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The myosin II motors are ATP-powered, force-generating machines driving cardiac and muscle contraction. Myosin II heavy chain isoform-beta (β-MyHC) is primarily expressed in the ventricular myocardium and slow-twitch muscle fibers, such as in M. soleus. M. soleus-derived myosin II (SolM-II) is often used as an alternative to the ventricular β-cardiac myosin (βM-II); however, the direct assessment of detailed biochemical and mechanical features of the native myosins is limited. By employing the optical trapping method, we examined the mechanochemical properties of the native myosins isolated from rabbit heart ventricle and M. soleus muscles at the single-molecule level. Contrary to previous reports, the purified motors from the two tissue sources, despite the same MyHC isoform, displayed distinct motile and ATPase kinetic properties. βM-II was ∼threefold faster in the actin filament-gliding assay than SolM-II. The maximum acto-myosin (AM) detachment rate derived in single-molecule assays was ∼threefold higher in βM-II. The stroke size for both myosins was comparable. The stiffness of the ‘AM rigor’ cross-bridge was also similar for both the motor forms. The stiffness of βM-II was found to be determined by the nucleotide state of the actin-bound myosin. Our analysis revealed distinct kinetic differences, i.e., a higher AM detachment rate for the βM-II, corresponding to the ADP release rates from the cross-bridge, thus elucidating the observed differences in the motility driven by βM-II and SolM-II. These studies have important implications for the future choice of tissue sources to gain insights into cardiomyopathies

The myosin II motors are ATP-powered force-generating machines driving cardiac and muscle contraction. Myosin II heavy chain isoform-beta (β-MyHC) is primarily expressed in the ventricular myocardium and in slow-twitch muscle fibers, such as M. soleus. M. soleus-derived myosin II (SolM-II) is often used as an alternative to the ventricular β-cardiac myosin (βM-II); however, the direct assessment of biochemical and mechanical features of the native myosins is limited. By employing optical trapping, we examined the mechanochemical properties of native myosins isolated from the rabbit heart ventricle and soleus muscles at the single-molecule level. We found purified motors from the two tissue sources, despite expressing the same MyHC isoform, displayed distinct motile and ATPase kinetic properties. We demonstrate βM-II was approximately threefold faster in the actin filament-gliding assay than SolM-II. The maximum actomyosin (AM) detachment rate derived in single-molecule assays was also approximately threefold higher in βM-II, while the power stroke size and stiffness of the "AM rigor" crossbridge for both myosins were comparable. Our analysis revealed a higher AM detachment rate for βM-II, corresponding to the enhanced ADP release rates from the crossbridge, likely responsible for the observed differences in the motility driven by these myosins. Finally, we observed a distinct myosin light chain 1 isoform (MLC1sa) that associates with SolM-II, which might contribute to the observed kinetics differences between βM-II and SolM-II. These results have important implications for the choice of tissue sources and justify prerequisites for the correct myosin heavy and light chains to study cardiomyopathies.

MicroRNAs (miRNAs) are short non-coding RNAs that function in post-transcriptional gene silencing and mRNA regulation. Although the number of nucleotides of miRNAs ranges from 17 to 27, they are mostly made up of 22 nucleotides. The expression of miRNAs changes significantly in cancer, causing protein alterations in cancer cells by preventing some genes from being translated into proteins. In this research, a structural analysis of 587 miRNAs that are differentially expressed in myeloid cancer was carried out. Length distribution studies revealed a mean and median of 22 nucleotides, with an average of 21.69 and a variance of 1.65. We performed nucleotide analysis for each position where Uracil was the most observed nucleotide and Adenine the least observed one with 27.8% and 22.6%, respectively. There was a higher frequency of Adenine at the beginning of the sequences when compared to Uracil, which was more frequent at the end of miRNA sequences. The purine content of each implicated miRNA was also assessed. A novel motif analysis script was written to detect the most frequent 3–7 nucleotide (3–7n) long motifs in the miRNA dataset. We detected CUG (42%) as the most frequent 3n motif, CUGC (15%) as a 4n motif, AGUGC (6%) as a 5n motif, AAGUGC (4%) as a 6n motif, and UUUAGAG (4%) as a 7n motif. Thus, in the second part of our study, we further characterized the motifs by analyzing whether these motifs align at certain consensus sequences in our miRNA dataset, whether certain motifs target the same genes, and whether these motifs are conserved within other species. This thorough structural study of miRNA sequences provides a novel strategy to study the implications of miRNAs in health and disease. A better understanding of miRNA structure is crucial to developing therapeutic settings.

ATR-FTIR Spectroscopy of aqueous cell culture samples is explained step-by-step. Infrared spectra acquisition, processing and analysis is included briefly.

Glucosylceramidase (GCase) is a lysosomal enzyme that catalyzes the cleavage of β-glucosidic linkage of glucocerebroside (GC) into glucose and ceramide; thereby, plays an essential function in the degradation of complex lipids and the turnover of cellular membranes. The growing list of 460 mutations in the gene coding for it—glucosylceramidase beta acid 1 (GBA1)—is reported to abolish its catalytic activity and decrease its enzyme stability, associating it with severe health conditions such as Gaucher disease (GD), Parkinson Disease (PD) and Dementia with Lewy bodies (DLB). Although the three-dimensional structure of wild type glucosylceramidase is elucidated, little is known about its features in human cells. Moreover, alternative sources of GCase that prove to be effective in the treatment of diseases with enzyme treatment therapies, impose the need for a simple and cost-effective procedure to study the enzyme behavior. This work, for the first time, shows a well-established, yet simple, cost- and time-efficient protocol for the study of GCase enzyme in human leukocytes by the artificial substrate p-Nitrophenyl-β-D-glucopyranoside (PNPG). Characterization of the enzyme in human leukocytes for activation parameters (optimal pH, Km, and Vmax) and enzyme inhibition was done. The results indicate that the optimum pH of GCase enzyme with PNPG is 5.0. The Km and Vmax values are 12.6mM and 333 U/mg, respectively. Gluconolactone competitively inhibits GCase, with a Ki value of 0.023 mM and IC50 of 0.047 mM. Glucose inhibition is uncompetitive with a Ki of 1.94 mM and IC50 of 55.3 mM. This is the first report for the inhibitory effect of glucose, δ-gluconolactone on human leukocyte GCase activity., Proteins; Biochemistry; Molecular Biology; Hematological System; Clinical Research; glucosylceramidase; glucosylceramide; PNPG; human leukocyte; enzyme kinetics; inhibition parameters; Gaucher Disease; glucose; gluconolactone.

Leukocytes are isolated by centrifugation after specific lysis of erythrocytes

Bu çalışmada, ATR-FTIR metodu ile baskılayan ve uyaran iki tip immünoterapötik oligodeoksinükleotitlerin, makrofaj hücrelerinde yol açtıkları yapısal ve fonksiyonel değişimlerin değerlendirilmesi yapılmıştır. Makrofaj hücreleri, bağışıklık sisteminde anahtar rol oynamaktadır. Sahip olduklari yüksek fleksibilite ile çevresel sinyallere kolayca alışabılen hücrelerdir. Bu anlamda mükemmel bir şekilde fiziyolojik degişikliklere mağruz kalabilme özelliğine sahiptirler. IR spektrumunda geniş çapta moleküler sinyaller verdiğinden dolayı, ATR-FTIR başarılı bir şekilde kullanılabilir. LPS ve CpG ODN bağışıklık sistemini hızlıca arttirabilirler ve çok etkili pro-enflamatuar sitokin, kemokin ve spesifik Ig ajanları üretirler. CpG ODN immünoterapötik bir ilaç aktivitesi gösterebildigi aciklanmistir. Ancak son zamanlarda çıkan klinik çalışmalarda, CpG'nin bazı ciddi sorunları beraberinde getirdiği gözlenmiştir, ki bunlardan bazıları alerji, toksik şok hatta organizmanın ölümüne kadar götürmektedir. CpG ODN ile sürdürülen makrofajlardaki bu aktivasyonun yan etkilerini durdurma ihtiyacı, baskılayıcı ODN'lerin bulunmasına yol açtmıstır, ki bunlardan birisi de A151 ODN'dir. A151 ODN'in çalısma mekanizması bilinmemektedir. Bu çalışmada, fare makrofaj RAW 264.7 hücreleri, PBS, CpG ODN, LPS, baskılayıcı ODNA151, ve ters K3-ODN tretmanları ile dört değisik zaman dilimlerinde aşılanmıştır. Homojen 2∙〖10〗^6/μL konsantrasyonlu hücre örnekleri hazırlanip üç replikada n=5 olmak üzere FTIR spektraları elde edilmiştir. Smoothing, baseline correction ve normalization adımlarından sonra spektralar kuantitativ incelemelere hazır olmuştur. Normal dağılımlı sonuçlar One-Way ANOVA ve Dunnet's test for multiple comparisons ile analizi yapılmıştır, normal dağılımı olmayan örnekler ise Kruskal–Wallis with Dunn's test for multiple comparisons ile incelenmiştir. Tüm sonuçlar ortalama±SEM olarak sunulmuştur. Sonuçlar, uyarıcı CpG ODN'nin IR spektrumunda yüksek değisimlere yol açtığı gözlenmiştir. Iki saate kadar ki zaman dilimlerinde, CpG ODN ve LPS, RNA sentezinin bariz bir şekilde arttığı gözlenmiştir. 6 saatten sonra ki zaman dilimlerinde, DNA sentezi/değisikliği başladiği bulunmuştur. LPS, anti-proleferativ bir ajan olduğu için, 6 saatten sonra, DNA sentezi yapmamıştır. Stimulasyon ajanlarının kullanıldığı örneklerde, protein seviyelerinin düştüğü bulunmuştur, ki bu aktivasyona bağli oksidantların oksidatif stres uygulamalari ile bağdaşmıstır. Bu etkileri düşürmek için, hücre içi proteozomlarin proteinleri degrade ettikleri bulunmuştur. Aktivasyona bağlı, membran akişkanlığında artiş gözlenmiştir, ki bu doymamis yağlarin düşüşü ile, artan lipid peroksidasyonu ile ve düsen kolesterol miktarlari ile doğru orantida olduğu belirlenmiştir. Son olarak, bu değişimlerin sinyalizasyon mekanizmalarinda, iyon geçirgenliğinde ve birçok membranlara bağlı hücre fonksiyonlarında değişimlere meydan açabilecegine sonuç bağlanmıştır.Anahatar Kelimeler: Makrofaj, CpG ODN, A151 ODN, FTIR spektroskopisi In the present study, ATR-FTIR was used to examine the structural and functional changes on murine macrophage cells upon activation and suppression by immunotherapeutic ODNs. Macrophage cells play a key role in innate and adaptive immunity. Their possession of high flexibility helps them to successfully respond to environmental signals. In this context, they can impressively alter their physiology in relation to their function and these changes have large molecular signals in the infrared spectral region that can be traced with FTIR spectroscopy. LPS and CpG ODN are used as well-known stimulants of macrophages to produce the powerful pro-inflammatory agents such as cytokines, chemokines and highly specific Ig's. CpG ODN is potentially used in many immunotherapeutic therapies, but is found to cause allergy, toxic shock and death. The need for a suppressive agent led to the innovation of suppressive ODN, whose mechanism is not elucidated. In the present study, murine macrophage-like RAW 264.7 cells are treated with PBS medium, CpG ODN, LPS, suppressive ODNA151, and K3 Flip-ODN in four different time scales, to monitor the changes on the course of time. Homogenous 2∙〖10〗^6/μL cell samples were prepared and FTIR spectra were recorded in triplicates. In total n=5 was performed. The spectra recorded underwent smoothing, baseline correction and normalization steps, and quantitative analysis was performed. One way-ANOVA with Dunnet's test for multiple comparisons and Kruskal–Wallis with Dunn's test for multiple comparisons was used for the analysis of normally and non-normally distributed data, respectively. All results were expressed as mean±SEM. The results clearly indicate that CpG-induced macrophage cells show prominent features in their IR spectra for their activation. The time period till 2h of stimulation seems to be the period of extensive RNA synthesis, and as it ceases, DNA synthesis starts from the 6th hour of stimulation upon both CpG ODN and LPS stimulation, except LPS at 6 hours, due to the property of LPS being an anti-proliferative agent. It is also found that stimulation of macrophage cells with CpG ODN and LPS decreases protein content. This is explained by the production of numerous oxidants, causing oxidative stress and in intention of protecting the cell; proteasomes start the degradation of oxidized proteins. The membrane fluidity increases during activation with CpG ODN and LPS, as a result of decreased unsaturated lipid content, increased lipid peroxidation and decreased cholesterol content. It was also noted that these changes may have altered the signal transduction processes, ion permeability and many other primary functions of cell membranes could be altered. Keywords: Macrophage, CpG ODN, A151 ODN, FTIR spectroscopy 112