Saduria entomon infected with Hysterothylacium aduncum found in situ in the stomach of cod (Gadus morhua) from the Baltic Sea.

The parasite fauna of cod (Gadus morhus) is well described, but the life cycles of Baltic cod parasites are known only in general terms. Invertebrates commonly found in the stomach of cod are recognized as intermediate hosts in the life cycles of nematodes or acanthocephalans. The aim of this study was to determine the source of infection of Baltic cod with parasites found in situ in invertebrates present in the cod stomach. Our results indicate that Saduria entomon is both a source of infection of Baltic cod with parasites and an intermediate host in the life cycle of Hysterothylacium aduncum in the Baltic Sea.

Correlative light and electron microscopy reveals discrepancy between gold and fluorescence labelling.

Electron microscopy (EM) is traditionally employed as a follow-up to fluorescence microscopy (FM) to resolve the cellular ultrastructures wherein fluorescently labelled biomolecules reside. In order to translate the information derived from FM studies to EM analysis, biomolecules of interest must be identified in a manner compatible with EM. Although fluorescent signals can serve this purpose when FM is combined with EM in correlative light and electron microscopy (CLEM), the traditional immunogold labelling remains commonly used in this context. In order to investigate how much these two strategies relate, we have directly compared the subcellular localization of on-section fluorescence labelling with on-section immunogold labelling. In addition to antibody labelling of LAMP-1, bioorthogonal click labelling was used to localize soluble cysteine cathepsins or membrane-associated sialylated glycans. We reveal and characterize the existence of inherent discrepancies between the fluorescence signal and the distribution of gold particles in particular in the case of membrane-associated antigens.

Postantibiotic effect and postantibiotic sub-MIC effect of LTX-109 and mupirocin on Staphylococcus aureus blood isolates.

The development of new synthetic antimicrobial peptides like LTX-109 provides a new class of drugs for the treatment of Staphylococcus aureus infections. We evaluated LTX-109 and mupirocin for pharmacodynamic parameters against 10 methicillin-resistant S. aureus isolates. The postantibiotic effect (PAE) is defined as the length of time that bacterial growth is suppressed following a brief exposure to an antibiotic. We also determined the sub-MIC effect (SME) which measures the direct effect of subinhibitory levels on strains that have not previously been exposed to antibiotics. The postantibiotic sub-MIC effect (PA-SME) is a combination of the PAE and SME. LTX-109 had an average PAE of 5·51 h vs 1·04 h for mupirocin. The PA-SME of LTX-109 ranged from 2·51 to 9·33 h as the concentration increased from 0·2 to 0·4 times the minimal inhibitory concentration (MIC). The PA-SME range for mupirocin was 0·93-2·58 h. LTX-109, as compared to mupirocin, demonstrated prolonged time of effect for these pharmacodynamic parameters, which supports persistent activity for several hours after the drug is no longer present or is below the MIC. The pharmacodynamic parameters studied here suggest that LTX-109 is less likely than mupirocin to generate resistance to S. aureus. SIGNIFICANCE AND IMPACT OF THE STUDY: Resistant bacterial infections continue to be a challenge for clinicians. Identification of antibiotics with pharmacodynamic advantages may be beneficial in the treatment of these infections. An antibiotic with a longer postantibiotic effect may be able to be administered less frequently resulting in improved adherence. In this study, a new synthetic antimicrobial peptide, LTX-109, demonstrated a more prolonged time for LTX-109 than mupirocin against methicillin-resistant Staphylococcus aureus.

Personality traits as an endophenotype in genetic studies on suicidality in bipolar disorder.

Introduction The influence of personality traits on suicidal behaviour risk has been well documented. Personality traits and suicidal behaviour are partially genetically determined and personality has been described as an endophenotype of suicidal behaviour. The aim of this study was to investigate a possible association between personality traits with suicidal behaviour and selected serotonergic gene polymorphisms. METHODS: In the study we included 156 patients meeting DSM-IV criteria for bipolar disorder (BP) and 93 healthy controls. The personality dimensions were assessed using the Temperament and Character Inventory (TCI). We genotyped two selected polymorphisms of the tryptophan hydroxylase 1 (TPH1) gene (rs1800532 218A>C and rs1799913 779A>C) and polymorphism in the promoter region of serotonin transporter gene (5-HTTLPR, rs25531) related to serotoninergic neurotransmission. Multiple poisson regression, logistic regression and Kruskal-Wallis tests were applied. RESULTS: We found numerous differences between the BP patients and the control group in terms of their TCI dimensions/subdimensions. Significant differences were found between patients with, and without, suicidal attempts in fatigability and asthenia (Ha4), as well as in harm avoidance (Ha). We also found that the interactions between TCI subdimensions (the interaction of disordiness (Ns4) and spiritual acceptance (St3), disordiness (Ns4) and integrated conscience (C5), extravagance (Ns3) and resourcefulness (Sd3)) were significantly contributing for suicidal behaviour risk. We found association between all studied genetic polymorphisms and several TCI dimensions and subdimensions. CONCLUSION: Our results confirm that personality traits are partially determined by genes. Both personality traits and the interactions between temperament and character traits, may be helpful in predicting suicidal behaviour.

Microbiological studies of the round goby (Neogobius melanostomus) from the Puck Bay, southern Baltic Sea.

In summer 2017 numerous dead round gobies ( Neogobius melanostomus) and individuals covered with white coating were observed in the Puck Bay. The aim of our research was to determine the microbiological composition of the round goby from the Puck Bay, focusing on the presence of pathogens. Bacteria were identified by biochemical methods and, by sequencing of 16S rRNA. The dominant bacterial species were Shewanella baltica, Pseudomonas spp. and Aeromonas spp. - opportunistic pathogens, commonly present in many fish species, which may become harmful for the organism in unfavorable conditions. It was the first trial to determine the composition of the bacterial flora of N. melanostomus from that area.

Isolation and structure of a covalent cross-link adduct between mitomycin C and DNA.

A DNA cross-link adduct of the antitumor agent mitomycin C (MC) to DNA has been isolated and characterized; the results provide direct proof for bifunctional alkylation of DNA by MC. Exposure of MC to Micrococcus luteus DNA under reductive conditions and subsequent nuclease digestion yielded adducts formed between MC and deoxyguanosine residues. In addition to the two known monoadducts, a bisadduct was obtained. Reductive MC activation with Na2S2O4 (sodium dithionite) leads to exclusive bifunctional alkylation. The structure of the bisadduct was determined by spectroscopic methods that included proton magnetic resonance, differential Fourier transform infrared spectroscopy, and circular dichroism. Formation of the same bisadduct in vivo was demonstrated upon injection of rats with MC. Computer-generated models of the bisadduct that was incorporated into the center of the duplex B-DNA decamer d(CGTACGTACG)2 indicated that the bisadduct fit snugly into the minor groove with minimal distortion of DNA structure. A mechanistic analysis of the factors that govern monofunctional and bifunctional adduct formation is presented.

Detection of Streptococcus pneumoniae colonisation in respiratory tract secretions of military personnel.

Respiratory tract infections with Streptococcus pneumoniae are an important cause of morbidity and mortality among military personnel. A sensitive method is needed to determine the prevalence of S. pneumoniae colonisation in respiratory secretions, as well as its role in pneumonia without an established aetiology. This study investigated the efficacy of two PCR assays in screening military personnel for S. pneumoniae colonisation. Nasopharyngeal swabs were obtained from 200 military personnel and tested for S. pneumoniae by culture and PCR. S. pneumoniae was cultured from three (1.5%) of the 200 samples. PCR for the lytA gene detected S. pneumoniae in 11% of the samples, while PCR for the pneumolysin gene detected S. pneumoniae in 3% of the samples. The sensitivity and negative predictive values were 100% for both PCR assays when compared to culture; the specificity and positive predictive values for the lytA PCR were 90.4% and 13.6%, respectively, compared with 98.5% and 50%, respectively, for the pneumolysin gene PCR. It was concluded that respiratory tract colonisation of military personnel with S. pneumoniae can be identified rapidly and reliably by PCR assays. The use of this technique may greatly enhance the ability to identify a microbial aetiology for pneumonia when compared with conventional culture methods.

Cardiovascular effects of exogenous adrenomedullin and CGRP in Ramp and Calcrl deficient mice.

Adrenomedullin (AM) and calcitonin gene-related peptide (CGRP) are potent vasodilator peptides and serve as ligands for the G-protein coupled receptor (GPCR) calcitonin receptor-like receptor (CLR/Calcrl). Three GPCR accessory proteins called receptor activity-modifying proteins (RAMPs) modify the ligand binding affinity of the receptor such that the CLR/RAMP1 heterodimer preferably binds CGRP, while CLR/RAMP2 and CLR/RAMP3 have a stronger affinity for AM. Here we determine the contribution of each of the three RAMPs to blood pressure control in response to exogenous AM and CGRP by measuring the blood pressure of mice with genetic reduction or deletion of the receptor components. Thus, the cardiovascular response of Ramp1-/-, Ramp2+/-, Ramp3-/-, Ramp1-/-/Ramp3-/- double-knockout (dKO), and Calcrl+/- mice to AM and CGRP were compared to wildtype mice. While under anesthesia, Ramp1-/- male mice had significantly higher basal blood pressure than wildtype males; a difference which was not present in female mice. Additionally, anesthetized Ramp1-/-, Ramp3-/-, and Calcrl+/- male mice exhibited significantly higher basal blood pressure than females of the same genotype. The hypotensive response to intravenously injected AM was greatly attenuated in Ramp1-/- mice, and to a lesser extent in Ramp3-/- and Calcrl+/- mice. However, Ramp1-/-/Ramp3-/- dKO mice retained some hypotensive response to AM. These results suggest that the hypotensive effect of AM is primarily mediated through the CLR/RAMP1 heterodimer, but that AM signaling via CLR/RAMP2 and CLR/RAMP3 also contributes to some hypotensive action. On the other hand, CGRP's hypotensive activity seems to be predominantly through the CLR/RAMP1 heterodimer. With this knowledge, therapeutic AM or CGRP peptides could be designed to cause less hypotension while maintaining canonical receptor-RAMP mediated signaling.

Recurrence of primary sclerosing cholangitis in patients after liver transplantation.

BACKGROUND: Primary sclerosing cholangitis (PSC) is a chronic cholestatic disease that progresses to end-stage liver disease. There are several specific problems related to the posttransplantation period in these patients. The aim of this study was to analyze a single center experience with 17 orthotopic liver transplantations (OLT) due to PSC. PATIENTS AND METHODS: Seventeen patients were included (10 men, 7 women). Actuarial patient and graft survival rates and the incidence of recurrent sclerosing cholangitis were determined at 1, 5, and 7 years. RESULTS: Fifteen patients received single grafts, whereas two patients required retransplants. Patients received either cyclosporine (n = 7) or tacrolimus (n = 10) based immunosuppression. The 1-, 5-, and 7-year patient survival rates were 80%, 60%, and 60%, respectively, whereas the graft survival rates were 88%, 65%, and 65%, respectively. Two patients had cholangiocarcinomas (CCA) diagnosed during OLT; both recurred within 6 months and had a fatal outcome. Two patients (12%) developed recurrent sclerosing cholangitis, as assessed by liver histology and imaging of biliary tree. CONCLUSIONS: Liver transplantation provides good patient and graft survival rates in cases affected with PSC. CCA is associated with poor recipient survival. Recurrent PSC occurs in approximately 12% of cases but does not significantly affect patient survival.

Thioesterase II, a new marker enzyme for human cells of breast epithelial origin.

Metabolic, enzymologic, and immunohistochemical techniques have been used to show that a human cell line of breast epithelial origin synthesized medium chain fatty acids via the ubiquitous fatty acid synthetase and a mammary-specific chain-terminating enzyme, thioesterase II. Previous studies in our laboratory with rodents indicated that thioesterase II is expressed exclusively in mammary epithelial cells, an observation consistent with the physiologic role of the enzyme in milk fat synthesis. Results of the present study suggest that the enzyme exhibits a similar cell specificity in its expression in humans and that the specificity is maintained in normal and neoplastic tissues. Thus thioesterase II was detected immunohistochemically in normal human breast epithelia derived from both lactating and nonlactating breast tissue, in cultured cells derived from both primary breast epithelial tumors and from a metastatic tumor of breast origin, and in several human breast epithelial cell lines; the enzyme could not be detected in HeLa cells, in a colon carcinoma, or in a mammary myoepithelial cell line. These findings raise the possibility that thioesterase II may be of use as a diagnostic tool to identify human tumors of breast epithelial origin.

Evaluation of thioesterase II as a serum marker for rat mammary cancer.

Thioesterase II, the key enzyme which regulates the production of medium-chain fatty acids by the mammary fatty acid synthetase, is expressed specifically in epithelial cells of the rat mammary gland, regardless of their state of differentiation, and we consider the enzyme to be a reliable marker for this cell type. The objective of this study was to determine whether this enzyme is expressed universally in tumors originating from rat mammary epithelial cells and whether it might be shed into the serum of host animals. Immunoreactive thioesterase II was detected in all of the epithelial derived mammary tumors tested, being highest in tumors that exhibited obvious epithelial morphology. Two of the tumors, R3230AC and DMBA 1, were transplanted into Fischer 344 rats and the levels of thioesterase II in the tumor and serum were monitored by enzyme immunoassay. Thioesterase II content of the transplanted tumors fell to, and remained at, a low level during the first week following transplantation. During this period the transplanted tumor established a new network of blood vessels; no thioesterase II was detectable in the serum. Subsequently, thioesterase II levels in the tumors returned to the values observed before transplantation and thioesterase II was detectable in the serum. Of 51 rats transplanted with the R3230AC tumor, 37 showed elevated serum thioesterase II levels; of 40 transplanted with the DMBA 1 tumor, 35 showed elevated serum thioesterase II. Furthermore, there was a statistically significant correlation between serum thioesterase II and tumor burden in both tumor model systems. The identity of the serum antigen reacting with anti-thioesterase II antibodies was confirmed, by Western immunoblotting, to be full-length thioesterase II polypeptide. Parallel studies with fatty acid synthetase, an enzyme with an ubiquitous tissue distribution, indicated as expected that serum levels of this enzyme were unlikely to provide a reliable index of mammary tumor status. These results indicate that thioesterase II may be a useful serum marker for mammary cancer.

Cytotoxic and antitumor activity of 1-nitroacridines as an aftereffect of their interstrand DNA cross-linking.

To determine whether the toxic effects and changes in many cell functions caused by antitumor 1-nitroacridines are related to their enzymatically mediated covalent interstrand DNA cross-linking (J. Konopa, J. W. Pawlak, and K. Pawlak. Chem.-Biol. Interact., 43: 175-197, 1983), the cross-linking potency of the derivatives with structural modifications in position 9 of the acridine nucleus was estimated as their in vitro threshold concentrations (0.3 to 4.5 microM), beyond which the first interstrand DNA cross-links could be detected in DNA of cultured HeLa S3 cells with a polyethylene glycol 6000-Dextran T500 assay. Statistically significant (p less than 0.05) correlations exist between the cross-linking potency of 1-nitroacridines and their in vivo antitumor activity and toxicity against mice with Sarcoma 180 tumors in solid form (3 to 1065 mumol/kg of body weight), as well as their in vitro cytotoxicity against cultured HeLa or HeLa S3 cells (0.0005 to 7.2 microM), indicating that the interstrand DNA cross-linking potency might be one of primary determinants of in vivo and in vitro biological activity of 1-nitroacridine antineoplastic drugs. Susceptibility of the parent agents to reduction does not appear to be a rate-limiting factor of DNA cross-linking potency of 1-nitroacridines and their metabolic transformations (J. W. Pawlak, and J. Konopa. Biochem. Pharmacol., 28: 3391-3402, 1979), because no significant differences were observed among the agents with respect to their polarographic half-wave potentials estimated under anaerobic conditions.

Synthesis and biological evaluation of a new series of sterols as potential hypocholesterolemic agents.

A new series of sterols was synthesized and tested in a CHO cell-based LDL receptor/luciferase (LDLR/Luc) assay to investigate the capability of derepressing the transcription of LDL receptor promoter in the presence of 25-hydroxycholesterol. The effect of various substitutions on antagonizing the repressing effect mediated by 25-hydroxycholesterol was also studied in terms of regio- and stereochemistry, lipophilicity, steric bulk, and pi-electron density. Except 12, compounds active in the primary LDLR/Luc assay were not active in the secondary simian virus 40/luciferase (SV40/Luc) assay, demonstrating the specificity of their in vitro activity. Eight active compounds of various structural types were selected and screened in a [1-14C-acetate]cholesterol biosynthesis inhibition assay; none has shown any interference with the cholesterol biosynthesis in CHO cells. In hypercholesterolemic hamsters, generally, compounds that were active in vitro were active in vivo and vice versa, with the exception of three in vitro inactive compounds: 3 beta-ols 3a' and 3c' as well as 3-ketone 2a. Experimental results from the livers of hamsters revealed that the in vivo conversion of 3a' or 2a to 3a has in part contributed to the observed in vivo activity, and it is also anticipated that 3c' may similarly be converted to 3c in hamsters.

The mode of action of cytotoxic and antitumor 1-nitroacridines. II. In vivo enzyme-mediated covalent binding of a 1-nitroacridine derivative, Ledakrin or Nitracrine, with dna and other macromolecules of mammalian or bacterial cells.

Earlier evidence, in Part I of this paper, has shown that cytotoxic and antitumor 1-nitroacridines did not primarily exert their potent inhibitory effects on cultured mammalian cells by physicochemical binding with DNA, although it undoubtedly occurred (Chem.-Biol. Interact., 43 (1983) 131). As a result it was investigated (i) whether 9-14C- or 1'-14C-labeled derivatives of their representative, 1-nitro-9-/3'-dimethylamino-n-propylamino/acridine (Ledakrin or Nitracrine), were capable of covalent binding with nucleic acids and other suitable macromolecules in target cells in vivo and/or (ii) whether activation of the agent in the cell was a necessary prerequisite for such binding. Using the criteria of resistance to exhaustive extractions with trichloroacetic acid and/or organic solvents, [14C]Ledakrin was found to bind covalently, with relatively little discrimination, with: (i) intracellular macromolecules, including DNA, of cultured tumor HeLa cells (370-2500 DNA base pairs/one Ledakrin molecule; (ii) experimental animal tumor Ehrlich ascites (Eat) cells in vivo (650-5880 DNA base pairs/one Ledakrin molecule); (iii) bacterial Bacillus subtilis SB 1058 cells (7000-33 000 Ledakrin links/one cell genome); (iv) NADPH-fortified rat liver homogenates in vitro (25.6 nmol/mg microsomal protein under air). These results far exceed the common levels reported for alkylating agents or chemical carcinogens. Unlike [ethyl-14C]quinacrine, compared in vitro, covalent macromolecules binding with Ledakrin in vitro, and most probably in vivo, can be equated to NADPH-dependent activation(s) by oxidoreductase systems and the presence of DNA alone was not satisfactory in itself to attain Ledakrin binding. Fractionation of the enzymatic digest of 14C-associated DNA, isolated from Eat cells exposed in vivo to [9-14C]Ledakrin, by Sephadex LH-20 column chromatography followed by mass spectrometry analyses of modified nucleosides, indicated that both mono- and dinucleosidical Ledakrin metabolites were the products of an in vivo reaction. This implied that the lethal reaction of the drug could be its cross-linking of the target macromolecules and/or its monofunctional attack on vitally important cellular components.

The mode of action of cytotoxic and antitumor 1-nitroacridines. III. In vivo interstrand cross-linking of DNA of mammalian or bacterial cells by 1-nitroacridines.

To define a critical lesion in presumable target DNA cause in vivo by the antitumor and cytotoxic 1-nitroacridines, Ehrlich ascites tumor (Eat) cells implanted into mice, HeLa cells grown in monolayer culture or Bacillus subtilis SB 1058 strain cells were exposed to Ledakrin [Nitracrine; 1-nitro-9-(3'-dimethylamino-n-propylamino)acridine], its non-antitumor congeners, or mitomycin C tested for comparison; total intracellular DNA was isolated from control or treated cells and denatured by heat, alkali or formamide, after which the chemically-induced fraction of interstrand cross-linked DNA molecules was assessed by thermal denaturation-renaturation curve analysis, hydroxylapatite column chromatography, or partitioning in a Dextran T500-polyethylene glycol 6000 biphasic system. Ledakrin, as compared to mitomycin C, was a very effective cross-linking agent, inducing one covalent cross-link per approx. 20 X 10(3) (B. subtilis), 56 X 10(3) (HeLa) or 80 X 10(3) (Eat) DNA base pairs. The first cross-links were introduced in B. subtilis cell genomes at minimal bactericidal concentrations of Ledakrin of mitomycin C. Ledakrin failed to induce discernible cross-linking of bihelical DNA when complexed with in cell-free system. Unlike the other two 1-nitroacridines which cross-linked DNA of cultured HeLa or B. subtilis cells, the non-antitumor 2-, 3- or 4-nitroacridines did not cause such effect and diminished cross-linking by Ledakrin or mitomycin C. Hence, upon metabolic activation in mammalian or bacterial cell Ledakrin and, most probably other 1-nitroacridines, become very effective DNA cross-linking agents and such effects appear to be responsible for the antitumor and potent cytotoxic activities of 1-nitroacridines.

The mode of action of cytotoxic and antitumor 1-nitroacridines. I. The 1-nitroacridines do not exert their cytotoxic effects by physicochemical binding with DNA.

The mode of action of cytotoxic and antitumor 1-nitroacridines and their isomeric derivatives was studied by comparing their effects in cell-free systems and towards cultured tumor HeLa cells, assuming that the nitroacridines considered exert cytotoxic effects by physicochemical binding with the DNA. All the nitroacridines impaired biosyntheses of DNA, RNA and protein in cultured HeLa cells and a causal relationship between nitroacridine inhibition of macromolecular biosyntheses and lethal effects of the agents appears likely. In cell-free systems, the nitroacridines bound with two independent sites on the DNA, forming complexes with enhanced resistance to DNA strand separation upon melting and inhibited the DNA polymerase reaction by altering activity of template and/or of enzyme. The 1-nitroacridines were poorly effective in cell-free systems and were the most potent inhibitors toward the growth of HeLa cells among the derivatives studied. It is concluded that the primary events responsible for cytotoxic effects of antitumor 1-nitroacridines and of their isomeric derivatives are different. The metabolic activation of 1-nitroacridines to more reactive intermediates which will attach to and alter the structure and/or function of DNA of sensitive cells is suggested.

Stereostructure of Rimocidin.

NMR studies of rimocidin, consisting of DQF-COSY, ROESY, HSQC, HMBC and 1D-TOCSY experiments, resulted in the assignment of the absolute configuration of the rimocidin chiral centers as 2S, 3R, 9S, 11R, 13S, 14R, 15S, 17R, and 27R. The geometry of tetraene chromophore was found to be all-trans.

Stereostructure of perimycin A.

The gross structure of perimycin A was revised: the position of the keto group was changed from C-13 to C-5. The stereostructure of perimycin A was established based upon NMR studies.

Stereostructure and NMR characterization of the antibiotic candidin.

The stereostructure of the heptaene macrolide antibiotic candidin was established on the basis of NMR studies: 13C, DQF-COSY, ROESY and C,H-COSY experiments. The absolute configuration of the candidin chiral centers were assigned as 3R, 5S, 10R, 11R, 13R, 15S, 16R, 17S, 19S, 34S, 35R, 36R and 37S.

The structure of amphotericin A. II. The complete structure of the antibiotic.

The structure of amphotericin A without the configuration of asymmetric carbon atoms has been elucidated. The stereochemistry of the sugar moiety has been determined. On the basis of homoscalar correlated 2D 1H spectra. of amphotericin A the position of the hemiketal moiety has been located, and the chemical shifts of all the protons in the antibiotic molecule have been determined.