Biology subjects

Pandey, H.

Publications and source records attributed to Pandey, H..

11 recordsLinked to original sources

bioRxiv2025

Dimeric Cin8 motors have an inherent plus-end bias and weak inter-head coordination

Kinesin-5 motors are bipolar tetramers that crosslink and slide antiparallel microtubules during mitotic spindle assembly. Fungal kinesin-5 motors, such as Cin8, exhibit bidirectional motility, switching between minus- and plus-end-directed stepping in response to environmental conditions; however, the molecular basis of this directional switching remains unclear. To better understand the origin of this bidirectional behavior, we investigated the motility and ATPase kinetics of two Cin8 dimers, created by fusing the motor domains to a stable coiled-coil domain from kinesin-1. To investigate the role of the proximal neck coiled-coil region in coordinating motor activity, we compared Cin8 dimers that included or lacked the first four heptads of the Cin8 neck-coil domain. By analyzing the stepping kinetics, microtubule residence times, and directional switching dynamics, we found that these Cin8 dimers move processively with a net plus-end directionality along with undirected movements, behaviors that mimic the plus-ended motility state of wild-type Cin8. However, fast minus-ended motility seen in wild-type Cin8 tetramers was not observed in the dimers. The instantaneous velocity distributions and ATPase rates were inconsistent with the undirected movement being solely due to passive diffusion, suggesting that they reflect random bidirectional stepping. Fewer undirected movements were seen on yeast microtubules, their native physiological substrate, compared to on bovine microtubules. Replacing the Cin8 neck-coil domain with a stable coiled-coil led to faster plus-end stepping, fewer undirected movements, a reduction in the microtubule binding duration, and enhanced coupling between ATP hydrolysis and plus-end stepping. Our results suggest that the native Cin8 neck coil confers flexibility between the two motor domains that contributes to bidirectional stepping, and that sustained minus-end movement requires regions outside the motor domain. Statement of SignificanceThe kinesin-5 family of motors, which contain two pairs of heads located on either end of a long stalk domain, power mitotic spindle formation by sliding antiparallel microtubules. The yeast kinesin-5, Cin8 moves to microtubule minus-ends under specific conditions, breaking the dogma that N-terminal kinesins move to microtubule plus-ends. To gain insight into how Cin8 changes its walking direction, we analyzed engineered Cin8 dimers, models of one half of full-length Cin8. The dimers step erratically, consistent with stepping in both directions, but have a plus-end bias. We find evidence that the two heads poorly coordinate, which contrasts with other kinesins, and suggest that the stepping direction may be regulated by altering the degree of inter-head coordination.

biophysics↗

bioRxiv2025

The acetylation of the histone-like protein HBsu at specific sites alters gene expression during sporulation in Bacillus subtilis

Sporulation is an adaptive response to starvation in bacteria that consists of a series of developmental changes in cellular morphology and physiology, leading to the formation of a highly resistant endospore. In Bacillus subtilis, there is an intricate developmental program which involves the precise coordination of gene expression and ongoing morphological changes to yield the mature spore. The histone-like protein HBsu is involved in proper spore packaging and compaction of the chromosomal DNA. Previously, we found that the acetylation of different lysine residues on HBsu impairs sporulation frequency and spore resistance properties. One mechanism by which HBsu influences the process of sporulation could be through the regulation of gene expression. To test this idea, we performed RT-qPCR to analyze gene expression throughout the sporulation process in wildtype and seven acetylation-mimicking (glutamine substitutions) mutant strains. Acetylation of HBsu at K41 increased the expression of key early and late sporulation genes, especially during the later stages. For example, overexpression of {sigma}F and {sigma}G drive expression of their regulon members at inappropriate times. These findings suggest that K41 acetylation activates gene expression and might represent an "on-off" switch for important regulatory factors as cells transition from early to late phases. The gene expression profiles of hbsK3Q, hbsK37Q, hbsK75Q, hbsK80Q, and hbsK86Q mutants were largely unchanged, but did have significant reductions of key late sporulation proteins, which could explain the observed defects in spore resistance properties. We propose that acetylation of HBsu at specific sites directly regulates gene expression during sporulation and this is required for proper timing and coordination.

microbiology↗

bioRxiv2024

Identifying the genetic and epigenetic basis for asymmetric bZIP expression in temperature-stressed bread wheat

Asymmetric expression in the bread wheat (Triticum aestivum) genome refers to the differential expression of genes from A, B, and D parental genomes. Bread wheat is a hexaploid crop with six copies of each chromosome. This complexity can result in unequal expression of genes from each parental genome, leading to asymmetry in gene expression. In other polyploid crops like cotton, transcription factors (TF) exhibit genome-biased expression; however, there are no comparable studies for bread wheat. One of the most prominent TFs families in plants is the basic Leucine Zippers (bZIP) which are eukaryote-specific proteins and regulate various biological processes, including stress-related responses. bZIP proteins are dimeric and several heptads long. They exhibit typical coiled-coil structures with strategically placed amino acids in each heptad, responsible for their stability and specificity. Here, we aim to decipher the structural basis of the asymmetric expression of the bZIP TFs in wheat under low and high-temperature conditions. Furthermore, 19 highly expressed stress-related TabZIP TFs were analysed for their asymmetric expression profiles as plants were exposed to temperature-stress conditions. Two benchmarks were used to analyse the asymmetric gene expression of bZIPs, i.e., a) the promoters occupancy by the epigenetic marker histones, namely, H3K4me3, H3k9ac (active) and H3K27me3 (repressive), b) density and diversity of cis-regulatory elements in the promoters. Notably, the genetic basis of the differences in protein sequences of bZIP triads was explored, which may impart structural stability to a specific homeolog enabling the plant to endure the stress conditions better.

plant biology↗

bioRxiv2021

The mechanochemical origins of the microtubule sliding motility within the kinesin-5 domain organization

The conserved kinesin-5 bipolar tetrameric motors slide apart microtubules during mitotic spindle assembly and elongation. Kinesin-5 bipolar organization originates from its conserved tetrameric helical minifilament, which position the C-terminal tail domains of two subunits near the N-terminal motor domains of two anti-parallel subunits (Scholey et al, 2014). This unique tetrameric structure enables kinesin-5 to simultaneously engage two microtubules and transmit forces between them, and for multiple kinesin-5 motors to organize via tail to motor interactions during microtubule sliding (Bodrug et al, 2020). Here, we show how these two structural adaptations, the kinesin-5 tail-motor domain interactions and the length of the tetrameric minifilament, determine critical aspects of kinesin-5 motility and sliding mechanisms. An x-ray structure of the 34-nm kinesin-5 minifilament reveals how the dual dimeric N-terminal coiled-coils emerge from the tetrameric central bundle. Using this structure, we generated active bipolar mini-tetrameric motors from Drosophila and human orthologs, which are half the length of native kinesin-5. Using single-molecule motility assays, we show that kinesin-5 tail domains promote mini-tetramers static pauses that punctuate processive motility. During such pauses, kinesin-5 mini-tetramers form multi-motor clusters mediated via tail to motor domain cross-interactions. These clusters undergo slow and highly processive motility and accumulate at microtubule plus-ends. In contrast to native kinesin-5, mini-tetramers require tail domains to initiate microtubule crosslinking. Although mini-tetramers are highly strained in initially aligning microtubules, they slide microtubules more efficiently than native kinesin-5, due to their decreased minifilament flexibility. Our studies reveal that the conserved kinesin-5 motor-tail mediated clustering and the length of the tetrameric minifilament are key features for sliding motility and are critical in organizing microtubules during mitotic spindle assembly and elongation.

cell biology↗

bioRxiv2020

Transient delivery of A-C/EBP protein perturbs differentiation of 3T3-L1 cells and induces preadipocyte marker genes

Transformation of committed 3T3-L1 preadipocytes to lipid-laden adipocytes involves timely appearance of numerous transcription factors (TFs), foremost among them C/EBP{beta}, is expressed during early phases of differentiation. Here we describe liposome-mediated protein transfection approach to rapidly downregulate C/EBP{beta} by A-C/EBP protein inhibitor. Signals from tagged A-C/EBP were observed in 3T3-L1 cells within 2hrs of protein inhibitor transfections whereas for gene transfection signals appeared in 48hrs. Following transient transfections, expression profiles of 24 marker genes belonging to pro- and anti-adipogenic, cell cycle, and preadipocytes pathways was analyzed. mRNA and protein expression profiles of adipocyte-marker genes showed lower expression in both A-C/EBP protein and gene transfected samples. Interestingly, for preadipocytes and cell fate determinant genes, striking differences were observed between protein and gene transfected samples. Preadipocyte differentiation factors Stat5a and Creb were downregulated in A-C/EBP protein samples. Five preadipocyte markers, namely, Pdgfr, Pdgfr{beta}, Ly6A, CD34 Itgb1 showed high expression in protein samples whereas only Ly6A and CD34 were expressed in gene transfected samples. Pdgfr and Pdgfr{beta}, two known cell fate markers were expressed in protein transfected samples 5-days post-differentiation suggesting a possible reversal of differentiation. Our study provides evidences for the robust and efficient knockdown of C/EBP{beta} protein to understand time-dependent gene regulation during adipogenesis.

molecular biology↗

bioRxiv2020

Flexibility of the neck-linker during docking is pivotal for function of bi directional kinesin

The role of the neck-linker (NL) element in regulating the functions of bi-directional kinesins is unknown. We report that replacing the NL of the bi-directional kinesin-5 Cin8 with sequences from plus-end directed kinesins produces non-functional Cin8 with defective spindle localization and abolished minus-end directionality and microtubule-crosslinking in vitro. Mutation of a single glycine in the NL of Cin8 to asparagine (proposed to serve as an N-latch that stabilizes the docked conformation of the NL in the plus-end directed kinesins) causes defects in the functions of Cin8. Strikingly, in a non-functional Cin8 containing the NL of the plus-end directed kinesin-5 Eg5, a single mutation of the N-latch asparagine back to glycine rescues the in vivo and in vitro defects. Since such replacement eliminates stabilizing interactions between the docked NL and the motor domain, we conclude that flexibility of NL during docking is pivotal for the function of bi-directional kinesin motors.

biophysics↗

bioRxiv2019

Nutritive diet becomes unhealthy if taken in a wrong ratio: Scientific validation of Ayurvedic concept of \"incompatible diet\" (Virrudh Aahar)

Honey and ghee is an important constituent of our diet. Honey contain about 200 components in its which play an important role as antioxidative, anti-inflammatory, antimicrobial, wound healing and sunburn, etc. Ghee has also many essential constituents such as phospholipids, PUFA, fat- soluble vitamins, etc. It is mention in Charak Samhita that equal ratio of honey and ghee when taken in food become toxic to health. This study was designed to explore the mechanism of toxicity through the biochemical and histological parameter. For this study, 24 Charles foster rats were taken and divided into four groups (n=6) normal, honey, ghee, and honey+ghee (1:1). After 60 days rats were sacrificed. Weight loss, Hair loss and red patches on ear were found on honey+ghee fed rats. LFT, amadori test, AGE, UV, glucose, DPP-4 and LPO were found significantly increased in honey+ghee group. SOD, Catalase, GSH, ABTS+, GLP-1, GIP-1, ACB and HB were found significantly decreased in honey+ghee group. No changes were found in RFT. H&E show normal morphology in kidney, intestine, and pancreas but mild inflammation was found in liver tissue. We conclude that the equal mixture of honey and ghee shows toxic effect. Increased formation of hydroxymethyl furfural, amadori product, DPP-4 activity and low incretins (GLP-1, GIP-1) activity resulting high postprandial hyperglycemic response are collectively responsible for oxidative stress-mediated toxicity of the mixture of honey and ghee in equal ratio.

pharmacology and toxicology↗

bioRxiv2019

Histopathological study of different organs of charles foster strain rat under the exposure of Pueraria tuberosa.

The present study was undertaken to investigate the safe doses of Pueraria tuberosa water extract (PTWE) on different organs. The OECD guidelines 407 of repeated toxicity was followed with respect to the selection of dose and days for different organs. The selected doses of PTWE were 250, 500, 1000 and 2000 mg/kg b wt for 7, 14, 21 and 28 days. Haematoxylin and eosin staining was used to study the morphological alterations in heart, intestine, testis, adrenal gland and spleen. In the present study, no adverse alterations in cardiac fibers of the heart, size and shapes in crypts and villi of intestine, seminiferous tubules and spermatozoa count in testis, three zones of adrenal gland, and spleen were seen in all treated groups of PTWE. There were no adverse morphological alterations found in described organs. The PTWE are safe at 1000 mg/kg b wt. up to 28 days and 2000 mg/ kg b. wt up to 21 days, respectively.

pharmacology and toxicology↗

bioRxiv2019

Herbal tablet of Pueraria tuberosa water extract suppresses the alloxan induced liver damage and hyperglycemia in rats

AimTo study the protective response of herbal formulation (tablets) of Pueraria tuberosa water extract (PTAB) on alloxan induced rat diabetic model.\n\nMethodologyAlloxan (120 mg/kg bw) was injected intraperitonially. Rats were divided into three groups: group 1 as normal, group 2 as diabetic control and group 3 were given PTAB upto 14 days. Blood glucose and liver function tests were done using their respective kits. Hematoxylene and eosin staining was done to evaluate the morphological changes in liver tissues. Through immunohistochemistry, we have checked the protein expression of VEGF, MMP9 and ki67.\n\nResultPTAB significantly decreases blood glucose level in a time dependent manner up to 14 days. As compared to diabetic control, PTAB decreases SGOT, SGPT and alkaline phosphates after 14 days of treatment. In diabetic control, the morphology of liver tissues were found damaged due to deformed hepatocytes and dilated lobules. Most of the hepatocytes after PTAB treatment were comparatively found similar to normal rats tissues, along with dilated blood vessels and normalized liver lobules. In addition to these results, PTAB suppresses the expressions of VEGF and MMP 9.\n\nConclusionPTAB was found to act as an effective hypoglycemic agent. In addition to this, PTAB also found to inhibit inflammation and apoptosis of liver. Thus, can be taken as a potential drug in liver diseases.

pharmacology and toxicology↗

bioRxiv2019

Anti-oxidant, anti-apoptotic, anti-hypoxic and anti-inflammatory conditions induced by PTY-2 against STZ-induced stress in islets

Background and AimEarlier assessment of Pueraria tuberosa tubers has shown anti-diabetic effects through incretin mimetic action and DPP-IV inhibition. The aim of this work was to further explore the protective role of aqueous extract of Pueraria tuberosa against streptozotocin (STZ)-induced pancreatic stress in rats.\n\nMethodsDiabetes was induced with STZ (65 mg/kg body weight) in Charles foster male rats. After 60 days of STZ administration, animals with blood glucose levels > 200 g/dL were considered as diabetic. All the rats were later divided into three groups: Group-1 (STZ untreated normal rats), Group-2 (Diabetic control), and Group-3 (PTY-2 [50 mg/100 g bw treatment for next 10 days to diabetic rats). The rats were then sacrificed at the 10th day of treatment accordingly.\n\nResultsSTZ treatment led to an increase in expression of MMP-9, Tnf , HIF-1, VEGF, IL-6, PKC {varepsilon}, NF-kB, and Caspase-3. Reverse transcriptase Polymerase Chain Reaction (PCR), IHC and western blot analysis showed an increase in the expressions of superoxide dismutase (SOD) and Nephrin, and a decrease in the expressions of NF-kB, PKC {varepsilon}, TNF- MMP-9, HIF-1, VEGF, Caspase 3 and IL-6 after 10 days of PTY-2 treatment.\n\nConclusionThe results show that PTY-2 favorably changed the expression of NF-kB, PKC {varepsilon}, TNF , MMP 9, HIF-1, VEGF, IL-6, Caspase3, Nephrin and SOD in cases of STZ-induced pancreatic stress. Further evaluation of PTY-2 might be helpful in establishing its role in the management of diabetes mellitus.\n\nHighlightsO_LIPTY 2 act as a protective herbal drug against STZ induced islet stress.\nC_LIO_LIPTY 2 upregulates protective and downregulates harmful markers.\nC_LIO_LIThis study composed of four pathway through which PTY 2 acts on pancreas.\nC_LI\n\nGRAPHICAL ABSTRACT\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=156 SRC=\"FIGDIR/small/670364v1_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (69K):\norg.highwire.dtl.DTLVardef@14b1f18org.highwire.dtl.DTLVardef@e6a1f2org.highwire.dtl.DTLVardef@19225b0org.highwire.dtl.DTLVardef@1e87bf5_HPS_FORMAT_FIGEXP M_FIG Mechanism of action of PTY 2 against STZ induced islet stress.\n\nC_FIG

pharmacology and toxicology↗

bioRxiv2019

PTY 2 As DPP-IV Inhibitor Prevents Intestinal Cell’s Apoptosis

Enhanced DPP-IV expression is found to get enhanced in various intestinal diseases. PTY-2 has already been known to have DPP-IV inhibitory potential. This inhibition has not yet been studied at mRNA level. Increased incretin secretion due to DPP-IV inhibition could lead to suppression of stress & intestinal cells apoptosis. Through histological analysis, we have found morphological damage of intestine after STZ injection (65 mg/kg bw) to male Charles foster rats. mRNA expressions were analyzed by PCR and apoptosis of cells was checked through tunnel assay & Bcl 2 expression. The number and length of villi get reduced in STZ induced diabetic control, but these damages have been getting reversed after the PTY-2 treatment for 10 days. The expression of SOD was found to get reduced while that of DPP-IV was enhanced in diabetic control group along with significant intestinal cell apoptosis. PTY-2 treatment reduces the stress by upregulating the expression of SOD and through downregulation of DPP-IV mRNA expression. These recoveries by PTY-2 leads to suppression of intestinal cells apoptosis. These short studies explain the protective action of PTY-2 against STZ induced intestinal damage. Hence, PTY-2 could be taken as an herbal treatment against intestinal disorders in case of chronic diabetes.

pharmacology and toxicology↗