Detection of protamine 2 in bovine spermatozoa and testicles.

BACKGROUND: Sperm DNA integrity is crucial for transmission of genetic information to future generations and DNA damage can occur during chromatin packaging. Chromatin packaging involves the replacement of somatic nucleosomal histones by nuclear proteins called protamines. Protamine 1 (PRM1) is transcribed and translated in spermatids of all mammals; however, protamine 2 (PRM2) is transcribed in low levels in spermatids and it is not yet described in bull mature spermatozoa. OBJECTIVES: The aim of this study was to assess gene and protein expression of PRM2 and corroborate gene and protein expression of PRM1 in bull spermatozoa and testis. MATERIALS AND METHODS: For this purpose, absolute q-RT-PCR was performed to calculate the number of copies of PRM1 and PRM2 mRNAs in bovine epididymal spermatozoa and testicular tissue. Western blot and mass spectrometry were performed to identify PRM1 and PRM2 in samples of bovine epididymal spermatozoa. Samples of bovine testicular tissue were collected to identify PRM1 and PRM2 by immunohistochemistry. RESULTS: We evaluated that the number of PRM1 mRNA copies was about hundred times higher than PRM2 mRNA copies in sperm and testicular samples (p < 0.0001). In addition, we estimated the PRM1: PRM2 ratio based on mRNA number of copies. In spermatozoa, the ratio was 1: 0.014, and in testicle, the ratio was 1: 0.009. We also evaluated the immunolocalization for PRM1 and PRM2 in bovine testis, and both proteins were detected in spermatids. Western blot and mass spectrometry in bovine epididymal spermatozoa confirmed these results. CONCLUSION: Our work identifies, for the first time, PRM2 in bovine epididymal spermatozoa and in testis. Further studies are still needed to understand the role of PRM2 on the chromatin of the spermatozoa and to verify how possible changes in PRM2 levels may influence the bull fertility.

Resistance of mice to Leptospira infection and correlation with chemokine response

Leptospirosis is globally widespread neglected disease, affecting most mammalian species. Clinical signs can be confused with other diseases which make the diagnosis and treatment difficult. Chemokines and cytokines are known for their role in the inflammatory and immune response to infections. The profile determination of chemokines' expressions in the course of infection may elucidate the defense mechanisms of the host and support the search for effective treatment strategies. We investigated the mechanisms of innate immunity through the comparison of chemokines induced during infection with L. interrogans in mice with different levels of susceptibility. We used lung and spleen tissues samples of mice from C3H/HeJ, C3H/HePas and Balb/c, respectively sensitive, intermediate susceptibility and resistant to the pathogen. The inoculation of L. interrogans in C3H/HeJ mice led a comparatively smaller change in chemokines expression in both spleen and lung tissues. In samples from spleens and lungs of C3H/HePas and Balb/c the higher increases occurred on CXCL9, CXCL16, CXCL5, CCL8 and CCL5 in Balb/c. Given the same genetic background, the differences in the responses of C3H/HePas compared to C3H/HeJ mice strongly suggest the role of chemokines for the survival of parental strain. Therefore, the greatest increase in CXC chemokines appears to be efficient to induce migration of cells to the secondary lymphoid organs and affected tissues, which is important to control infection. Overall, CXC chemokines are important for the activation and attraction of T cell and may influence the course and control of the infection in resistant Balb/c mice.(AU) i

Resistance of mice to Leptospira infection and correlation with chemokine response.

Leptospirosis is globally widespread neglected disease, affecting most mammalian species. Clinical signs can be confused with other diseases which make the diagnosis and treatment difficult. Chemokines and cytokines are known for their role in the inflammatory and immune response to infections. The profile determination of chemokines' expressions in the course of infection may elucidate the defense mechanisms of the host and support the search for effective treatment strategies. We investigated the mechanisms of innate immunity through the comparison of chemokines induced during infection with L. interrogans in mice with different levels of susceptibility. We used lung and spleen tissues samples of mice from C3H/HeJ, C3H/HePas and Balb/c, respectively sensitive, intermediate susceptibility and resistant to the pathogen. The inoculation of L. interrogans in C3H/HeJ mice led a comparatively smaller change in chemokines expression in both spleen and lung tissues. In samples from spleens and lungs of C3H/HePas and Balb/c the higher increases occurred on CXCL9, CXCL16, CXCL5, CCL8 and CCL5 in Balb/c. Given the same genetic background, the differences in the responses of C3H/HePas compared to C3H/HeJ mice strongly suggest the role of chemokines for the survival of parental strain. Therefore, the greatest increase in CXC chemokines appears to be efficient to induce migration of cells to the secondary lymphoid organs and affected tissues, which is important to control infection. Overall, CXC chemokines are important for the activation and attraction of T cell and may influence the course and control of the infection in resistant Balb/c mice.

Prediction of time to delivery by transperineal ultrasound in second stage of labor.

OBJECTIVE: To investigate whether the transperineal sonographic (TPS) parameters angle of progression (AoP) and midline angle (MLA) can predict the time remaining in the second stage of labor. METHODS: We evaluated prospectively women with a singleton pregnancy in cephalic presentation at term between October 2013 and September 2014. TPS volumes were obtained immediately after confirmation by digital vaginal examination of a fully dilated cervix. AoP and MLA were measured offline by analyzing the ultrasound volumes. Progression of labor was evaluated every hour during the second stage. The associations of AoP and MLA with the interval between TPS assessment and delivery were evaluated using multivariable Cox proportional hazards analyses in nulliparous and parous women separately. RESULTS: A total of 557 women were evaluated. An AoP ≥ 160° (adjusted hazard ratio (aHR), 2.52 (95% CI, 1.98-3.19)) and MLA ≤ 10° (aHR, 1.79 (95% CI, 1.35-2.34)) in nulliparous women and an AoP ≥ 150° (aHR, 1.86 (95% CI, 1.34-2.57)) and MLA ≤ 20° (aHR, 1.69 (95% CI, 1.21-2.34)) in parous women were significantly associated with the remaining time in labor. The positive/negative likelihood ratios of AoP, MLA, clinical station (fetal head descent as observed by digital examination) and clinical rotation (fetal head rotation as observed by digital examination) at these cut-off points were 3.6/0.6, 2.0/0.6, 1.6/0.6 and 1.6/0.8, respectively, in nulliparous women, and 2.4/0.6, 1.3/0.7, 7.6/0.5 and 5.2/0.7, respectively, in parous women. CONCLUSION: TPS assessment of AoP and MLA in the second stage of labor was useful for predicting the time remaining in labor and had higher predictive value than did digital vaginal examination in nulliparous women. Copyright © 2016 ISUOG. Published by John Wiley & Sons Ltd.

Activation of the mitochondrial caspase pathway and subsequent calpain activation in monkey RPE cells cultured under zinc depletion.

PURPOSE: Decreased zinc levels in the macula are reported in patients with age-related macular degeneration, and the zinc chelator N,N,N',N'-tetrakis (2- pyridylmethyl) ethylenediamine) (TPEN) causes death of human retinal pigment epithelial (RPE) cells. The purpose of the present study was to investigate signal transduction pathways during cell death initiated by TPEN, using monkey RPE cells. METHODS: RPE cells were cultured with TPEN. Activation of calpains and caspases, and proteolysis of their substrates were detected by immunoblotting. Incubation of calpain inhibitor SNJ-1945 or caspase inhibitor z-VAD-fmk was used to confirm activation of specific proteases. RESULTS: TPEN caused a time-dependent decrease in viable RPE cells. Cell death was accompanied by activation of calpain-1, caspase-9, and caspase-3. SNJ-1945 inhibited calpain activation and slightly inhibited caspase-9 activation. z-VAD-fmk inhibited caspases and calpain-1 activation. TPEN did not activate caspase-12. CONCLUSIONS: Relative zinc deficiency in RPE cells causes activation of cytosolic calpain and mitochondrial caspase pathways without ER stress.

EGFR regulation by microRNA in lung cancer: correlation with clinical response and survival to gefitinib and EGFR expression in cell lines.

BACKGROUND: Allelic loss in chromosome 3p is one of the most frequent and earliest genetic events in lung carcinogenesis. We investigated if the loss of microRNA-128b, a microRNA located on chromosome 3p and a putative regulator of epidermal growth factor receptor (EGFR), correlated with response to targeted EGFR inhibition. Loss of microRNA-128b would be equivalent to losing a tumor suppressor gene because it would allow increased expression of EGFR. PATIENTS AND METHODS: We initially showed that microRNA-128b is a regulator of EGFR in non-small-cell lung cancer (NSCLC) cell lines. We tested microRNA-128b expression levels by quantitative RT-PCR, genomic copy number by quantitative PCR, and mutations in the mature microRNA-128b by sequencing. We determined whether microRNA-128b loss of heterozygosity (LOH) in 58 NSCLC patient samples correlated with response to gefitinib and evaluated EGFR expression and mutation status. RESULTS: We determined that microRNA-128b directly regulates EGFR. MicroRNA-128b LOH was frequent in tumor samples and correlated significantly with clinical response and survival following gefitinib. EGFR expression and mutation status did not correlate with survival outcome. CONCLUSION: Identifying microRNA regulators of oncogenes could have far-reaching implications for lung cancer patients including improving patient selection for targeted agents, development of novel therapeutics, or development as early biomarkers of disease.

Amelioration of retinal degeneration and proteolysis in acute ocular hypertensive rats by calpain inhibitor ((1S)-1-((((1S)-1-benzyl-3-cyclopropylamino-2,3-di-oxopropyl)amino)carbonyl)-3-methylbutyl)carbamic acid 5-methoxy-3-oxapentyl ester.

BACKGROUND: Our recent study suggested involvement of calpain-induced proteolysis in retinal degeneration and dysfunction in acute ocular hypertensive rats. The purpose of the present study was to determine if an orally available form of calpain inhibitor, ((1S)-1-((((1S)-1-benzyl-3-cyclopropylamino-2,3-di-oxopropyl)amino)carbonyl)-3-methylbutyl)carbamic acid 5-methoxy-3-oxapentyl ester (SNJ-1945), ameliorated retinal degeneration induced by acute hypertension in rats. To help extrapolate the effect of SNJ-1945 from the rat model to the human glaucomatous patient, in vitro inhibition of calpain-induced proteolysis by SNJ-1945 in monkey and human retinal proteins was compared with proteolysis in rat proteins. METHODS: Intraocular pressure (IOP) in rats was elevated to 110 mm Hg for 50 min. SNJ-1945 was administrated i.p. or orally before ocular hypertension. Retinal degeneration was evaluated by hematoxylin and eosin (H&E) staining and cell counting. Transcripts for calpains and calpastatin in rat, monkey, and human retinas were measured by quantitative RT-PCR. Calpain activities were determined by casein zymography. Soluble retinal proteins from rat, monkey, and humans were incubated with calcium to activate calpains, with or without SNJ-1945. Proteolysis of calpain substrate alpha-spectrin was analyzed by immunoblotting. RESULTS: Elevated IOP caused retinal degeneration and proteolysis of alpha-spectrin. Both i.p. and oral administration of SNJ-1945 inhibited proteolysis of alpha-spectrin and ameliorated retinal degeneration. Transcript levels for calpain 1 and calpastatin were similar in rat, monkey, and human retinas. Calpain 2 transcript levels were higher in rats compared with monkey and human. Appreciable caseinolytic activities due to calpains were observed in monkey and human retinas. Incubation of retinal soluble proteins with calcium led to proteolysis of alpha-spectrin due to calpains in rat, monkey, and human samples. SNJ-1945 similarly inhibited proteolysis in all species. CONCLUSION: Our results suggested that orally available calpain inhibitor SNJ-1945 might be a possible candidate drug for testing in preventing progression of glaucomatous retinal degeneration.

Low activity by the calpain system in primate lenses causes resistance to calcium-induced proteolysis.

The human genome contains 14 genes for 80 kDa catalytic subunit of the calcium-activated protease calpain (EC 34.22.17), yet no calpain-like cleavage sites have been detected on human lens crystallins in vivo. The purpose of the present study was to provide a comprehensive study of calpain activation in human and macaque lenses developing experimental cataract due to lens culture in ionophore A23187. Zymography was used to measure calpain activity; SDS-PAGE and immunoblotting were used to detect hydrolysis of potential lens protein substrates. Quantitative PCR was used to measure transcripts for calpains and the endogenous inhibitor calpastatin. We found that the lack of appreciable calpain-induced proteolysis in primate lenses is most likely due to relatively low levels of endogenous calpain activity compared to the high levels of endogenous calpain inhibitor, calpastatin.

Biochemical properties of lens-specific calpain Lp85.

Lens-specific Lp82 and ubiquitous m-calpain are neutral, calcium-activated, cysteine proteases. Both calpains are activated during rodent lens maturation and cataract formation. Lp85 calpain (Lens protein with MW=85 kDa) is a slightly larger splice variant of Lp82. Lp85 contains a 28 amino acid insert peptide (IS3) in calcium binding domain IV. Theoretically, the insert could alter the properties of Lp85 and influence proteolytic activity. The purpose of the present experiment was to compare the biochemical properties of Lp85 to Lp82 and m-calpain. Recombinant Lp85 and Lp82 were separately expressed using the baculovirus system and partially purified using Co2+ affinity and DEAE chromatographies. Calcium activation, pH dependency, and susceptibility to calpain inhibitors were assessed in a protease assay using BODIPY fluorescence-labeled casein substrate. Hydrolysis of lens proteins was assessed by SDS-PAGE and immunoblotting. Cleavage site analysis was performed by mass spectroscopy and Edman sequencing. Computer-based homology modeling was used to predict the influence of the IS3 region on the 3-dimensional structure of Lp85. Compared to m-calpain, Lp85 showed a lower calcium-activation requirement (K(50%act)=20 microM), marked insensitivity to, and cleavage of, the endogenous tissue inhibitor of calpains-calpastatin, and different preferred cleavage sites on alphaA-crystallin (five amino acid C-terminal truncation) and on aquaporin 0 (G239 and N246). Although the IS3 insert was predicted to form a loop protruding from the calcium binding region of Lp85, the biochemical properties of Lp85 studied were nearly identical to those of Lp82. Lp85 and Lp82 did not catalyze hydrolysis of each other, but both hydrolyzed m-calpain. Lp85 seems to be the enzymatic equivalent of Lp82. Both calpains could become active at lower cellular calcium levels than m-calpain. Lp85/Lp82 may have different functions than m-calpain since they cleave substrates at different sites. Lp85/Lp82 may regulate m-calpain activity by catalyzing the hydrolysis of calpastatin. The function of the IS3 insert on Lp85 remains unknown but is speculated to control subcellular distribution.

Expression of calpain small subunit 2 in mammalian tissues.

PURPOSE: The purpose of the current experiments was to more closely define the distribution and the function of calpain small subunit 2 (css2). Css2 is a newly discovered regulatory protein for the calcium activated proteases, mu- and m-calpains. METHODS: Tissues from rat, monkey, and man of various ages were used to determine expression patterns of css2 by relative quantitative RT-PCR using 18S rRNA as an endogenous standard. Recombinant css2 and the 80 kDa catalytic subunit of m-calpain (80 kDa/css2) were co-expressed in Escherichia coli. Casein zymography was used to measure the enzymatic activity of 80 kDa/css2 proteins. Lens alpha-crystallin and beta B1-crystallin were used as substrates to determine proteolysis by 80 kDa/css2. Computer-based homology modeling was used to predict interactions between the traditional small subunit (css1) or css2 with the 80 kDa catalytic subunit. RESULTS: Css2 appears to be a functional equivalent of css1 in vitro in that the calcium-dependent proteolytic activity of 80 kDa/css2 was similar to recombinant m-calpain (80 kDa/css1). In rat and human lens, css2 transcripts increased with age, whereas css1 transcripts decreased with age. Human beta B1-crystallin and rat alpha A-crystallin were cleaved similarly by 80 kDa/css2 and 80 kDa/css1. Interestingly, alpha A-insert crystallin was not hydrolyzed when css2 was substituted for css1 in the calpain dimer, suggesting that css2 may perform different functions from css1 in terms of proteolysis of lens crystallins during maturational growth of the lens. Css2 may also assist in the proper folding of the 80 kDa subunit and regulate protease activity in the absence of calcium. CONCLUSIONS: The wide distribution of css2 transcripts in rat and monkey suggested that css2 is a second, widely distributed (rather than tissue-specific) calpain small subunit, in addition to the long-recognized css1. Further studies at the protein level will indicate if css2 has unique functions apart from css1.

Three types of polymorphisms in exon 14 in porcine Mx1 gene.

Much is known about the antiviral activity of Mx proteins in species such as mouse and human. In the mouse, loss of resistability to influenza virus has been shown to be due to specific polymorphisms in the Mx gene. This gene is therefore an interesting candidate gene for disease resistance in farm animals. The porcine Mx1 gene has already been identified and characterized based on its homology with mouse Mx1; however, until now no evidence of polymorphisms in the porcine gene has been reported. In this study, we have found two new polymorphisms in exon 14 of porcine Mx1 by DNA sequencing and confirmed their presence in different breeds, using polymerase chain reaction (PCR)-restriction fragment length polymorphisms (RFLP) with NarI and NaeI restriction enzymes. On the basis of the deduced amino acid sequence, one allele contains a deletion that may result in a frameshift to yield several amino acid substitutions and extension of the carboxyl terminal region of Mx1 protein. The deletion allele, Mx1c, was found to be segregating in Landrace, Berkshire, Duroc, Hampshire, and Yucatan miniature pig. A second point mutation, Mx1b, was detected in Meishan and two Vietnamese native pig breeds. All other breeds tested were fixed for the Mx1a allele that is identical to the sequence reported previously. It will be interesting to determine if the Mx1c deletion is associated with variation in resistance to the myxovirus family in the pig.

Establishment of enzyme immunoassay for measuring serum sultopride levels.

Measurement of serum sultopride levels was performed using an enzyme immunoassay. Little or no cross-reactivity with metabolites of sultopride and other drugs was found. The results of reproducibility, recovery, and dilution testing were all good enough for clinical use. A comparison between the measurement values of this method (y) with that of high-performance liquid chromatography (x) showed high correlation (n = 211, r = 0.991, p < 0.0001, y = 0.99x + 107.5). In a comparison between the sultopride dose and serum levels in 161 patients, interindividual differences were large (19 times for same doses), implying that the serum level cannot be predicted from the dosage. The method was found to be reliable for serum level measurements of sultopride and useful for monitoring compliance and assessing the optimal dose.

Radical cyclization in heterocycle synthesis. 12. Sulfanyl radical addition-addition-cyclization (SRAAC) of unbranched diynes and its application to the synthesis of A-ring fragment of 1alpha,25-dihydroxyvitamin D3.

Sulfanyl radical addition-addition-cyclization (SRAAC) of unbranched diynes proceeded smoothly to give cyclized exo-olefins, while the sulfanyl radical addition-cyclization-addition (SRACA) of diynes having a quaternary carbon gave cyclized endo-olefins. This method was successfully applied to the synthesis of A-ring fragment of 1alpha,25-dihydroxyvitamin D3.

K-ras mutation in sputum of primary lung cancer patients does not always reflect that of cancerous cells.

K-ras mutation in sputum was examined using mutant-allele-specific amplification method among 100 primary lung cancer and 15 non-oncological patients. K-ras mutation was detected in 11 out of 59 adenocarcinoma cases (18.6%), 5 out of 32 squamous cell carcinoma cases (15.6%), 2 out of 4 large cell carcinoma cases (50.0%) and 3 out of 15 non-oncological disease cases (20.0%). In the 18 cases of primary lung cancer K-ras mutation was examined in both sputum and the resected specimen of the primary lesion. In 5 cases K-ras mutation in sputum was detected without K-ras mutation in primary lesion. Therefore, these findings suggested that K-ras mutation in sputum may not be directly related to that of the primary lesion.

Specific inhibition of HIV-1 gene expression by double-stranded RNA.

RNA interference (RNAi) is phenomenon in which introduced double-stranded RNAs (ds-RNAs) silence gene expression through specific degradation of their cognate mRNAs. RNAi has been observed in a wide variety of organisms and is considered to be a feature of nearly all eukaryotes. The mediators of sequence specific messenger RNA degradation are 21-23 nucleotide small interfering RNAs generated by ribonucleaseIII cleavage from longer dsRNAs. To investigate the potential of dsRNA to interfere with the function of HIV-1 genes, we have designed six longer dsRNAs containing the HIV-1 gag and env genes. Double-stranded RNAs were tested for inhibitory effects using monkey COS cells. In these anti-HIV-1 activity tests, the dsRNAs targeted to the HIV-1 env sequence showed more than 90% anti-HIV-1 efficiency. Our results show that dsRNA interference can be used as a powerful method for the inhibition of HIV-1 gene expression.

Association between single nucleotide polymorphisms in the hMSH3 gene and sporadic colon cancer with microsatellite instability.

The association between three single nucleotide polymorphisms (SNPs) in the hMSH3 gene and sporadic colon cancer with microsatellite instability (MSI) was analyzed. Of the three SNPs observed in this population, SNPs at residues 235 and 693 were novel, while that at residue 3133 was previously described. The SNPs at residues 235 and 3133 caused amino acid substitutions, V79I and T1045A, respectively. We analyzed the allele frequencies of the three SNPs in samples from 19 patients with sporadic colon cancer with MSI and 90 healthy controls. We found that the V79 allele frequency was significantly higher in the tumor samples than in controls. In addition, the frequency of the G693 allele showed a higher trend in the tumor samples than in controls. These results indicated that some SNPs in the hMSH3 gene were associated with colon cancer with MSI.