surface plasmon resonance spectroscopy and in vitro reconstitution analysis. The results provide, for the first time, a direct and quantitative assessment of the stability of the rotor. The data indicated very tight binding between the c(11)ring and the gammaepsilon complex, with an apparent K(d) value of approximately 7.4nm. The rotor assembly was primarily dependent on the interaction of the cring with the gammasubunit, and binding of the cring to the free epsilon subunit was not observed. Mutagenesis of selected conserved amino acid residues of all three rotor components (cR45, cQ46, gammaE204, gammaF203 and epsilonH38) severely affected rotor assembly. The interaction kinetics between the gammaepsilon complex and c(11)ring mutants suggested that the assembly of the c(11)gammaepsiloncomplex was governed by interactions of low and high affinity. Low-affinity binding was observed between the polar loops of the cring subunits and the bottom part of the gamma subunit. High-affinity interactions, involving the two residues gammaE204 and epsilonH38, stabilized the holo-c(11)gammaepsilon complex. NMR experiments indicated the acquisition of conformational order in otherwise flexible C- and N-terminal regions of the gamma subunit on rotor assembly. The results of this study suggest that docking of the central stalk of the F(1)complex to the rotor ring of F(o) to form tight, but reversible, contacts provides an explanation for the relative ease of dissociation and reconstitution of F(1)F(o)complexes.     

SARS-CoV-2 S2P spike ages through distinct states with altered immunogenicity

The SARS-CoV-2 spike is the primary target of virus-neutralizing antibodies and critical to the development of effective vaccines against COVID-19. Here, we demonstrate that the prefusion-stabilized two-proline “S2P” spike—widely employed for laboratory work and clinical studies—unfolds when stored at 4 °C, physiological pH, as observed by electron microscopy (EM) and differential scanning calorimetry, but that its trimeric, native-like conformation can be reacquired by low pH treatment. When stored for approximately 1 week, this unfolding does not significantly alter antigenic characteristics; however, longer storage diminishes antibody binding, and month-old spike elicits virtually no neutralization in mice despite inducing high ELISA-binding titers. Cryo-EM structures reveal the folded fraction of spike to decrease with aging; however, its structure remains largely similar, although with varying mobility of the receptor-binding domain. Thus, the SARS-CoV-2 spike is susceptible to unfolding, which affects immunogenicity, highlighting the need to monitor its integrity.     

Fluorine level in the bone and cartilage in children treated with ciprofloxacin (data obtained from the femur distal part]

Comparative data on the content of fluorine in various sections of the left femur distal part, i.e. spongy bone, process zone and articular cartilage are presented. The examination was performed on postmortem sections from 10 pediatric cases (the average age of 9.5 years) with mucoviscidosis or aplastic anemia treated before the death for up to 12 months with ciprofloxacin in a dose of 20 to 40 mg/kg body weight. The control postmortem sections were from 10 practically healthy children (the average age of 10.4 years) killed in a car accident. The sections were subjected to 2-stage dry ashing with ionometric determination of the fluorine ions. The results were treated according to the V.Yu. Urbach procedure. It was shown that the content of fluorine in the sponge bone and process zone in both the groups did not significantly differ. A comparatively high content of fluorine was detected only in the postmortem sections of the articular cartilage from the children of the main group who however had no chondrotoxic symptoms while alive. That means that the difference is statistically and clinically insignificant.     

Two types of store-operated Ca2+ channels with different activation modes and molecular origin in LNCaP human prostate cancer epithelial cells

The one or more coupling mechanisms of store-operated channels (SOCs) to endoplasmic reticulum (ER) Ca2+ store depletion as well as the molecular identity of SOCs per se still remain a mystery. Here, we demonstrate the co-existence of two populations of molecular distinct endogenous SOCs in LNCaP prostate cancer epithelial cells, which are preferentially activated by either active inositol 1,4,5-trisphosphate (IP3)-mediated or passive thapsigargin-facilitated store depletion and have different ER store content sensitivity. The first population, called SOC(CC) (for "conformational coupling"), is characterized by preferential IP3 receptor-dependent mode of activation, as judged from sensitivity to cytoskeleton modifications, and dominant contribution of transient receptor potential (TRP) TRPC1 within it. The second one, called SOC(CIF) (for "calcium influx factor"), depends on Ca(2+)-independent phospholipase A2 for activation with probable CIF involvement and is mostly represented by TRPC4. The previously identified SOC constituent in LNCaP cells, TRPV6, seems to play equal role in both SOC populations. These results provide new insight into the nature of SOCs and their representation in the single cell type as well as permit reconciliation of current SOC activation hypotheses.     

Potato glycoalkaloids: true safety or false sense of security?

As one of the major agricultural crops, the cultivated potato is consumed each day by millions of people from diverse cultural backgrounds. A product of global importance, the potato tuber contains toxic glycoalkaloids (GAs) that cause sporadic outbreaks of poisoning in humans, as well as many livestock deaths. This article will discuss some aspects of the potato GAs, including their toxic effects and risk factors, methods of detection of GAs and biotechnological aspects of potato breeding. An attempt has been made to answer a question of vital importance - are potato GAs dangerous to humans and animals and, if so, to what extent?     

Transgene-induced CCWGG methylation does not alter CG methylation patterning in human kidney cells

Several reports suggest that C^mCWGG methylation tends not to co-exist with ^mCG methylation in human cells. We have asked whether or not methylation at CCWGG sites can influence CG methylation. DNA from cells expressing an M.EcoRII–GFP fusion was actively methylated at CCWGG sites. CG methylation as measured by R.HpaII/R.MspI ratios was unchanged in cells expressing the transgene. Cloned representatives of C^mCWGG methylated DNA often contained, or were adjacent to an ALU repeat, suggesting that M.EcoRII-GFP actively methylated gene-rich R-band DNA. The transgenic methyltransferase applied C^mCWGG methylation to a representative human promoter that was heavily methylated at CG dinucleotides (the SERPINB5 promoter) and to a representative promoter that was essentially unmethylated at CG dinucleotides (the APC promoter). In each case, the CG methylation pattern remained in its original state, unchanged by the presence of neighboring C^mCWGG sites. Q-PCR measurements showed that RNA expression from the APC gene was not significantly altered by the presence of C^mCWGG in its promoter. Kinetic studies suggested that an adjacent C^mCWGG methylation site influences neither the maintenance nor the de novo methylation activities of purified human Dnmt1. We conclude that C^mCWGG methylation does not exert a significant effect on CG methylation in human kidney cells.     

Interdomain Stabilization Impairs CD4 Binding and Improves Immunogenicity of the HIV-1 Envelope Trimer

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Role of cell wall components in yeasts of the genus Candida in the morphology of the cell

The object of this work was to study the function of the mannan and glucan layers in the cell wall of Candida yeasts. When the outer mannan layer was removed with proteinases, the cells became round and their volume increased by a factor of 2.2. The contribution of the above layers into the elastic properties of the cell wall was estimated in qualitative terms. Apparently, mannans play a more important role in cell morphogenesis than it was supposed earlier.