Photon and fluorescence correlation spectroscopy and light scattering of eye-lens proteins at moderate concentrations

The bovine eye-lens protein, alpha L-crystallin, has been studied with photon correlation spectroscopy to obtain the mutual diffusion coefficient, Dm, with fluorescence correlation spectroscopy to determine the tracer diffusion coefficient, DT, and with light scattering to get the isothermal osmotic compressibility (delta pi/delta c) P,T. The concentration dependence of Dm, DT, and (delta pi/delta c) P,T up to a volume fraction phi of the protein of 2.5 x 10(-2) has been interpreted on the basis of four different interaction potentials: (a) an extended hard-sphere potential; (b) a shielded Coulomb potential; (c) a shielded Coulomb interaction where the effect of counterions is included; (d) a simple mixed potential. The three parameters Dm, DT, and (delta pi/delta c) P,T have also been combined in the generalized Stokes-Einstein equation, Dm = [(delta pi/delta c)P,T . (1--phi) . (DT)]/(kappa B . T). Our results indicate that, in the case that photon correlation spectroscopy gives the mutual diffusion coefficient Dm, the applicability of the Stokes-Einstein equation can be questioned; or that, when one assumes the Stokes-Einstein equation to be valid, there is significant discrepancy between the result of photon correlation spectroscopy and Dm.     

Comparative effects of trichothecene mycotoxins on bovine platelet function: Acetyl T-2 toxin,a more potent inhibitor than T-2 toxin

The effects of the trichothecene mycotoxins (acetyl T-2 toxin, T-2 toxin, HT-2 toxin, palmityl T-2 toxin, diacetoxyscirpenol (DAS), deoxynivalenol (DON), and T-2 tetraol) on bovine platelet function were examined in homologous plasma stimulated with platelet activating factor (PAF). The mycotoxins inhibited platelet function with the following order of potency: acetyl T-2 toxin > palmityl T-2 toxin = DAS > HT-2 toxin = T-2 toxin. While T-2 tetraol was completely ineffective as an inhibitor, DON exhibited minimal inhibitory activity at concentrations above 10×10^?4M. The stability of the platelet aggregates formed was significantly reduced in all mycotoxin treated platelets compared to that of the untreated PAF controls. It is suggested that the increased sensitivity of PAF stimulated bovine platelets to the more lipophilic mycotoxins may be related to their more efficient partitioning into the platelet membrane compared to the more hydrophilic compounds.     

An extensive review on antifungal approaches in the treatment of mucormycosis

During the period of COVID-19, the occurrences of mucormycosis in immunocompromised patients have increased significantly. Mucormycosis (black fungus) is a rare and rapidly progressing fungal infection associated with high mortality and morbidity in India as well as globally. The causative agents for this infection are collectively called mucoromycetes which are the members of the order Mucorales. The diagnosis of the infection needs to be performed as soon as the occurrence of clinical symptoms which differs with types of Mucorales infection. Imaging techniques magnetic resonance imaging or computed tomography scan, culture testing, and microscopy are the approaches for the diagnosis. After the diagnosis of the infection is confirmed, rapid action is needed for the treatment in the form of antifungal therapy or surgery depending upon the severity of the infection. Delaying in treatment declines the chances of survival. In antifungal therapy, there are two approaches first-line therapy (monotherapy) and combination therapy. Amphotericin B ( 1 ) and isavuconazole ( 2 ) are the drugs of choice for first-line therapy in the treatment of mucormycosis. Salvage therapy with posaconazole ( 3 ) and deferasirox ( 4 ) is another approach for patients who are not responsible for any other therapy. Adjunctive therapy is also used in the treatment of mucormycosis along with first-line therapy, which involves hyperbaric oxygen and cytokine therapy. There are some drugs like VT-1161 ( 5 ) and APX001A ( 6 ), Colistin, SCH 42427, and PC1244 that are under clinical trials. Despite all these approaches, none can be 100% successful in giving results. Therefore, new medications with favorable or little side effects are required for the treatment of mucormycosis.     

Quasi-elastic light scattering of Artemia ribosomes sedimented in a CsCl density gradient

It is impossible to measure the diffusion coefficient of macromolecules directly and accurately by quasi—elastic light scattering, when aggregates cannot be eliminated from the solutions to be investigated. Nevertheless, a simple method can be applied to overcome this problem in many cases. Aggregates are separated from the monomeric macromolecules by rate-zonal sedimentation in a CsCl density gradient in a transparent centrifugation tube; the monomers are then located by laser light scattering intensity measurements; photon correlation spectroscopy of the scattered light finally yields their diffusion coefficient. The viscosity of aqueous CsCl solutions at different temperatures and concentrations allows a good separation by centrifugation and a low uncertainty in the reduction of the measured diffusion coefficient to standard conditions.The application of the method to eukaryotic large ribosomal subunits is described as an example.     

The molecular weight of Artemia ribosomes, as determined from their refractive-index increment and light-scattering intensity

Cytoplasmic ribosomes were isolated from the cryptobiotic embryos of the brine shrimp Artemia salina. Measurements of their refractive-index increments and light-scattering intensities give a value for their molecular weight of (3.4±0.2)×10⁶.     

Monoclonal antibodies to rabbit liver cytochrome P-448

Cytochrome P-448 (mol wt 55,000 Daltons) from rabbit liver was purified to a specific content of 16.6 nmol/mg. Mice were immunised with this preparation, their spleens removed and dissociated lymphocytes hybridised with myeloma cells. Four monoclonal antibodies against cytochrome P-448 were raised and partially characterised. All four antibodies interacted with cytochrome P-448 in intact microsomal fractions and selectively immunoadsorbed cytochrome P-448 from solubilised microsomal preparations. One of the antibodies inhibited benzo[a] pyrene hydroxylase activity in a reconstituted system, one had no effect on activity and two increased activity. The possible applications of such antibodies are discussed.     

Saikosaponin-d,a novel SERCA inhibitor,induces autophagic cell death in apoptosis-defective cells

Autophagy is an important cellular process that controls cells in a normal homeostatic state by recycling nutrients to maintain cellular energy levels for cell survival via the turnover of proteins and damaged organelles. However, persistent activation of autophagy can lead to excessive depletion of cellular organelles and essential proteins, leading to caspase-independent autophagic cell death. As such, inducing cell death through this autophagic mechanism could be an alternative approach to the treatment of cancers. Recently, we have identified a novel autophagic inducer, saikosaponin-d (Ssd), from a medicinal plant that induces autophagy in various types of cancer cells through the formation of autophagosomes as measured by GFP-LC3 puncta formation. By computational virtual docking analysis, biochemical assays and advanced live-cell imaging techniques, Ssd was shown to increase cytosolic calcium level via direct inhibition of sarcoplasmic/endoplasmic reticulum Ca²⁺ ATPase pump, leading to autophagy induction through the activation of the Ca²⁺/calmodulin-dependent kinase kinase–AMP-activated protein kinase–mammalian target of rapamycin pathway. In addition, Ssd treatment causes the disruption of calcium homeostasis, which induces endoplasmic reticulum stress as well as the unfolded protein responses pathway. Ssd also proved to be a potent cytotoxic agent in apoptosis-defective or apoptosis-resistant mouse embryonic fibroblast cells, which either lack caspases 3, 7 or 8 or had the Bax-Bak double knockout. These results provide a detailed understanding of the mechanism of action of Ssd, as a novel autophagic inducer, which has the potential of being developed into an anti-cancer agent for targeting apoptosis-resistant cancer cells.     

Litre-scale microbial fuel cells operated in a complete loop

Using the anode effluent to compensate the alkalinization in a bio-cathode has recently been proposed as a way to operate a microbial fuel cell (MFC) in a continuous and pH neutral way. In this research, we successfully demonstrated that the operation of a MFC without any pH adjustments is possible by completing the liquid loop over cathode and anode. During the complete loop operation, a stable current production of 23.2 ± 2.5 A m⁻³ MFC was obtained, even in the presence of 3.2–5.2 mg O₂ L⁻¹ in the anode. The use of current collectors and subdivided electrical circuitries for relative large 2.5-L-scale MFCs resulted in ohmic cell resistances in the order of 1.4–1.7 mΩ m³ MFC, which were comparable to values of ten times smaller MFCs. Nevertheless, the bio-cathode activity still needs to be improved significantly with a factor 10–50 in order achieve desirable current densities of 1,000 A m⁻³ MFC. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Interaction of detergents with bovine lens α-crystallin: evidence for an oligomeric structure based on amphiphilic interactions

We have studied the quaternary structure of α-crystallin in the presence of increasing concentrations of amphiphilic and neutral detergents using gel filtration, light-scattering, boundary and equilibrium sedimentation. We observed a continuous reduction of the molar mass of the polymeric α-crystallin on increasing the concentration of sodium dodecyl sulphate from 0.1 mM to 5 mM, ending up with the monomeric peptides. Dodecyltrimethylammonium bromide also disrupts the oligomeric structure of α-crystallin but the interaction appears to be cooperative: in the sharp transition region (for a 1 mg/ml protein solution) from 3 to 8 mM of the detergent, only the native protein and a mixture of monomeric and dimeric peptide-DTAB complexes can be observed. Concomitant studies of the circular dichroism in the far UV revealed a substantial decrease of the β-sheet and increase of the α-helix secondary structure. The latter can be related to the presence of amphiphilic polypeptide sequences in the constituent αA and αB peptides. These studies reveal for the first time a direct relation between changes in the secondary structure of the αA and αB peptides and the formation of the oligomeric α-crystallin structure: the binding of the amphiphilic detergent reduces the β-sheet content, induces the formation of α-helix secondary structure and reduces the tendency of the peptide to form large aggregates. The different mechanisms for reducing the oligomeric size by anionic and cationic detergents with identical apolar parts stresses the importance of charge interactions. Our findings support some aspects of the micelle model of α-crystallin and can be related to its chaperone activity. Accepted: 18 October 1996