Dopaminergic influence on thyrotropin secretion in primary hypothyroidism.

This study was conducted to assess the influence of dopamine on thyrotropin secretion in patients with primary hypothyroidism before and after optimized L-thyroxin replacement therapy. Thyrotropin responses to dopamine infusion (4 microg/kg/min over 3 hours) and IV metoclopramide (10 mg bolus), a dopamine receptor blocker were studied in 25 consecutive patients with primary hypothyroidism before and after achieving stable euthyroid state and compared with 15 normal age-matched controls. Thyrotropin response to both dopamine infusion (decremental) and IV metoclopramide bolus (incremental) was greater in patients with primary hypothyroidism than that in the control subjects. Thyrotropin response was greater in women than in men. The magnitude of decremental thyrotropin response to dopamine infusion and the incremental response to IV metoclopramide bolus significantly correlated with the basal T3 and T4 levels. Thyrotropin response was blunted to dopamine infusion but not to metoclopramide at follow-up after six-month replacement with L-thyroxin, and both the responses were comparable in women and men in patient group. We conclude that modulation of dopaminergic system by dopamine or by dopamine receptor blocker has a greater influence on thyrotropin secretion in patients with primary hypothyroidism than euthyroid normal subjects.     

Fenugreekine,a new steroidal sapogenin-peptide ester of Trigonella foenum-graecum

A new C₂₇-steroidal sapogenin-peptide ester, fenugreekine, has been isolated from seeds of Trigonella foenum-graecum. On acid hydrolysis, it afforded diosgenin, yamogenin, (25R)-spirosta-3,5-diene, a mixture of three isomeric (2S,3R,4R-, 2S,3R,4S-, 2S,3S,4R-)-4-hydroxyisoleucine lactones, 4′-hydroxyisoleucyl-4-hydroxyisoleucine lactone, and a C₁₄-dipeptide which was partially characterized. On the basis of this chemical transformation and spectral (UV, IR, PMR, MS) evidence of fenugreekine and its transformation products, the steroidal sapogenin-peptide ester is assigned structure (1). The two dipeptides also have not been encountered before in nature or prepared synthetically. The compound shows a number of interesting pharmacological and virological activities.     

Protein Profiles of Somatic Embryos and Regenerated Plants from NaCl Selected and Control Cultures of Orchardgrass

The protein profile of cells of control somatic embryos was compared to that of embryos that have become selected and maintained on 200 mM NaCl in order to detect salt inducible proteins. Two proteins (60 and 51.5 kDa) were more abundant in the selected embryos and one protein with molecular mass 18 kDa was unique to the selected embryos. Enhanced content of 27 kDa protein was observed in all somatic embryos indicating its involvement in the embryonal state. Similar pattern of salt inducible proteins in selected somatic embryos and the plantlets regenerated from such embryos was found. This revised version was published online in July 2006 with corrections to the Cover Date.     

The 80s loop of the catalytic chain of Escherichia coli aspartate transcarbamoylase is critical for catalysis and homotropic cooperativity.

The X-ray structure of the Escherichia coli aspartate transcarbamoylase with the bisubstrate analog phosphonacetyl-L-aspartate (PALA) bound shows that PALA interacts with Lys84 from an adjacent catalytic chain. To probe the function of Lys84, site-specific mutagenesis was used to convert Lys84 to alanine, threonine, and asparagine. The K84N and K84T enzymes exhibited 0.08 and 0.29% of the activity of the wild-type enzyme, respectively. However, the K84A enzyme retained 12% of the activity of the wild-type enzyme. For each of these enzymes, the affinity for aspartate was reduced 5- to 10-fold, and the affinity for carbamoyl phosphate was reduced 10- to 30-fold. The enzymes K84N and K84T exhibited no appreciable cooperativity, whereas the K84A enzyme exhibited a Hill coefficient of 1.8. The residual cooperativity and enhanced activity of the K84A enzyme suggest that in this enzyme another mechanism functions to restore catalytic activity. Modeling studies as well as molecular dynamics simulations suggest that in the case of only the K84A enzyme, the lysine residue at position 83 can reorient into the active site and complement for the loss of Lys84. This hypothesis was tested by the creation and analysis of the K83A enzyme and a double mutant enzyme (DM) that has both Lys83 and Lys84 replaced by alanine. The DM enzyme has no cooperativity and exhibited 0.18% of wild-type activity, while the K83A enzyme exhibited 61% of wild-type activity. These data suggest that Lys84 is not only catalytically important, but is also essential for binding both substrates and creation of the high-activity, high-affinity active site. Since low-angle X-ray scattering demonstrated that the mutant enzymes can be converted to the R-structural state, the loss of cooperativity must be related to the inability of these mutant enzymes to form the high-activity, high-affinity active site characteristic of the R-functional state of the enzyme.     

Reactive extraction of cephalosporin antibiotics in hollow fiber membrane

Non-dispersive reactive extraction of cephalosporin antibiotics has been studied using hollow fiber membrane modules. Extraction as well as stripping has been studied using a pH swing procedure. Cephalosporin was extracted from an aqueous solution of cephalosporin having a pH above the pK_a2 value to an organic phase containing Aliquat-336 as the extractant and n-heptane as the diluent. The solute was stripped from the loaded organic phase to another aqueous phase of pH maintained well below the pK_a2 value of the cephalosporin. The extraction cum stripping relies on pH dependance of the distribution coefficient of cephalosporin in aqueous phase. Reasonably high solute recovery and mass transfer rate have been achieved in the hollow fiber module. Mass transfer performance of a single module has been evaluated and experimentally observed low value of height of transfer unit (HTU) indicates good prospect of hollow fiber membrane for the extraction duty.     

MicroDNA levels are dependent on MMEJ,repressed by c-NHEJ pathway,and stimulated by DNA damage

Extrachromosomal circular DNA (eccDNA) are present within all eukaryotic organisms and actively contribute to gene expression changes. MicroDNA (200-1000bp) are the most abundant type of eccDNA and can amplify tRNA, microRNA, and novel si-like RNA sequences. Due to the heterogeneity of microDNA and the limited technology to directly quantify circular DNA molecules, the specific DNA repair pathways that contribute to microDNA formation have not been fully elucidated. Using a sensitive and quantitative assay that quantifies eight known abundant microDNA, we report that microDNA levels are dependent on resection after double-strand DNA break (DSB) and repair by Microhomology Mediated End Joining (MMEJ). Further, repair of DSB without resection by canonical Non-Homologous End Joining (c-NHEJ) diminishes microDNA formation. MicroDNA levels are induced locally even by a single site-directed DSB, suggesting that excision of genomic DNA by two closely spaced DSB is not necessary for microDNA formation. Consistent with all this, microDNA levels accumulate as cells undergo replication in S-phase, when DNA breaks and repair are elevated, and microDNA levels are decreased if DNA synthesis is prevented. Thus, formation of microDNA occurs during the repair of endogenous or induced DNA breaks by resection-based DNA repair pathways.     

Proteomic analysis and protein structure prediction of Shigella phage Sfk20 based on a comparative study using structure prediction approaches

Bacteriophages are the natural predators of bacteria and are available abundantly everywhere in nature. Lytic phages can specifically infect their bacterial host (through attachment to the receptor) and use their host replication machinery to replicate rapidly, a feature that enables them to kill a disease-causing bacteria. Hence, phage attachment to the host bacteria is the first important step of the infection process. It is reported in this study that the receptor could be an LPS which is responsible for the attachment of the Sfk20 phage to its host (Shigella flexneri 2a). Phage Sfk20 bacteriolytic activity was examined for preliminary optimization of phage titer. The phage Sfk20 viability at different saline conditions was conducted. The LC–MS/MS technique used here for detecting and identifying 40 Sfk20 phage proteins helped us to get an initial understanding of the structural landscape of phage Sfk20. From the identified proteins, six structurally significant proteins were selected for structure prediction using two neural network systems: AlphaFold2 and ESMFold, and one homology modeling software: Phyre2. Later the performance of these modeling systems was compared using various metrics. We conclude from the available and generated information that AlphaFold2 and Phyre2 perform better than ESMFold for predicting Sfk20 phage protein structures.     

Design and synthesis of new piperidone grafted acetylcholinesterase inhibitors

Alzheimer’s disease (AD) is a neurodegenerative disorder affecting 35 million people worldwide. A common strategy to improve the well-being of AD patients consists on the inhibition of acetylcholinesterase with the concomitant increase of the neurotransmitter acetylcholine at cholinergic synapses. Two series of unreported N-benzylpiperidines 5(a–h) and thiazolopyrimidines 9(a–q) molecules were synthesized and evaluated in vitro for their acetylcholinesterase (AChE) inhibitory activities. Among the newly synthesized compounds, 5h, 9h, 9j, and 9p displayed higher AChE enzyme inhibitory activities than the standard drug, galantamine, with IC₅₀ values of 0.83, 0.98, and 0.73 μM, respectively. Cytotoxicity studies of 5h, 9h, 9j, 9n and 9p on human neuroblastoma cells SH-SY5Y, showed no toxicity up to 40 μM concentration. Molecular docking simulations of the active compounds 5h and 9p disclosed the crucial role of π-π-stacking in their binding interaction to the active site AChE enzyme. The presented compounds have potential as AChE inhibitors and potential AD drugs.     

Point mutations in the DNA binding domain of p53 contribute to glioma progression and poor prognosis

TP53 mutations play a significant role in glioma tumorigenesis. When located in in the DNA binding domain, these mutations can perturb p53 protein conformation and its function, often culminating in altered downstream signaling. Here we describe prevalent pattern of TP53 point mutations in a cohort of 40 glioma patients and show their relevance to gliomagenesis. Point mutations in exon 5–9 of TP53 gene were detected by DNA sequencing. Possible influence of identified mutations at the function of p53 was studied computationally and correlated with the survival. Point mutations in TP53 were detected in 10 glioma samples (25%), out of which 70% were from high grade glioma. A total of 19 TP53 point mutations were identified, out of which 42% were found to be in the DNA binding region of p53. Computational analysis predicted 87.5% of these mutations to be “probably damaging”. In three patients with tumors possessing point mutations R273H, R248Q, Y163H and R175H and poor survival times, structural analysis revealed the nature of these mutations to be disruptive and associated with high risk for cancer progression. In high grade glioma, recurrent TP53 point mutations may be the key to tumor progression, thus, emphasizing their significance in gliomagenesis.     

SAC and Berberine Mediated Repression of Reactive Species and Hepatoprotection After DEN + CCl<Subscript>4</Subscript> Exposure

Oxidative stress is proposed to play a pivotal role in the development of hepatocellular carcinoma. With an accelerated metabolism cancer cells demand high reactive species accumulation to maintain their indiscriminate cell growth and proliferation. Here we wanted to see the status of reactive species in the chemically induced liver cancer. For this purpose swiss albino mice were exposed to DEN and CCl₄ to develop an in vivo model of hepatocarcinoma. Depletion of cellular antioxidants regulated accretion of reactive species during the development of DEN + CCl₄ induced tumor formation in hepatocytes. Currently available therapeutics for heptatocellular carcinoma is costly and coupled with certain bystander effects to the surrounding control cells. Therefore considering the antioxidant properties of SAC and berberine we treated DEN + CCl₄ exposed mice after the development of liver tumor. Results effectively pointed out the usefulness of the alternative treatment with SAC and berberine in hepatoprotection. Replenishment of both enzymatic and non enzymatic antioxidant efficiently reduced accumulation of reactive species and that eventually closely associated with effective reduction in tumor number and size after drug treatment in DEN + CCl₄ exposed mice.