The structure of model and peptide disulfides markedly affects their reactivity and products formed with singlet oxygen.

Disulfide bonds are critical structural elements in proteins and stabilize folded structures. Modification of these linkages is associated with a loss of structure and function. Previous studies have reported large variations in the rate of disulfide oxidation by hypohalous acids, due to stabilization of reaction intermediates. In this study we hypothesized that considerable variation (and hence selective oxidation) would occur with singlet oxygen (1O2), a key intermediate in photo-oxidation reactions. The kinetics of disulfide-mediated 1O2 removal were monitored using the time-resolved 1270 nm phosphorescence of 1O2. Stern-Volmer plots of these data showed a large variation (∼103) in the quenching rate constants kq (from 2 × 107 for α-lipoic acid to 3.6 × 104 M-1s-1 for cystamine). The time course of disulfide loss and product formation (determined by LC-MS) support a role for 1O2, with mono- and di-oxygenated products detected. Elevated levels of these latter species were generated in D2O- compared to H2O buffers, which is consistent with solvent effects on the 1O2 lifetime. These data are interpreted in terms of the intermediacy of a zwitterion [-S+(OO-)-S-], which either isomerizes to a thiosulfonate [-S(O)2-S-] or reacts with another parent molecule to give two thiosulfinates [-S(O)-S-]. The variation in quenching rates and product formation are ascribed to zwitterion stabilization by neighboring, or remote, lone pairs of electrons. These data suggest that some disulfides, including some present within or attached to proteins (e.g., α-lipoic acid), may be selectively modified, and undergo subsequent cleavage, with adverse effects on protein structure and function.

Effect of Glycan Shift on Antibodies against Hepatitis C Virus E2 412-425 Epitope Elicited by Chimeric sHBsAg-Based Virus-Like Particles.

Two of the most important mechanisms of hepatitis C virus (HCV) immune evasion are the high variability of the amino acid sequence and epitope shielding via heavy glycosylation of the envelope (E) proteins. Previously, we showed that chimeric sHBsAg (hepatitis B virus [HBV] small surface antigen)-based virus-like particles (VLPs) carrying highly conserved epitope I from the HCV E2 glycoprotein (sHBsAg_412-425) elicit broadly neutralizing antibodies (bnAbs). However, many reports have identified escape mutations for such bnAbs that shift the N-glycosylation site from N417 to N415. This shift effectively masks the recognition of epitope I by antibodies raised against the wild-type glycoprotein. To investigate if glycan-shift-mediated immune evasion could be overcome by targeted vaccination strategies, we designed sHBsAg-based VLPs carrying epitope I with an N417S change (sHBsAg_N417S). Studies in BALB/c mice revealed that both sHBsAg_412-425 and sHBsAg_N417S VLPs were immunogenic, eliciting antibodies that recognized peptides encompassing epitope I regardless of the N417S change. However, we observed substantial differences in E1E2 glycoprotein binding and cell culture-derived HCV (HCVcc) neutralization between the sera elicited by sHBsAg_412-425 and those elicited by sHBsAg_N417S VLPs. Our results suggest a complex interplay among antibodies targeting epitope I, the E1E2 glycosylation status, and the epitope or global E1E2 conformation. Additionally, we observed striking similarities in the E1E2 glycoprotein binding patterns and HCVcc neutralization between sHBsAg_412-425 sera and AP33, suggesting that the immunization of mice with sHBsAg_412-425 VLPs can elicit AP33-like antibodies. This study emphasizes the role of antibodies against epitope I and represents an initial effort toward designing an antigen that elicits an immune response against epitope I with a glycan shift change. IMPORTANCE Epitope I, located within amino acids 412 to 423 of the HCV E2 glycoprotein, is an important target for an epitope-based HCV vaccine. One interesting feature of epitope I is the N417 glycosylation site, where a single change to S417 or T417 can shift the glycosylation site to position N415. This shift can effectively prevent the binding of broadly neutralizing antibodies targeting epitope I. Aiming to overcome glycan-shift-mediated immune evasion, we constructed sHBsAg_N417S VLPs carrying E2 epitope I, with N417S, and compared them with VLPs carrying wild-type epitope I. We show that antibodies elicited by the sHBsAg-based VLPs presenting two variants of the 412-425 epitope targeted two distinct glycan variants of the HCV E1E2 heterodimer. Our study suggests that due to the conformational flexibility of the E2 glycoprotein and epitope I, future vaccine antigens should elicit antibodies targeting more than one conformation and glycosylation variant of the 412-423 epitope.

Implementation of the Patient Counselling Service at the Cancer Hospital in Radom, Poland.

Background: Non-adherence occurs in various groups of patients, including those with chronic diseases. One strategy to increase adherence among oncological patients is to individualise treatment and expand pharmaceutical care. Pharmaceutical labels that remind patients how they should take their medications are of great importance in this respect. Objective: The main objective of this study was to evaluate medication adherence in oncological patients, and to gather their opinions on the individual medication labelling system as an element of effective treatment. Methods: The study was conducted in 2021 among 82 patients of the oncological department of the Centre of Oncology in Radom. The research tool was a questionnaire consisting of personal data and two parts relating to the patient's disease and the medication labelling system. Results: Nearly half of the respondents reported that they forget to take medications and how they should take them. These problems increased with the age of the patient and the number of administered medications. Of the respondents, 89% stated that the labels with dosing information are helpful. Over 67% agreed that these labels should be affixed to all medications. Nearly 90% of the respondents believed the labels should be available in all pharmacies. Conclusions: Non-adherence is a common phenomenon among oncological patients. Pharmacists providing a labelling service for medicinal products can play a significant role in reducing this phenomenon.

Novel Short PEG Chain-Substituted Porphyrins: Synthesis, Photochemistry, and In Vitro Photodynamic Activity against Cancer Cells.

This work presents the synthesis and characterization of metal-free, zinc (II), and cobalt (II) porphyrins substituted with short PEG chains. The synthesized compounds were characterized by UV-Vis, 1H and 13C NMR spectroscopy, and MALDI-TOF mass spectrometry. The origin of the absorption bands for tested compounds in the UV-Vis range was determined using a computational model based on the electron density functional theory (DFT) and its time-dependent variant (TD-DFT). The photosensitizing activity was evaluated by measuring the ability to generate singlet oxygen (ΦΔ), which reached values up to 0.54. The photodynamic activity was tested using bladder (5637), prostate (LNCaP), and melanoma (A375) cancer cell lines. In vitro experiments clearly showed the structure-activity relationship regarding types of substituents, their positions in the phenyl ring, and the variety of central metal ions on the porphyrin core. Notably, the metal-free derivative 3 and its zinc derivative 6 exerted strong cytotoxic activity toward 5637 cells, with IC50 values of 8 and 15 nM, respectively. None of the tested compounds induced a cytotoxic effect without irradiation. In conclusion, these results highlight the potential value of the tested compounds for PDT application.

Sensitized photoreduction of selected benzophenones. Mass spectrometry studies of radical cross-coupling reactions.

The hydrogen atom transfer reaction (HAT) between selected benzophenones (benzophenone BP, 3-carboxybenzophenone 3CB, and 4-carboxybenzophenone 4CB) and 2-propanol was reinvestigated focusing on stable product analysis. As expected, the primary species of these HAT's are the respective diphenyl and dimethyl ketyl radicals that eventually undergo several radical coupling reactions leading to stable photoproducts. However, the mechanisms of these free radical reactions remain unclear and open to question. In this report, we focus on the detailed analysis of the stable photoproducts of these reactions using liquid chromatography coupled with high-resolution mass spectrometry (LC-ESI-QTOF-MS/MS). Products of photopinacolization (benzpinacol and two diastereoisomers of 4CB and 3CB dimers) and isomeric radical cross-coupling adducts of respective diphenyl and dimethyl ketyl radicals were separated chromatographically, and their structures were determined by high-resolution MS/MS, and the mechanisms of the reactions are discussed.

Immune response against SARS-CoV-2 variants: the role of neutralization assays.

Since the emergence of the novel coronavirus SARS-CoV-2 in late 2019, the COVID-19 pandemic has hindered social life and global economic activity. As of July 2021, SARS-CoV-2 has caused over four million deaths. The rapid spread and high mortality of the disease demanded the international scientific community to develop effective vaccines in a matter of months. However, unease about vaccine efficacy has arisen with the spread of the SARS-CoV-2 variants of concern (VOCs). Time- and cost-efficient in vitro neutralization assays are widely used to measure neutralizing antibody responses against VOCs. However, the extent to which in vitro neutralization reflects protection from infection remains unclear. Here, we describe common neutralization assays based on infectious and pseudotyped viruses and evaluate their role in testing neutralizing responses against new SARS-CoV-2 variants. Additionally, we briefly review the recent findings on the immune response elicited by available vaccines against major SARS-CoV-2 variants, including Alpha, Beta, Gamma, and Delta.

Immunization with Leishmania tarentolae-derived norovirus virus-like particles elicits high humoral response and stimulates the production of neutralizing antibodies.

BACKGROUND: Noroviruses are a major cause of epidemic and sporadic acute non-bacterial gastroenteritis worldwide. Unfortunately, the development of an effective norovirus vaccine has proven difficult and no prophylactic vaccine is currently available. Further research on norovirus vaccine development should be considered an absolute priority and novel vaccine candidates are needed. One of the recent approaches in safe vaccine development is the use of virus-like particles (VLPs). VLP-based vaccines show great immunogenic potential as they mimic the morphology and structure of viral particles without the presence of the virus genome. RESULTS: This study is the first report showing successful production of norovirus VLPs in the protozoan Leishmania tarentolae (L. tarentolae) expression system. Protozoan derived vaccine candidate is highly immunogenic and able to not only induce a strong immune response (antibody titer reached 104) but also stimulate the production of neutralizing antibodies confirmed by receptor blocking assay. Antibody titers able to reduce VLP binding to the receptor by > 50% (BT50) were observed for 1:5-1:320 serum dilutions. CONCLUSIONS: Norovirus VLPs produced in L. tarentolae could be relevant for the development of the norovirus vaccine.

Radiation- and Photo-Induced Oxidation Pathways of Methionine in Model Peptide Backbone under Anoxic Conditions.

Within the reactive oxygen species (ROS) generated by cellular metabolisms, hydroxyl radicals (HO•) play an important role, being the most aggressive towards biomolecules. The reactions of HO• with methionine residues (Met) in peptides and proteins have been intensively studied, but some fundamental aspects remain unsolved. In the present study we examined the biomimetic model made of Ac-Met-OMe, as the simplest model peptide backbone, and of HO• generated by ionizing radiation in aqueous solutions under anoxic conditions. We performed the identification and quantification of transient species by pulse radiolysis and of final products by LC-MS and high-resolution MS/MS after γ-radiolysis. By parallel photochemical experiments, using 3-carboxybenzophenone (CB) triplet with the model peptide, we compared the outcomes in terms of short-lived intermediates and stable product identification. The result is a detailed mechanistic scheme of Met oxidation by HO•, and by CB triplets allowed for assigning transient species to the pathways of products formation.

Early Events of Photosensitized Oxidation of Sulfur-Containing Amino Acids Studied by Laser Flash Photolysis and Mass Spectrometry.

The mechanism of photooxidation of methionine (N-Ac-Met-NH-CH3, 1) and methyl-cysteine (N-Ac-MeCys-NH-CH3, 2) analogues by 3-carboxybenzophenone triplet (3CB*) in neutral aqueous solution was studied using techniques of nanosecond laser flash photolysis and steady-state photolysis. The short-lived transients derived from 3CB and sulfur-containing amino acids were identified, and their quantum yields and kinetics of formation and decay were determined. The stable photoproducts were analyzed using liquid chromatography coupled with high-resolution mass spectrometry. Substantial differences in the mechanisms were found for methionine and S-methyl-cysteine analogues for both primary and secondary photoreactions. A new secondary reaction channel (back hydrogen atom transfer from the ketyl radical to the carbon-centered α-thioalkyl radical yielding reactants in the ground states) was suggested. The detailed mechanisms of 3CB* sensitized photooxidation of 1 and 2 are proposed and discussed.

Specific Antibodies Induced by Immunization with Hepatitis B Virus-Like Particles Carrying Hepatitis C Virus Envelope Glycoprotein 2 Epitopes Show Differential Neutralization Efficiency.

Hepatitis C virus (HCV) infection with associated chronic liver diseases is a major health problem worldwide. Here, we designed hepatitis B virus (HBV) small surface antigen (sHBsAg) virus-like particles (VLPs) presenting different epitopes derived from the HCV E2 glycoprotein (residues 412-425, 434-446, 502-520, and 523-535 of isolate H77C). Epitopes were selected based on their amino acid sequence conservation and were previously reported as targets of HCV neutralizing antibodies. Chimeric VLPs obtained in the Leishmania tarentolae expression system, in combination with the adjuvant Addavax, were used to immunize mice. Although all VLPs induced strong humoral responses, only antibodies directed against HCV 412-425 and 523-535 epitopes were able to react with the native E1E2 glycoprotein complexes of different HCV genotypes in ELISA. Neutralization assays against genotype 1-6 cell culture infectious HCV (HCVcc), revealed that only VLPs carrying the 412-425 epitope induced efficient HCV cross-neutralizing antibodies, but with isolate specific variations in efficacy that could not necessarily be explained by differences in epitope sequences. In contrast, antibodies targeting 434-446, 502-520, and 523-535 epitopes were not neutralizing HCVcc, highlighting the importance of conformational antibodies for efficient virus neutralization. Thus, 412-425 remains the most promising linear E2 epitope for further bivalent, rationally designed vaccine research.

Immunogenicity and functional characterization of Leishmania-derived hepatitis C virus envelope glycoprotein complex.

Hepatitis C virus (HCV) envelope glycoproteins E1 and E2 are the main inducers of a cross-neutralizing antibody response which plays an important role in the early phase of viral infection. Correctly folded and immunologically active E1E2 complex can be expressed in mammalian cells, though the production process might still prove restrictive, even if the immunological response of a vaccine candidate is positive. Here, we report a characterization and immunogenicity study of a full-length (fE1E2) and soluble version of the E1E2 complex (tE1E2) from genotype 1a, successfully expressed in the cells of Leishmania tarentolae. In a functional study, we confirmed the binding of both Leishmania-derived E1E2 complexes to the CD-81 receptor and the presence of the major epitopes participating in a neutralizing antibody response. Both complexes were proved to be highly immunogenic in mice and elicited neutralizing antibody response. Moreover, cross-reactivity of the mouse sera was detected for all tested HCV genotypes with the highest signal intensity observed for genotypes 1a, 1b, 5 and 6. Since the development of a prophylactic vaccine against HCV is still needed to control the global infection, our Leishmania-derived E1E2 glycoproteins could be considered a potential cost-effective vaccine candidate.

Immunogenicity of Leishmania-derived hepatitis B small surface antigen particles exposing highly conserved E2 epitope of hepatitis C virus.

BACKGROUND: Hepatitis C virus (HCV) infection is a major health problem worldwide, affecting an estimated 2-3 % of human population. An HCV vaccine, however, remains unavailable. High viral diversity poses a challenge in developing a vaccine capable of eliciting a broad neutralizing antibody response against all HCV genotypes. The small surface antigen (sHBsAg) of hepatitis B virus (HBV) has the ability to form highly immunogenic subviral particles which are currently used as an efficient anti-HBV vaccine. It also represents an attractive antigen carrier for the delivery of foreign sequences. In the present study, we propose a bivalent vaccine candidate based on novel chimeric particles in which highly conserved epitope of HCV E2 glycoprotein (residues 412-425) was inserted into the hydrophilic loop of sHBsAg. RESULTS: The expression of chimeric protein was performed in an unconventional, Leishmania tarentolae expression system resulting in an assembly of particles which retained immunogenicity of both HCV epitope and sHBsAg protein. Direct transmission electron microscopy observation and immunogold staining confirmed the formation of spherical particles approximately 22 nm in diameter, and proper foreign epitope exposition. Furthermore, the sera of mice immunized with chimeric particles proved reactive not only to purified yeast-derived sHBsAg proteins but also HCV E2 412-425 synthetic peptide. Most importantly, they were also able to cross-react with E1E2 complexes from different HCV genotypes. CONCLUSIONS: For the first time, we confirmed successful assembly of chimeric sHBsAg virus-like particles (VLPs) in the L. tarentolae expression system which has the potential to produce high-yields of properly N-glycosylated mammalian proteins. We also proved that chimeric Leishmania-derived VLPs are highly immunogenic and able to elicit cross-reactive antibody response against HCV. This approach may prove useful in the development of a bivalent prophylactic vaccine against HBV and HCV and opens up a new and low-cost opportunity for the production of chimeric sHBsAg VLPs requiring N-glycosylation process for their proper functionality and immunogenicity.

Purification and stability of octameric mitochondrial creatine kinase isoform from herring (Clupea harengus) organ of vision.

Creatine kinases (CKs) constitute a large family of isoenzymes that are involved in intracellular energy homeostasis. In cells with high and fluctuating energy requirements ATP level is maintained via phosphocreatine hydrolysis catalyzed by creatine kinase. In contrast to invertebrates and higher vertebrates, in poikilothermic vertebrates the adaptations for the regulation of energy metabolism by changes in the oligomeric state of CK isoforms are not well known. The present study aimed at identification of herring eye CK isoforms and focuses on factors affecting the CK-octamer stability. In addition to the CK octamer, three different dimeric isoforms of CK were detected by cellulose acetate native electrophoresis. Destabilization of octamer was studied in the presence of TSAC substrates and about 50% of octamers dissociated into dimers within 24h. Moreover, we found that the increase of temperature from 4 °C to 30 °C caused rapid inactivation of dimers in TSAC-treated samples but did not affect octameric structures. In a thermostability assay we demonstrated that octamers retain their activity even at 50 °C. Our results indicate that destabilization of the octameric structure can lead to loss of enzyme activity at higher temperatures (above 30 °C). Furthermore, our results based on N-terminal sequence analysis suggest that probably the mitochondrial s-type CK, rather than u-type, is predominantly expressed in herring eye. In conclusion the existence of four various CK isoforms in one organ may reflect complex regulation of energy metabolism in the phototransduction process in teleost fishes.

Novel human SR-BI antibodies prevent infection and dissemination of HCV in vitro and in humanized mice.

BACKGROUND & AIMS: Hepatitis C virus (HCV)-induced end-stage liver disease is currently the major indication for liver transplantation in the Western world. After transplantation, the donor liver almost inevitably becomes infected by the circulating virus and disease progression is accelerated in immune suppressed transplant patients. The current standard therapy, based on pegylated interferon and ribavirin, induces severe side effects and is often ineffective in this population. Therefore, new strategies to prevent graft re-infection are urgently needed. We have previously shown that monoclonal antibodies (mAbs) against the HCV co-receptor scavenger receptor class B type I (SR-BI/Cla1) inhibit infection by different HCV genotypes in cell culture. METHODS: Using phage display libraries, we have generated a large set of novel human mAbs against SR-BI and evaluated their effectiveness in preventing HCV infection and direct cell-to-cell spread in vitro and in vivo using uPA-SCID mice with a humanized liver. RESULTS: Eleven human monoclonal antibodies were generated that specifically recognize SR-BI. Two antibodies, mAb8 and mAb151, displayed the highest binding and inhibitory properties and also interfered with direct cell-to-cell spread in vitro. Studies in humanized mice showed that both antibodies were capable of preventing HCV infection and could block intrahepatic spread and virus amplification when administered 3 days after infection. Interestingly, anti-SR-BI therapy was effective against an HCV variant that escaped the control of the adaptive immune response in a liver transplant patient. CONCLUSIONS: The anti-SR-BI mAbs generated in this study may represent novel therapeutic tools to prevent HCV re-infection of liver allografts.

A human monoclonal antibody targeting scavenger receptor class B type I precludes hepatitis C virus infection and viral spread in vitro and in vivo.

UNLABELLED: Endstage liver disease caused by chronic hepatitis C virus (HCV) infection is the leading indication for liver transplantation in the Western world. However, immediate reinfection of the grafted donor liver by circulating virus is inevitable and liver disease progresses much faster than the original disease. Standard antiviral therapy is not well tolerated and usually ineffective in liver transplant patients, whereas anti-HCV immunotherapy is hampered by the extreme genetic diversity of the virus and its ability to spread by way of cell-cell contacts. We generated a human monoclonal antibody against scavenger receptor class B type I (SR-BI), monoclonal antibody (mAb)16-71, which can efficiently prevent infection of Huh-7.5 hepatoma cells and primary hepatocytes by cell-culture-derived HCV (HCVcc). Using an Huh7.5 coculture system we demonstrated that mAb16-71 interferes with direct cell-to-cell transmission of HCV. Finally we evaluated the in vivo efficacy of mAb16-71 in "human liver urokinase-type plasminogen activator, severe combined immune deficiency (uPA-SCID) mice" (chimeric mice). A 2-week anti-SR-BI therapy that was initiated 1 day before viral inoculation completely protected all chimeric mice from infection with serum-derived HCV of different genotypes. Moreover, a 9-day postexposure therapy that was initiated 3 days after viral inoculation (when viremia was already observed in the animals) suppressed the rapid viral spread observed in untreated control animals. After cessation of anti-SR-BI-specific antibody therapy, a rise of the viral load was observed. CONCLUSION: Using in vitro cell culture and human liver-chimeric mouse models, we show that a human mAb targeting the HCV coreceptor SR-BI completely prevents infection and intrahepatic spread of multiple HCV genotypes. This strategy may be an efficacious way to prevent infection of allografts following liver transplantation in chronic HCV patients, and may even hold promise for the prevention of virus rebound during or following antiviral therapy.

[Creatine kinase isoenzymes--characterization and functions in cell]. / Izoenzymy kinazy kreatynowej--charakterystyka i funkcje w komórce.

Creatine kinase (CK, EC 2.7.3.2) is a key enzyme of cellular bioenergetics. The tissue-specific distribution, subcellular localization and function of CK isoenzymes in tissues and cells with high energy requirements, as well as the molecular structure of mitochondrial CK, point to an important physiological role of CK system for cellular energetics. The extensive studies about properties of mitochondrial creatine kinase isoforms gave a new perspective to create a functional model of CK isoforms action called "phosphocreatine shuttle". In this model the CK isoforms together with easily diffusible compounds like creatine and phosphocreatine, maintain a cellular energy buffer and intracellular energy transport system, where the "high-energy" phosphate is transferred between site of ATP synthesis and site of ATP utilization. Mt-CK octamer is able to bridge two mitochondrial membranes and interacts functionally with porin VDAC and ANT in cardiolipin vicinity. Mt-CK with its substrates also activates oxidative phosphorylation and effectively inhibits formation of mitochondrial permeability transition pore. Any destabilization of octamer structure would induce an apoptosis process.

Purification and some properties of two creatine kinase isoforms from herring (Clupea harengus) spermatozoa.

Creatine kinase (CK, EC 2.7.3.2) isoforms play important role in energy homeostasis and together with easily diffusible compounds like creatine and phosphocreatine maintain a cellular energy buffer and intracellular energy transport system. The CK activity in spermatozoa is the highest from all studied tissues in herring. It was detected that the two CK isoforms, CK1 and CK2, are characteristic only for spermatozoa and are not expressed in other herring tissues. Isolation and purification procedures allowed obtaining purified enzymes with specific activity of the 345 micromol/min/mg for CK1 and 511 micromol/min/mg for CK2. Native Mr's of the CK1 and CK2 determined by gel permeation chromatography were about 330,000 and 90,000, respectively. These results indicate that CK1 form has octameric structure and CK2 is a dimer mostly characteristic for cytosolic CK enzymes. In immunoblotting studies with antisera against CK2, the response was observed for CK2 and there was no response for CK1 and two other isoforms from herring skeletal muscle. These findings make the herring isoforms an interesting model for studies on the fish CK biochemical properties.

Characterization of creatine kinase isoforms in herring (Clupea harengus) skeletal muscle.

It is known that mitochondrial creatine kinase (MtCK) in mammals is always expressed in conjunction with one of the cytosolic forms of creatine kinase (CK), either muscle-type (MM-CK) or brain-type (BB-CK) in tissues of high, sudden energy demand. The two creatine kinase (CK) isoforms were detected in herring (Clupea harengus) skeletal muscle: cytosolic CK and mitochondrial CK (MtCK) that displayed the different electrophoretic mobility. These isoforms differ in molecular weight and some biochemical properties. Isolation and purification procedures allowed to obtain purified enzymes with specific activity of the 206 micromol/min/mg for cytosolic CK and 240 micromol/min/mg for MtCK. Native M(r)s of the cytosolic CK and MtCK determined by gel permeation chromatography were 86.000 and 345.000, respectively. The results indicate that one of isoforms found in herring skeletal muscle is a cytosolic dimer and the other one, is a mitochondrial octamer. Octamerization of MtCK is not an advanced feature and also exists in fish. These values correspond well with published values for MtCKs and cytosolic CK isoforms from higher vertebrate classes and even from lower invertebrates.

Quantitative determination of creatine kinase release from herring (Clupea harengus) spermatozoa induced by tributyltin.

Creatine kinase (CK, ATP creatine phosphotransferase, EC 2.7.3.2) is an enzyme participating in ATP regeneration, which is the primary source of energy in living organisms. We demonstrated that CK from herring spermatozoa has high activity ( approximately 452 micromol/min/g of fresh semen) and has a different electrophoretic mobility from isoenzymes present in skeletal muscle. In our study, we investigated toxic effect of tributyltin (TBT) on herring spermatozoa using a specific sperm viability kit to observe live and dead sperm cells with a confocal microscope. Treatment of herring spermatozoa with TBT caused a time-dependent decrease of viability: 35% nonviable cells with 5 microM TBT and more than 90% nonviable cells with 10 microM TBT after 6 h exposure. We also monitored CK release from damaged spermatozoa into surrounding medium containing different concentrations of TBT. The higher concentration of TBT was used the more CK release from spermatozoa was observed. We suggest that CK could be a good biomarker of sperm cell membranes degradation in the case when lactate dehydrogenase release from permeabilized cells is not possible for rapid determination of the effect of TBT.