Search

S-glutathionylation (SSG) is increasingly recognized as a critical signaling mechanism in the heart, yet SSG modifications in cardiac sarcomeric proteins remain understudied. Here we identified SSG of the ventricular isoform of myosin light chain 1 (MLC-1v) in human, swine, and mouse cardiac tissues using top-down mass spectrometry (MS)-based proteomics. Our results enabled the accurate identification, quantification, and site-specific localization of SSG in MLC-1v across different species. Notably, the endogenous SSG of MLC-1v was observed in human and swine cardiac tissues but not in mice. Treating non-reduced cardiac tissue lysates with GSSG elevated MLC-1v SSG levels across all three species.

Native top-down mass spectrometry (nTDMS) allows characterization of protein structure and noncovalent interactions with simultaneous sequence mapping and proteoform characterization. The majority of nTDMS studies utilize purified recombinant proteins, with significant challenges hindering application to endogenous systems. To perform native top-down proteomics (nTDP), where endogenous proteins from complex biological systems are analyzed by nTDMS, it is essential to separate proteins under nondenaturing conditions. However, it remains difficult to achieve high resolution with MS-compatible online chromatography while preserving protein tertiary structure and noncovalent interactions. Herein, we report the use of online mixed-bed ion exchange chromatography (IEC) to enable separation of endogenous proteins from complex mixtures under nondenaturing conditions, preserving noncovalent interactions for nTDP analysis. We have successfully detected large proteins (>146 kDa) and identified endogenous metal-binding and oligomeric protein complexes in human heart tissue lysate. The use of a mixed-bed stationary phase allowed retention and elution of proteins over a wide range of isoelectric points without altering the sample or mobile phase pH. Overall, our method provides a simple online IEC-MS platform that can effectively separate proteins from complex mixtures under nondenaturing conditions and preserve higher-order structure for nTDP applications.

Native top-down mass spectrometry (nTDMS) has emerged as a powerful structural biology tool that can localize post-translational modifications (PTMs), explore ligand-binding interactions, and elucidate the three-dimensional structure of proteins and protein complexes in the gas-phase. Fourier-transform ion cyclotron resonance (FTICR) MS offers distinct capabilities for nTDMS, owing to its ultrahigh resolving power, mass accuracy, and robust fragmentation techniques. Previous nTDMS studies using FTICR have mainly been applied to overexpressed recombinant proteins and protein complexes. Here, we report the first nTDMS study that directly analyzes human heart tissue lysate by direct infusion FTICR MS without prior chromatographic separation strategies. We have achieved comprehensive nTDMS characterization of cardiac contractile proteins that play critical roles in heart contraction and relaxation. Specifically, our results reveal structural insights into ventricular myosin light chain 2 (MLC-2v), ventricular myosin light chain 1 (MLC-1v), and alpha-tropomyosin (α-Tpm) in the sarcomere, the basic contractile unit of cardiac muscle. Furthermore, we verified the calcium (Ca 2+ ) binding domain in MLC-2v. In summary, our nTDMS platform extends the application of FTICR MS to directly characterize the structure, PTMs, and metal-binding of endogenous proteins from heart tissue lysate without prior separation methods.

To improve the coverage in bottom-up proteomics, S-aminoethylation of cysteine residues (AE-Cys) was carried out with 2-bromoethylamine, followed by cleavage with lysyl endopeptidase (Lys-C) or Lys-C/trypsin. A model study with bovine serum albumin showed that the C-terminal side of AE-Cys was successfully cleaved by Lys-C. The frequency of side reactions at amino acids other than Cys was less than that in the case of carbamidomethylation of Cys with iodoacetamide. Proteomic analysis of A549 cell extracts in the data-dependent acquisition mode after AE-Cys modification afforded a greater number of identified protein groups, especially membrane proteins. In addition, label-free quantification of proteins in mouse nonsmall cell lung cancer (NSCLC) tissue in the data-independent acquisition mode after AE-Cys modification showed improved NSCLC pathway coverage and greater reproducibility. Furthermore, the AE-Cys method could identify an epidermal growth factor receptor peptide containing the T790 M mutation site, a well-established lung-cancer-related mutation site that has evaded conventional bottom-up methods. Finally, AE-Cys was found to fully mimic Lys in terms of collision-induced dissociation fragmentation, ion mobility separation, and cleavage by Lys-C/trypsin, except for sulfoxide formation during sample preparation.

Motivation: Native top-down proteomics (nTDP) integrates native mass spectrometry (nMS) with top-down proteomics (TDP) to provide comprehensive analysis of protein complexes together with proteoform identification and characterization. Despite significant advances in nMS and TDP software developments, a unified and user-friendly software package for analysis of nTDP data remains lacking., Results: We have developed MASH Native to provide a unified solution for nTDP to process complex datasets with database searching capabilities in a user-friendly interface. MASH Native supports various data formats and incorporates multiple options for deconvolution, database searching, and spectral summing to provide a "one-stop shop" for characterizing both native protein complexes and proteoforms., Availability and Implementation: The MASH Native app, video tutorials, written tutorials, and additional documentation are freely available for download at https://labs.wisc.edu/gelab/MASH_Explorer/MASHSoftware.php. All data files shown in user tutorials are included with the MASH Native software in the download .zip file., (© The Author(s) 2023. Published by Oxford University Press.)

The coronavirus nucleocapsid (N) protein is known to bind to nucleic acids and facilitate viral genome encapsulation. Here we report that the N protein can mediate RNA or DNA entering neighboring cells through ACE2-independent, receptor (STEAP2)-mediated endocytosis, and achieve gene expression. The effect is more pronounced for the N protein of wild-type SARS-CoV-2 than that of the Omicron variant and other human coronaviruses. This effect is enhanced by RANTES (CCL5), a chemokine induced by N protein, and lactate, a metabolite produced in hypoxia, to cause more damage. These findings might explain the clinical observations in SARS-CoV-2-infected cases. Moreover, the N protein-mediated function can be inhibited by N protein-specific monoclonal antibodies or p38 mitogen-activated protein kinase inhibitors. Since the N-protein-mediated nucleic acid endocytosis involves a receptor commonly expressed in many types of cells, our findings suggest that N protein may have an additional role in SARS-CoV-2 pathogenesis., Competing Interests: The authors declare no competing interests., (© 2023 The Author(s).)

Despite advancements of data-independent acquisition mass spectrometry (DIA-MS) to provide comprehensive and reproducible proteome profiling, its utility in very low-input samples is limited. Due to different proteome complexities and corresponding peptide ion abundances, the conventional LC-MS/MS acquisition and widely used large-scale DIA libraries may not be suitable for the micro-nanogram samples. In this study, we report a sample size-comparable library-based DIA approach to enhance the proteome coverage of low-input nanoscale samples (i.e., nanogram cells, ∼5-50 cells). By constructing sample size-comparable libraries, 2380 and 3586 protein groups were identified from as low as 0.75 (∼5 cells) and 1.5 ng (∼10 cells), respectively, highlighting one of the highest proteome coverage with good reproducibility (86%-99% in triplicate results). For the 0.75 ng sample (∼5 cells), significantly superior identification (2380 proteins) was achieved by small-size library-based DIA, compared to 1908, 1749, and 107 proteins identified from medium-size and large-size libraries and a lung cancer resource spectral library, respectively. A similar trend was observed using a different instrument and data analysis pipeline, indicating the generalized conclusion of the approach. Furthermore, the small-size library uniquely identified 518 (22%) proteins in the low-abundant region and spans over a 5-order dynamic range. Spectral similarity analysis revealed that the fragmentation ion pattern in the DIA-MS/MS spectra of the dataset and spectral library play crucial roles for mapping low abundant proteins. With these spectral libraries made freely available, the optimized library-based DIA strategy and DIA digital map will advance quantitative proteomics applications for mass-limited samples.

Objective: Hepatic steatosis causes nonalcoholic fatty liver disease and may progress to fibrosis. Ultrasound is the first-line approach to examining hepatic steatosis. Fatty droplets in the liver parenchyma alter ultrasound radiofrequency (RF) signal statistical properties. This study proposes using sample entropy, a measure of irregularity in time-series data determined by the dimension [Formula: see text] and tolerance [Formula: see text], for ultrasound parametric imaging of hepatic steatosis and fibrosis. Methods: Liver donors and patients were enrolled, and their hepatic fat fraction (HFF) ([Formula: see text]), steatosis grade ([Formula: see text]), and fibrosis score ([Formula: see text]) were measured to verify the results of sample entropy imaging using sliding-window processing of ultrasound RF data. Results: The sample entropy calculated using [Formula: see text] 4 and [Formula: see text] was highly correlated with the HFF when a small window with a side length of one pulse was used. The areas under the receiver operating characteristic curve for detecting hepatic steatosis that was [Formula: see text]mild, [Formula: see text]moderate, and [Formula: see text]severe were 0.86, 0.90, and 0.88, respectively, and the area was 0.87 for detecting liver fibrosis in individuals with significant steatosis. Discussion/Conclusions: Ultrasound sample entropy imaging enables the identification of time-series patterns in RF signals received from the liver. The algorithmic scheme proposed in this study is compatible with general ultrasound pulse-echo systems, allowing clinical fibrosis risk evaluations of individuals with developing hepatic steatosis.

Alzheimer's disease (AD) is a progressive debilitating neurodegenerative disease and the most common form of dementia in the older population. At present, there is no definitive effective treatment for AD. Therefore, researchers are now looking at stem cell therapy as a possible treatment for AD, but whether stem cells are safe and effective in humans is still not clear. In this narrative review, we discuss both preclinical studies and clinical trials on the therapeutic potential of human stem cells in AD. Preclinical studies have successfully differentiated stem cells into neurons in vitro, indicating the potential viability of stem cell therapy in neurodegenerative diseases. Preclinical studies have also shown that stem cell therapy is safe and effective in improving cognitive performance in animal models, as demonstrated in the Morris water maze test and novel object recognition test. Although few clinical trials have been completed and many trials are still in phase I and II, the initial results confirm the outcomes of the preclinical studies. However, limitations like rejection, tumorigenicity, and ethical issues are still barriers to the advancement of stem cell therapy. In conclusion, the use of stem cells in the treatment of AD shows promise in terms of effectiveness and safety.

The long-term stability of affinity-based protein labeling probes is crucial to obtain reproducible protein labeling results. However, highly stable probes generally suffer from low protein labeling efficiency and pose significant challenges when labeling low abundance native proteins in living cells. In this paper, we report that protein labeling probes based on an ortho-difluorophenyl ester reactive module exhibit long-term stability in DMSO stock solution and aqueous buffer, yet they can undergo rapid and selective labeling of native proteins. This novel electrophile can be customized with a wide range of different protein ligands and is particularly well-suited for the labeling and imaging of transmembrane proteins. With this probe design, the identity and relative levels of basal and hypoxia-induced transmembrane carbonic anhydrases were revealed by live cell imaging and in-gel fluorescence analysis. We believe that the extension of this difluorophenyl ester reactive module would allow for the specific labeling of various endogenous membrane proteins, facilitating in-depth studies of their distribution and functions in biological processes., (© 2020 Wiley-VCH GmbH.)

Entropy is a quantitative measure of signal uncertainty and has been widely applied to ultrasound tissue characterization. Ultrasound assessment of hepatic steatosis typically involves a backscattered statistical analysis of signals based on information entropy. Deep learning extracts features for classification without any physical assumptions or considerations in acoustics. In this study, we assessed clinical values of information entropy and deep learning in the grading of hepatic steatosis. A total of 205 participants underwent ultrasound examinations. The image raw data were used for Shannon entropy imaging and for training and testing by the pretrained VGG-16 model, which has been employed for medical data analysis. The entropy imaging and VGG-16 model predictions were compared with histological examinations. The diagnostic performances in grading hepatic steatosis were evaluated using receiver operating characteristic (ROC) curve analysis and the DeLong test. The areas under the ROC curves when using the VGG-16 model to grade mild, moderate, and severe hepatic steatosis were 0.71, 0.75, and 0.88, respectively; those for entropy imaging were 0.68, 0.85, and 0.9, respectively. Ultrasound entropy, which varies with fatty infiltration in the liver, outperformed VGG-16 in identifying participants with moderate or severe hepatic steatosis ( p < 0.05). The results indicated that physics-based information entropy for backscattering statistics analysis can be recommended for ultrasound diagnosis of hepatic steatosis, providing not only improved performance in grading but also clinical interpretations of hepatic steatosis.

Background: The issue of suboptimal influenza vaccination coverage among pregnant women remains relevant. Our study aimed to explore the determinants and coverage of influenza vaccination among pregnant women in the United States using a nationally representative sample. Materials and Methods: This study was conducted with the 2012-2016 U.S. National Health Interview Survey. The Andersen's Health Behavior Model was applied as the conceptual framework to explore potential factors that may influence the influenza vaccination rate. A series of individual determinants, categorized into predisposing, enabling, and need factors, were compared using logistic regressions between women who received an influenza vaccination before or during pregnancy and those who did not. Results: An average of 36% women received an influenza vaccination before or during pregnancy among an estimated five million pregnant women. Even though the percentage increased from 31% in 2012 to 40% in 2016, it remained lower than the Healthy People 2020 target of 80%. The odds of receiving an influenza vaccination before or during pregnancy were lower among women who had public or no insurance coverage (odds ratio [OR]; 95% confidence interval, 0.510 [0.323-0.806] and 0.351 [0.175-0.705], respectively), lived in South (0.546 [0.336-0.887]), ever smoked 100 cigarettes (0.622 [0.419-0.923]), and had infrequent to light alcohol consumption in the past year (0.670 [0.457-0.983], reference: no alcohol consumption in the past year). Having a bachelor's degree increased the odds of getting an influenza vaccine compared to a high school diploma or less (2.086 [1.353-3.215]). Conclusions: Our study found that the influenza vaccination coverage among pregnant women remains suboptimal, and disparities may still exist across women with different sociodemographic and socioeconomic status. Clinicians should actively recommend influenza vaccination for pregnant women, and policy makers may consider developing interventions to improve the vaccination rate.

Background: The safety of Laser-assisted in situ keratomileusis (LASIK) in aircrew was unclear, in addition, LASIK was not yet approved for aircrew of Taiwan Air Force. This study was aimed to evaluate visual performance in LASIK eyes in hypoxic and twilight environment., Methods: 48 myopic eyes of 24 subjects enrolled in this study were divided into LASIK group and control group. Subjects were exposed in hypoxic (15% O 2 ) and mesopic (3 cd/m 2 ) environment. Visual performance was evaluated using the Early Treatment of Diabetic Retinopathy Study (ETDRS) visual chart, and Functional Acuity Contrast Test (FACT) before and after the expirement. Physiological parameters of all subjects were measured and recorded throughout the experiment., Results: There was no significant difference of the two groups regarding their age, height, weight, and BMI. There is significant difference of preoperative spherical refractive error between the two groups. The results of physiological parameters were similar between two groups. Under normoxic conditions, there were no significant difference regarding distant vision in photopic and mesopic environments, so as for near vision. As a whole, the contrast sensitivity of the LASIK group were lowered than that of the control group about 35%, under whether normoxic or hypoxic conditions; photopic or mesopic circumstances. Under normoxic conditions, the measured accommodation of the LASIK group were 21% lowered than that of the control group and 31% lowered under hypoxic circumstances., Conclusion: There was no significant difference of visual acuity between the two groups regarding hypoxic and mesopic environment, but reduced contrast sensitivity was significant in LASIK group as compared to those of the control group. Accommodation was significantly lowered in LASIK group, compared with control group, in hypoxic environment. Whether postoperative visual performance after LASIK in aircrew during flying duty is safe might need further investigation., (Copyright © 2018. Published by Elsevier Taiwan LLC.)

Aromatase and estrogen receptor α (ER) are two key proteins for the proliferation of endocrine-responsive and -resistant breast cancers. Aromatase is an enzyme involved in the conversion of androgen (such as testosterone) to estrogen (such as 17β-estradiol). It is also a very effective therapeutic target for the treatment of endocrine-responsive breast cancer. Comparing endocrine-responsive and -resistant breast cancer, aromatase protein levels do not change significantly. Aromatase activity; however, can be increased via PI3K/Akt/IGFR signaling pathways in endocrine resistant cells. The activity of aromatase has been reported to be modulated by phosphorylation. The ER is an important steroid nuclear receptor in the proliferation of both endocrine-responsive and -resistant cells. Although the mutation or amplification of ER can cause endocrine resistance, it is not commonly found. Some point mutations and translocation events have been characterized and shown to promote estrogen-independent growth. Phosphorylation by cross-talk with growth factor pathways is one of the main mechanisms for ligand-independent activation of ER. Taken together, both ER and aromatase are important in ER-dependent breast cancer and the development of endocrine resistance., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Of all breast cancer patients, about 70% are ER+ and 10% are ER+/HER2+. The ER+/HER2+ patients have a worse outcome compared to ER+/HER2- patients. Currently there is a lack of effective prognosis biomarkers for the prediction of outcome in ER+/HER2+ patients. Genome-wide differences in ER binding between the endocrine-responsive and endocrine-resistant cells were discovered using ChIP-seq, and combined with gene expression microarray data to identify direct ER target genes. These genes were correlated to survival outcome using publicly available breast cancer patient cohorts. We found the expression of the gene SERPINA1 to have a significant predictive value for the overall survival (OS) of ER+ patients in the TCGA cohort, and validated this finding in the Curtis cohort. SERPINA1 also has a significant predictive value for the OS of ER+/HER2+ patients in the TCGA cohort, with validation in the Bild cohort. The expression of SERPINA1 can be suppressed by fulvestrant and HER2 siRNA. Our results indicate that ER is constitutively activated, resulting in an E2-independent ER binding to the SERPINA1 gene and upregulation of SERPINA1 expression. Importantly, results of survival correlation suggests that high expression of SERPINA1 could be predictive for a better clinical outcome of ER+ and ER+/HER2+ patients.

Progression or recurrence due to resistance to aromatase inhibitors (AIs) is a significant clinical problem for a considerable number of patients with breast cancer. Programmed cell death 4 (PDCD4), a tumor suppressor protein, is targeted for degradation during tumor progression. In the current study, we aimed to examine PDCD4 expression and regulation in AI-resistant breast cancer cells, and its association with survival in patients with estrogen receptor (ER)-positive breast cancer. We determined PDCD4 expression levels in AI-resistant breast cancer cell lines and ER-positive breast cancer tumors, investigated the regulation of PDCD4 in AI-resistant breast cancer cell lines, and carried out a Kaplan-Meier survival analysis in two independent cohorts that included a total of 420 patients with ER-positive breast cancer. We found that PDCD4 expression was down-regulated in AI-resistant breast cancer cells, and this down-regulation was inversely correlated with activation of HER2 signaling. Moreover, lower expression of PDCD4 was significantly associated with HER2 positive status in ER-positive breast tumors. Down-regulation of PDCD4 was mediated through up-regulation of HER2 via the mitogen-activated protein kinase (MAPK), protein kinase B (PKB/AKT), and miR-21 in AI-resistant breast cancer cells. MiR-21 inhibitor and the ER down-regulator fulvestrant induced PDCD4 expression and decreased cell proliferation in AI-resistant breast cancer cells. Furthermore, forced overexpression of PDCD4 resensitized AI-resistant cells to AI or hormone deprivation. Finally, we identified that down-regulation of PDCD4 was associated with a lower rate of disease-free survival in patients with ER-positive breast cancer and high histologic grade of breast tumors. In summary, our study shows that expression of PDCD4 is down-regulated by HER2 signaling in AI-resistant breast cancer. Down-regulation of PDCD4 is associated with AI resistance and a poor prognosis in patients with ER-positive breast cancer.

Aim: The study investigated the three symptoms of burnout among hospital nurses and examined the buffering effects of optimism and proactive coping in relation to burnout., Background: Nursing is a profession that can easily lead to burnout. Burnout has been one of the most investigated work outcomes in current research. Previous research has largely ignored the positive influence of individuals on job outcomes and has not tested a constructive framework that might facilitate interventions to prevent burnout., Method: A cross-sectional survey of 314 staff nurses in general hospitals in Taiwan. Participants completed a set of questionnaires with demographic information., Finding: The findings suggested that higher levels of proactive coping behaviours and optimism were associated with lower levels of burnout. Optimism was found to have the strongest relationship with the decreased personal accomplishment of burnout., Conclusion: The findings of this study confirmed the importance of optimism and proactive coping in prevention of symptoms of burnout., Implications for Nursing Management: The results of this study provided important recommendations regarding stress management interventions for health-care managers, nurses, psychologists and human resource staff in the reduction of burnout to promote mental health in an organisation., (© 2013 John Wiley & Sons Ltd.)

Background: Endothelin-1 (ET-1) is elevated and participates in the regulation of several brain inflammatory disorders. The deleterious effects of ET-1 on endothelial cells may aggravate brain inflammation mediated through the upregulation of cyclooxygenase-2 (COX-2) gene expression. However, the signaling mechanisms underlying ET-1-induced COX-2 expression in brain microvascular endothelial cells remain unclear., Objective: The goal of this study was to examine whether ET-1-induced COX-2 expression and prostaglandin E2 (PGE2) release were mediated through a c-Src-dependent transactivation of epidermal growth factor receptor (EGFR) pathway in brain microvascular endothelial cells (bEnd.3 cells)., Methods: The expression of COX-2 induced by ET-1 was evaluated by Western blotting and RT-PCR analysis. The COX-2 regulatory signaling pathways were investigated by pretreatment with pharmacological inhibitors, short hairpin RNA (shRNA) or small interfering RNA (siRNA) transfection, chromatin immunoprecipitation (ChIP), and promoter activity reporter assays. Finally, we determined the PGE2 level as a marker of functional activity of COX-2 expression., Results: First, the data showed that ET-1-induced COX-2 expression was mediated through a c-Src-dependent transactivation of EGFR/PI3K/Akt cascade. Next, we demonstrated that ET-1 stimulated activation (phosphorylation) of c-Src/EGFR/Akt/MAPKs (ERK1/2, p38 MAPK, and JNK1/2) and then activated the c-Jun/activator protein 1 (AP-1) via Gq/i protein-coupled ETB receptors. The activated c-Jun/AP-1 bound to its corresponding binding sites within COX-2 promoter, thereby turning on COX-2 gene transcription. Ultimately, upregulation of COX-2 by ET-1 promoted PGE2 biosynthesis and release in bEnd.3 cells., Conclusions: These results demonstrate that in bEnd.3 cells, c-Src-dependent transactivation of EGFR/PI3K/Akt and MAPKs linking to c-Jun/AP-1 cascade is essential for ET-1-induced COX-2 upregulation. Understanding the mechanisms of COX-2 expression and PGE2 release regulated by ET-1/ETB system on brain microvascular endothelial cells may provide rational therapeutic interventions for brain injury and inflammatory diseases.

Introduction: This study was undertaken to assess whether 3-dimensional computed tomography (CT) can be used to evaluate the relationships between the mandibular muscles and craniofacial morphology in children with different underlying vertical facial patterns., Methods: Twenty children (mean age, 11.9 +/- 1.6 years) underwent cranial CT examination. Three-dimensional CT images were reconstructed for the evaluation of the cross-sectional size, volume, and spatial orientation of the masseter, medial pterygoid, and lateral pterygoid muscles. These muscle factors were also assessed in relation to vertical and transverse craniofacial form., Results: Positive correlations were found between the muscles' cross-sectional area and volume, and between muscle size and transverse facial width. Despite the limited sample size, differences were also found in the orientation of the masseter and medial pterygoid muscles in growing patients with different underlying vertical facial patterns., Conclusions: Three-dimensional CT can be used for the assessment of soft- and hard-tissue dentofacial forms. Clinicians should note the potential differences in muscle cross-sectional area, volume, and orientation in subjects with different underlying vertical facial patterns.

Using a large consortium of undergraduate students in an organized program at the University of California, Los Angeles (UCLA), we have undertaken a functional genomic screen in the Drosophila eye. In addition to the educational value of discovery-based learning, this article presents the first comprehensive genomewide analysis of essential genes involved in eye development. The data reveal the surprising result that the X chromosome has almost twice the frequency of essential genes involved in eye development as that found on the autosomes.

Aims: To determine the potential uses and limitations of 3DCT craniofacial imaging in contemporary clinical practice., Methods: The relevant historical and contemporary literature was reviewed., Results: There would seem to be many potential uses for 3D-CT craniofacial imaging in clinical practice. Significant limitations may, however, relate to the availability and cost of such imaging and the delivered radiation dose., Conclusions: With continued research and development, the use of such 3D-CT craniofacial imaging techniques may become appropriate for future orthodontic and clinical diagnostic applications.

The Rev peptide that binds to nucleophosmin/B23 with the highest affinity exhibited the greatest cytotoxicity on Ras-3T3 cells and inhibited tumor growth most effectively in nude mice. The efficiency of colony formation in soft agar of Ras-3T3 cells was significantly inhibited by treatment with Rev peptide. In addition, Rev peptide could potentiate the doxorubicin-induced decrease of cellular viability in U1 bladder cancer cells and inhibition of tumor growth in nude mice. Treatment of Rev peptide increased protein expression and transcriptional activity of p53 and inhibited the nucleophosmin/B23-mediated PCNA promoter activation. Peptides having high affinity of binding to molecular targets such as nucleophosmin/B23 represent a potentially useful approach to anti-cancer biotherapeutics.

Rana catesbeiana ribonuclease (RC-RNase) is a pyrimidine-guanine sequence-specific ribonuclease found only in R. catesbeiana (bullfrog) oocytes, but not in other organs. The protein is localized in the yolk granules of oocytes but not in other organelles, as detected by immunohistochemistry. More than 99% of RC-RNase was found in the yolk granule pellet when a mild separation method was employed under physiological conditions. The ribonuclease was purified by precipitation of yolk granules, extraction of RC-RNase with 0.09 M NaCl, selective removal of impurities by Hepes buffer, and chromatographies on phosphocellulose and carboxymethyl cellulose columns. Three milligrams of RC-RNase was purified from a 1-g pellet of yolk granules prepared from 2 g of ovary tissue. Therefore, 150 milligrams of RC-RNase could be obtained from a mature female bullfrog (600 g in weight) which had 100 g of ovary tissue. The properties of RC-RNase isolated from yolk granules tested so far are identical to those of RC-RNase isolated from the cytosolic fraction and similar to those of a sialic acid-binding lectin from bullfrog oocytes. To investigate the possible role of RC-RNase in the regulation of cell growth and differentiation during embryogenesis, its cytotoxic activity against various cell lines was examined. The degradation of ribosomal RNA was found in RC-RNase-treated HeLa cells. However, both events were not found in RNase A-treated HeLa cells. Therefore, RC-RNase is proposed to have both ribonucleolytic and cytotoxic activity and a specific receptor on the tumor cell surface is suspected to be involved in the recognition and binding, and possibly entry of RC-RNase.

Microwave fixed liver and kidney tissues were examined by electron microscopy. It was found that the preservation of fine structure of these tissues by this method is equal to that processed by routine methods. No difficulty was encountered in sectioning microwave fixed tissue blocks. It is obvious that microwave fixation is a faster and more efficient method.