Dimerization of the p53 oligomerization domain: identification of a folding nucleus by molecular dynamics simulations

Dimerization of the p53 oligomerization domain involves coupled folding and binding of monomers. To examine the dimerization, we have performed molecular dynamics (MD) simulations of dimer folding from the rate-limiting transition state ensemble (TSE). Among 799 putative transition state structures that were selected from a large ensemble of high-temperature unfolding trajectories, 129 were identified as members of the TSE via calculation of a 50% transmission coefficient from at least 20 room-temperature simulations. This study is the first to examine the refolding of a protein dimer using MD simulations in explicit water, revealing a folding nucleus for dimerization. Our atomistic simulations are consistent with experiment and offer insight that was previously unobtainable.     

Metalloids in plants: A systematic discussion beyond description

Metalloids represent a wide range of elements with intermediate physiochemical properties between metals and non-metals. Many of the metalloids, like boron, selenium, and silicon are known to be essential or quasi-essential for plant growth. In contrast, metalloids viz. arsenic and germanium are toxic to plant growth. The toxicity of metalloids largely depends on their concentration within the living cells. Some elements, at low concentration, may be beneficial for plant growth and development; however, when present at high concentration, they often exert negative effects. In this regard, understanding the molecular mechanisms involved in the uptake of metalloids by roots, their subsequent transport to different tissues and inter/intra-cellular redistribution has great importance. The mechanisms of metalloids' uptake have been well studied in plants. Also, various transporters, as well as membrane channels involved in these processes, have been identified. In this review, we have discussed in detail the aspects concerning the positive/negative effects of different metalloids on plants. We have also provided a thorough account of the uptake, transport, and accumulation, along with the molecular mechanisms underlying the response of plants to these metalloids. Additionally, we have brought up the previous theories and debates about the role and effects of metalloids in plants with insightful discussions based on the current knowledge.     

A New Extracellular β-Galactosidase Producing Kluyveromyces sp. PCH397 from Yak Milk and Its Applications for Lactose Hydrolysis and Prebiotics Synthesis

β-Galactosidase is a crucial glycoside hydrolase enzyme with potential applications in the dairy, food, and pharmaceutical industries. The enzyme is produced in the intracellular environment by bacteria and yeast. The present study reports yeast Kluyveromyces sp. PCH397 isolated from yak milk, which has displayed extracellular β-galactosidase activity in cell-free supernatant through the growth phase. To investigate further, cell counting and methylene blue staining of culture collected at different growth stages were performed and suggested for possible autolysis or cell lysis, thereby releasing enzymes into the extracellular medium. The maximum enzyme production (9.94 ± 2.53U/ml) was achieved at 37 °C in a modified deMan, Rogosa, and Sharpe (MRS) medium supplemented with lactose (1.5%) as a carbon source. The enzyme showed activity at a wide temperature range (4–50 °C), maximum at 50 °C in neutral pH (7.0). In addition to the hydrolysis of lactose (5.0%), crude β-galactosidase also synthesized vital prebiotics (i.e., lactulose and galacto-oligosaccharides (GOS)). Additionally, β-fructofuranosidase (FFase) activity in the culture supernatant ensued the synthesis of a significant prebiotic, fructo-oligosaccharides (FOS). Hence, the unique features such as extracellular enzymes production, efficient lactose hydrolysis, and broad temperature functionality by yeast isolate PCH397 are of industrial relevance. In conclusion, the present study unrevealed for the first time, extracellular production of β-galactosidase from a new yeast source and its applications in milk lactose hydrolysis and synthesis of valuable prebiotics of industrial importance.Supplementary InformationThe online version contains supplementary material available at 10.1007/s12088-021-00955-1.Keyword: β-Galactosidase, Lactulose, Galacto-oligosaccharides, Fructo-oligosaccharides, Milk-microbes

β-Galactosidase (EC 3.2.1.23) hydrolyzes the glycosidic bond in β-galactosides and finds applications in the food industry [1, 2]. The trans-glycosylation property of β-galactosidase (β-gal) is widely used to produce various galactosylated products and prebiotics such as GOS and lactulose [3–7]. The β-gal enzyme is produced intracellularly by many bacteria and yeast, a major constraint for industrial production [1, 8]. Therefore, extracellular β-gal producing bacteria/yeast are of huge relevance. Hence, the present work revealed an efficient extracellular β-gal producing microbe from dairy products of the Indian Himalaya and evaluated its applications in lactose hydrolysis and prebiotics’ synthesis.In this study, twenty milk and four curd samples were collected from the Lahaul and Pangi valleys of Himachal Pradesh, India. The samples were plated on MRS and Elliker agar medium (Himedia, India) for 2–7 days at 28 °C and 37 °C until visible microbial growth. Morphologically distinct isolates were screened for β-gal activity using X-Gal and IPTG plate assay [6, 9]. The positive isolates were screened for β-gal production in liquid MRS medium. The β-gal activity was expressed as U/mg dcw (dry cell weight) for whole cells and U/ml for cell-free supernatant [10, 11]. Yeast isolate PCH397 showing the highest and extracellular enzymatic activity was selected. The culture and reaction conditions for maximum β-gal activity were optimized. FFase activity of whole cells and cell-free supernatant was estimated as described by Lincoln and More [12].The cell-free supernatant (β-gal) was employed for applications in lactose hydrolysis and prebiotic synthesis. The enzyme was incubated with lactose solution (5%, w/v) at 37 °C for lactose hydrolysis followed by thin layer chromatography (TLC) [13] analysis and quantification using the ImageJ program (http://rsbweb.nih.gov/ij/). Further, the cell-free supernatant was incubated with milk at 4 °C for milk lactose hydrolysis. Samples were withdrawn at different time intervals and analyzed for residual lactose concentration using ultra-high performance liquid chromatography-quadrupole-time of flight-ion mobility mass spectrometry (UHPLC-Q-TOF-IMS) [14]. Prebiotic production was carried out by mixing an equal volume of the enzyme with a sugar solution i.e., lactose (40%, w/v) for GOS, and lactose (20%, w/v) + fructose (20%, w/v) for lactulose and FOS production, respectively at 50 °C for 24 h [6]. Samples were analyzed by TLC for GOS, UHPLC-Q-TOF-IMS for FOS and lactulose synthesis.The study resulted in the isolation of 203 morphologically distinct microbes, 62 of which were tested positive for β-gal. Based on quantitative screening, eight isolates showing maximum β-gal activity were selected and examined for the intracellular and extracellular enzymatic activities (Table S1). Yeast isolate PCH397 exhibited maximum extracellular β-gal activity (9.94 ± 2.53 U/ml) along with FFase activity (0.59 ± 0.155) after 48 h of incubation. Isolate PCH397 was identified as Kluyveromyces marxianus by its morphological and molecular characterization (Fig. S1). Phylogenetic tree based on ITS DNA sequence showed similarity (99.63%) with Kluyveromyces marxianus CBS712. To the best of our knowledge, the genus Kluyveromyces has not been reported earlier for extracellular β-gal production. In the past, efforts were made to produce β-gal extracellularly through permeabilization or incorporation of signal peptide to β-gal gene in a fusion construct [15, 16]. The isolate PCH397 was selected due to its generally regarded as safe (GRAS) status and the novel feature of extracellular enzyme production.Highest β-gal activity in the extracellular environment was observed when PCH397 was grown in MRS medium supplemented with 1.5% (w/v) lactose as a substrate and incubated at 37 °C for 48 h (Fig. S2). PCH397 produced extracellular β-gal at lower lactose concentration (1.5%) as compared to various Kluyveromyces spp. [15] where 3% lactose has been used in the growth medium for intracellular β-gal production. Further, whether the extracellular enzyme activity is due to the secretion or cell lysis, the CFU count and cell viability were checked by the methylene blue test. The decreased cell count in the late stationary phase for live cells (Fig. S3) and increased number of methylene blue stained cells indicated cell death (Fig S4). These results suggested that cell lysis in the late stationary phase leads to the secretion of enzymes in extracellular medium. The extracellular production of enzyme would lead to a lower production costs of the enzyme.Cell-free supernatant showed the highest β-gal activity at pH 7.0 in 10 mM sodium phosphate buffer at 50 °C in 5 min (Fig S2). The β-gal enzyme from the current finding holds promise in the sweet whey and milk lactose hydrolysis [1] due to its neutral pH optima. Also, β-gal, which is functional at high temperatures, is used in the synthesis of oligosaccharides [1, 3]. High temperature increases the reaction rate as well as lactose solubility, thus, facilitating transgalactosylation reactions [17]. The β-gal activity (9 U/ml) in cell-free supernatant of PCH397 completely hydrolyzed 5.0% of lactose within 8 h at 37 °C (Fig. 1a, S5a). In a recent study, 5.0% lactose was also hydrolyzed by purified β-gal (5 U/ml) of Paenibacillus barengoltzii CAU904 within 8 h at 40 °C [13]. Under refrigerated conditions (4 °C), the cell free supernatant hydrolyzed ~ 50% milk lactose within 36 h and ~ 80% in 72 h (Fig. 1b, S5b). Since β-gal of PCH397 is active at 4 °C, the enzyme could be utilized to hydrolyze lactose in dairy products under refrigerated conditions. Lactose-free milk products or low-lactose milk products are important dietary constituents for lactose intolerant individuals and deliver essential nutrients to combat nutritional deficiencies [18]. Even with commercially purified enzymes, 100% milk-lactose hydrolysis could not be achieved at a low temperature [19]. However, the crude enzyme from the present investigation can efficiently hydrolyze milk lactose at ambient and refrigerated conditions, reducing the cost associated with enzyme purification. Additionally, the source of enzyme is Kluyveromyces sp. which has GRAS status, therefore, can be used in food applications.Open in a separate windowFig. 1Lactose hydrolysis by crude β-gal of PCH397. a Relative quantification of the hydrolysed products from lactose (5%, w/v) at 37 °C for 24 h. b Relative decrease in lactose concentration (%) at refrigerated conditions obtained by UHPLC-QTOF-IMSFurther, the enzyme was evaluated for its ability to catalyze transgalactosylation reactions at 50 °C. The crude enzyme was incubated with different substrate mixture viz. lactose and fructose. After 8 h of incubation, 50% of lactose was hydrolyzed into glucose, galactose, and GOS (Fig. S6a). Maximum GOS production was achieved after 12 h (Fig. 2a). The purified β-gal from Paenibacillus barengoltzii synthesized GOS from 350 g/L of lactose within 4 h [13]. Though GOS synthesis was faster in comparison to the current study, it is to be noted that we used a crude enzyme mixture instead of a purified enzyme. The crude enzyme has also shown FFase activity (Table S1), and was used for the synthesis of FOS from lactose and fructose mixture. UHPLC-Q-TOF-IMS analysis confirmed the formation of FOS (Fig. 2b). Multiple peaks were observed in the sample containing lactulose, one of which was identical with the peak of lactulose standard (Fig. 2c) as confirmed by HPAEC-PAD (Fig. S6b). The lactulose formation was maximum at 20 h of incubation (Fig. S6c).Open in a separate windowFig. 2Hydrolysis and transgalactosylation of lactose by crude enzyme from PCH397 having β-gal and FFase activity. a Relative quantification of the hydrolyzed and transgalactosylated products. UHPLC-QTOF-IMS detection of prebiotics b FOS and c lactulose with their respective standardIt is the first report of simultaneous co-synthesis of multiple prebiotics i.e., GOS, FOS, and lactulose using a yeast strain. Similar reports for GOS and FOS synthesis have been attempted by enzymatic means from fungal sources in the past [6]. The synthesis of multiple prebiotics is very advantageous. Numerous studies have shown that blended consumption of multiple prebiotics including GOS and FOS has many health benefits [20–24]. The combination of GOS, FOS, and lactulose can be of considerable importance for their prebiotic applications. In conclusion, our findings revealed a yeast source for the cost-effective production of β-galactosidase and a strategy for co-synthesis of valuable prebiotics, which is not reported in the past. The utilization of a yeast source with GRAS status for lactose hydrolysis and co-synthesis of prebiotics promises various health benefits and commercial relevance.     

Lipid fluidity and composition of the erythrocyte membrane from healthy dogs and labrador retrievers with hereditary muscular dystrophy

Erythrocyte membranes and their liposomes were prepared from clinically normal dogs and Labrador retrievers with hereditary muscular dystrophy. The static and dynamic components of fluidity of each membrane were then assessed by steady-state fluorescence polarization techniques using limiting hindered fluorescence anisotropy and order parameter values of 1,6-diphenyl-1,3,5-hexatriene (DPH) and fluorescence anisotropy values ofdl-2-(9-anthroyl)-stearic acid anddl-12-(9-anthroyl)-stearic acid, respectively. Membrane lipids were extracted and analyzed by thin-layer chromatography and gas chromatography. The results of these studies demonstrated that the lipid fluidity of erythrocyte membranes, and their liposomes, prepared from dystrophic dogs were found to possess significantly lower static and dynamic components of fluidity than control counterparts. Analysis of the composition of membranes from dystrophic dogs revealed a higher ratio of saturated fatty acyl chain/unsaturated chains (w/w) and lower double-bond index. Alterations in the fatty acid composition such as decrease in levels of linoleic (18:2) and arachidonic (20:4) acids and increase in palmitic (16:0) and stearic (18:0) acids were also observed in the membranes of dystrophic animals. These associated fatty acyl alterations could explain, at least in part, the differences in membrane fluidity between dystrophic and control dogs.     

Spontaneous demonstration of counterclockwise right atrial activation following successful isthmus ablation

We describe an uncommon case of typical flutter with symptomatic sinus node dysfunction, in which a permanent junctional rhythm developed following ablation of the cavo-tricuspid isthmus. This rhythm activated the right atrium in counter clockwise manner thus providing spontaneous proof of unidirectional isthmus block, a phenomenon that is usually demonstrated by proximal coronary sinus pacing.     

Modification of chromium (VI) phytotoxicity by exogenous gibberellic acid application in <Emphasis Type="Italic">Pisum sativum</Emphasis> (L.) seedlings

Effects of exogenous gibberellic acid (GA; 10 and 100 μM) application on growth, protein and nitrogen contents, ammonium (NH₄ ⁺) content, enzymes of nitrogen assimilation and antioxidant system in pea seedlings were investigated under chromium (VI) phytotoxicity (Cr VI; 50, 100 and 250 μM). Exposure of pea seedlings to Cr and 100 μM GA resulted in decreased seed germination, fresh and dry weight and length of root and shoot, and protein and nitrogen contents compared to control. Compared to control, Cr and 100 μM GA led to the significant alteration in nitrogen assimilation in pea. These treatments decreased root and shoot nitrate reductase (NR), glutamine synthetase (GS) and glutamine 2-oxoglutarate aminotransferase (GOGAT) activities (except 50 μM Cr alone for GOGAT) while glutamate dehydrogenase (GDH) activity and NH₄ ⁺ content increased. Compared to control, the root and shoot activities of superoxide dismutase (SOD) and ascorbate peroxidase (APX) increased (except APX activity at 250 μM Cr + 100 μM GA) while catalase (CAT), glutathione reductase (GR) and dehydroascorbate reductase (DHAR) activities were decreased (except GR at 100 μM GA alone) following exposure of Cr and 100 μM GA. Total ascorbate and total glutathione in root and shoot decreased by the treatments of Cr and 100 μM GA while their levels were increased by the application of 10 μM GA compared to Cr treatments alone. It has been reported that application of 10 μM GA together with Cr alleviated inhibited levels of growth, nitrogen assimilation and antioxidant system compared to Cr treatments alone. This study showed that application of 10 μM GA counteracts some of the adverse effects of Cr phytotoxicity with the increased levels of antioxidants and sustained activities of enzymes of nitrogen assimilation; however, 100 μM GA showed apparently reverse effect under Cr phytotoxicity.     

Endophytic Fungal Flora from Roots and Fruits of an Indian Neem Plant <Emphasis Type="Italic">Azadirachta indica</Emphasis> A. Juss., and Impact of Culture Media on their Isolation

Azadirachta indica A. Juss. (neem), native to India, is well known worldwide for its insecticidal and ethanopharmacological properties. Although endophytic microbes are known from this plant as only leaves and stems were the subjects of past reports. Now, a variety of procedures and a number of different media were used to isolate the maximum number of endophytic fungi from unripe fruits and roots. A total of 272 isolates of 29 filamentous fungal taxa were isolated at rate of 68.0% from 400 samples of three different individual trees (at locations-Az1, Az2, Az3). Mycological agar (MCA) medium yielded the highest number of isolates (95, with a 14.50% isolation rate) with the greatest species richness. Mycelia Sterilia (1, 2, 3) accounted for 11.06%, Coelomycetes 7.25%, while Hyphomycetes showed the maximum number of representative isolates (81.69%). Mycelia-Sterilia (1, 2, 3), based on their 5.8S ITS 1, ITS2 and partial 18S and 28S rDNA sequences were identified as Fusarium solani (99%), Chaetomium globosum (93%) and Chaetomium globosum (93%) respectively. Humicola, Drechslera, Colletotrichum, and Scytalidium sp. were some of the peculiar fungal endophytes recovered from this plant.     

Structure of AscE and induced burial regions in AscE and AscG upon formation of the chaperone needle-subunit complex of type III secretion system in Aeromonas hydrophila

In the type III secretion system (T3SS) of Aeromonas hydrophila, the putative needle complex subunit AscF requires both putative chaperones AscE and AscG for formation of a ternary complex to avoid premature assembly. Here we report the crystal structure of AscE at 2.7 A resolution and the mapping of buried regions of AscE, AscG, and AscF in the AscEG and AscEFG complexes using limited protease digestion. The dimeric AscE is comprised of two helix-turn-helix monomers packed in an antiparallel fashion. The N-terminal 13 residues of AscE are buried only upon binding with AscG, but this region is found to be nonessential for the interaction. AscE functions as a monomer and can be coexpressed with AscG or with both AscG and AscF to form soluble complexes. The AscE binding region of AscG in the AscEG complex is identified to be within the N-terminal 61 residues of AscG. The exposed C-terminal substrate-binding region of AscG in the AscEG complex is induced to be buried only upon binding to AscF. However, the N-terminal 52 residues of AscF remain exposed even in the ternary AscEFG complex. On the other hand, the 35-residue C-terminal region of AscF in the complex is resistant to protease digestion in the AscEFG complex. Site-directed mutagenesis showed that two C-terminal hydrophobic residues, Ile83 and Leu84, of AscF are essential for chaperone binding.