The serum resistance of gonococci in the majority of urethral exudates is due to sialylated lipopolysaccharide seen as a surface coat

Examined before subculture, gonococci in 18 urethral exudates collected from different patients were serum-resistant. For 15 exudates, the resistance was drastically reduced by treatment with neuraminidase and by one subculture on laboratory media. It was restored by incubation with cytidine 5'-monophospho-N-acetyl neuraminic acid (CMP-NANA). Electron microscopic examination of gonococci in eight exudates showed a surface structure stained by Ruthenium red which disappeared in most samples when they were treated with neuraminidase. These results were identical with those of previous studies on in vitro grown gonococci which had shown that serum resistance is due to sialylation of a 4.5-kDa conserved component of gonococcal lipopolysaccharide (LPS) by host CMP-NANA, which masks the target site for bactericidal IgM and renders surface LPS stainable by Ruthenium red. The serum resistance of gonococci in the remaining three exudates was not reduced by neuraminidase nor by subculture. The mechanism of this stable resistance is unknown.     

Construction of isogenic gonococci with variable porin structure: effects on susceptibility to human serum and antibiotics

Protein I (PI) is the most abundant protein on the gonococcal cell surface and besides its porin function it may have important properties contributing to pathogenicity. By allelic exchange using cloned PI genes from FA19 (PIA) and MS11 (PIB) and a selectable marker introduced closely downstream of these genes, we constructed sets of isogenic gonococcal strains that differ only in their PI gene. Analysis revealed that PI has a major effect on stable resistance to normal human serum, and a slight effect on low-level resistance to antibiotics. All PIA/B hybrids were hypersusceptible to serum, suggesting a possible explanation for why such hybrids do not occur in nature.     

Recombinant human arginase toxicity in mice is reduced by citrulline supplementation

Human recombinant arginase I cobalt coupled to polyethylene glycol 5000 (HuArg I [Co]-PEG5000) achieved potent in vitro depletion of arginine from tissue culture medium and cytotoxicity to many cancer cell lines. The recombinant enzyme also produced tumor growth inhibition of hepatocellular carcinoma and pancreatic carcinoma xenografts. Although these results were promising, the therapeutic index was narrow. Toxicities were seen in normal cells in tissue culture. In vivo normal tissue injury occurred at doses twice the effective dose. The current study was conducted to define, in greater detail, the maximum tolerated dose (MTD), pharmacodynamics, and dose-limiting toxicities (DLTs) of twice-weekly intraperitoneal HuArg I [Co]-PEG5000 in Balb/c mice. Animal weight and survival were monitored, serum arginine levels measured, and complete blood cell counts, chemistries, necropsies, and histologies were performed. In addition, methods to ameliorate the HuArg I [Co]-PEG5000 adverse effects were tested. Supplemental l-citrulline was given concurrently with the arginase drug. The HuArg I [Co]-PEG5000 MTD in mice was 5 mg/kg twice weekly, and DLTs included weight loss and marrow necrosis. No other organ damage or changes in blood cell counts or chemistries were observed. Arginase reduced serum arginine levels from 60 μM to 4 to 6 μM. Supplemental l-citrulline given per os or daily subcutaneously reduced and delayed toxicities, and l-citrulline given twice daily subcutaneously completely prevented animal toxicities. On the basis of these results, we hypothesize that HuArg I [Co]-PEG5000, particularly with supplemental l-citrulline, may be an attractive therapeutic agent for argininosuccinate synthetase-deficient tumors.     

The perturbation of thrombin binding to human platelets by trypsin and neuraminidase

The initial step in the interaction of thrombin with human platelets in binding of the enzyme to the platelet surface. The effects of digestion of isolated platelets with trypsin and neuraminidase on aggregation, release of serotonin and binding of thrombin have been examined.Trypsin is a powerful inducer of platelet aggregation as well as the release reaction. The aggregation effect of trypsin may be blocked with disodium ehtylenediaminetatraacetate (EDTA). Further, in the presence of EDTA, trypsin-induced release of [¹⁴C]serotonin is 15–20% lower compared to controls and the initial lag period is prolonged. Conditions were developed under which trypsin did neither aggregate nor release serotonin from platelets. Even under these conditions, trypsin caused a profound loss in the thrombin binding capacity of platelets. Thus, the trypsin-induced fall in the thrombin binding capacity and the platelet response are dissociated. This loss in the thrombin binding by trypsin is due to a lower number of binding sites available on the platelet surface and is not due to an altered affinity.Neuraminidase did not induce platelet aggregation or the release reaction. The ability of platelets to bind thrombin was also unimpaired by prior digestion with neuraminidase. Thus, the sialic acid at the platelet surface is not essential in the function of thrombin recognition by the receptor. This moiety may nontheless be a constituent of a glycoprotein which might act as the thrombin receptor.     

Activity of human dihydrolipoamide dehydrogenase is largely reduced by mutation at isoleucine-51 to alanine

Dihydrolipoamide dehydrogenase (E3) belongs to the pyridine nucleotide-disulfide oxidoreductase family including glutathione reductase and thioredoxin reductase. It catalyzes the reoxidation of dihydrolipoyl moiety of the acyltransferase components of three alpha-keto acid dehydrogenase complexes and of the hydrogen-carrier protein of the glycine cleavage system. Isoleucine-51 of human E3, located near the active disulfide center Cys residues, is highly conserved in most E3s from several sources. To examine the importance of this highly conserved Ile-51 in human E3 function, it was substituted with Ala using site-directed mutagenesis. The mutant was expressed in Escherichia coli and highly purified using an affinity column. Its E3 activity was decreased about 100-fold, indicating that the conservation of the Ile-51 residue in human E3 was very important to the efficient catalytic function of the enzyme. Its altered spectroscopic properties implied that conformational changes could occur in the mutant.     

Opsonic activity of guinea pig serum is reduced by in vivo administration of carcinogenic nitrosamines

The administration of each of four carcinogenic nitrosamines to normal guinea pigs resulted in decreased opsonic activity of their sera but did not affect the capacity of their peritoneal macrophages to phagocytize particles opsonized with normal serum. Diphenylnitrosamine, a noncarcinogenic analogue, had no significant effect on opsonic or phagocytic activity.     

Resistance to apoptosis is correlated with the reduced caspase-3 activation and enhanced expression of antiapoptotic proteins in human cervical multidrug-resistant cells

Recent studies have indicated that induction of apoptosis is the primary cytotoxic mechanism of most cancer chemotherapeutic agents, and abnormalities in the control of apoptosis can affect the sensitivity of malignant cells to multiple drugs. Here, we treated cells with cisplatin and other apoptotic stimuli and found that multidrug-resistant (MDR) endocervical HEN-16-2/CDDP cells, compared with drug-sensitive parental cells, were significantly more resistant to apoptosis and exhibited decreased proteolytic activation of caspase-3. The latter was further demonstrated by decreased cleavage of its substrate poly(ADP-ribose) polymerase (PARP). Further, Western blot analysis showed that MDR HEN-16-2/CDDP cells had significantly higher levels of the apoptosis-inhibiting proteins BAG-1 p50 and p33 isoforms and Bcl-X(L). This study provided the first evidence that overexpression of antiapoptotic BAG-1 p50 and p33 and Bcl-X(L) may cause resistance to apoptosis through reduction of caspase-3 activity in human cervical cells having an MDR phenotype.     

Species-specific uptake of DNA by gonococci is mediated by a 10-base-pair sequence.

Piliated Neisseria gonorrhoeae are known to be transformed less readily if transforming DNA competes with DNA containing the 10-bp sequence GCCGTCTGAA. It has been postulated that the 10-bp sequence is a recognition sequence which is required for efficient DNA uptake. We show that the presence of various forms of this 10-bp sequence results in increased uptake of double-stranded DNA into a DNase-resistant state and allows genetic transformation by an otherwise nontransformable plasmid.     

Alzheimer-like plaque formation by human macrophages is reduced by fibrillation inhibitors and lovastatin

The cerebral deposition of Abeta-peptide as amyloid fibrils and plaques represents a hallmark characteristic of Alzheimer's disease (AD). AD plaques are defined by their green birefringence after Congo red staining, their spherulite-like superstructure and their association with specific secondary components. Here we show that primary human macrophages promote the formation of amyloid plaques that correspond in all aforementioned characteristics to typical amyloid plaques from diseased tissues: they consist of aggregated Abeta-peptide, they reveal the typical 'Maltese cross" structure and they are associated with the secondary components glycosaminoglycanes, apolipoprotein E (apoE) and the raft lipids cholesterol and sphingomyelin. Plaque formation can be impaired in this cell system by addition of small molecules, such as Congo red, melantonine and lovastatin, suggesting potential applications for the study of cellular amyloid formation and for the identification or validation of drug candidates.     

A surface polysaccharide forms when gonococci are converted to serum resistance by cytidine 5''-monophospho-N-acetyl neuraminic acid

A serum-susceptible, guinea-pig chamber-passaged, laboratory strain (BS4 (agar)) of Neisseria gonorrhoeae was converted to serum resistance by incubation with cytidine 5-monophospho-N-acetyl neuraminic acid (CMP-NANA) and examined by electron microscopy after staining with ruthenium-red. In contrast to serum susceptible gonococci incubated without CMP-NANA, the majority (60-70%) of the serum resistant organisms showed a surface accumulation of polysaccharide. This surface polysaccharide was enhanced on all the resistant gonococci after incubation with fresh human serum. Control susceptible gonococci were devoid of the polysaccharide after incubation with heated human serum. Identical results were obtained with a fresh gonococcal isolate which had lost serum resistance on subculture but which, in common with 3 other isolates, was restored to serum resistance by incubation with CMP-NANA.     

Annexin 2 expression is reduced in human osteosarcoma metastases

Osteosarcoma is an aggressive primary bone cancer affecting primarily children and young adults. The development of valuable diagnostic indicators and therapeutic agents will be enhanced by the identification and characterization of genes that contribute to its aggressive behavior. We used representational difference analysis to isolate genes differentially expressed between primary human osteosarcoma tumors and subsequent metastatic lung lesions to identify genes potentially involved in metastatic potential. Several genes were differentially expressed between the two tumor populations, including annexin2. The levels of annexin2 mRNA and protein inversely correlated with metastatic potential in a subset of human osteosarcoma tumor specimens, as well as in a human osteosarcoma cell line selected for increased metastatic potential. Annexin2 has been described in several cellular localizations with various functional implications, many of which may be relevant to metastatic potential. Therefore, the subcellular localization of endogenous annexin2 protein was evaluated biochemically by subcellular fractionation and immunologically by flow cytometry and immunofluorescence in osteoblastic cells. Annexin2 was localized to the cytoplasm and intracellular aspect of the plasma membrane, excluded from the nucleus and undetectable on the cell surface or in the conditioned medium. Overexpression of annexin2 in osteosarcoma cells did not alter several in vitro phenotypes often used to assess metastatic potential including motility, adhesion, and proliferation. However, our previous data have implicated annexin2 in the mineralization process of osteoblastic cells in vitro. Consistent with an increase in differentiation-induced mineralization, there was diminished tumorigenicity and experimental metastatic potential of osteosarcoma cells overexpressing annexin2. These data suggest that annexin2 may downregulate osteosarcoma aggressiveness by inducing a more differentiated state in osteoblastic cells.     

Properties of human chorion neuraminidase

Some properties of human chorion neuraminidase were studied. Using n-butanol, a solubilized preparation of neuraminidase with specific activity considerably exceeding the initial activity of the chorion homogenate was obtained. The pH-dependence and substrate specificity of the enzyme towards low molecular weight (sialylglycolipids and sialylglycoproteins) native substrates were examined. These properties of solubilized neuraminidase from human chorion were found to be similar to those of the lysosomal enzyme from other animal tissues. The results abtained are consistent with the properties of neuraminidase from native chorion and amniotic fluid cell cultures. Based on the substrate specificity of the solubilized enzyme, it was found that chorion biopsy specimens could be used for prenatal diagnosing of sialidoses and mucolipidoses IV. Some properties of solubilized human chorion beta-galacotosidase were studied.     

Lipid oxidation is reduced in obese human skeletal muscle

The purpose of this study was to discern cellular mechanisms that contribute to the suppression of lipid oxidation in the skeletal muscle of obese individuals. Muscle was obtained from obese [body mass index (BMI), 38.3 +/- 3.1 kg/m(2)] and lean (BMI, 23.8 +/- 0.9 kg/m(2)) women, and fatty acid oxidation was studied by measuring (14)CO(2) production from (14)C-labeled fatty acids. Palmitate oxidation, which is at least partially dependent on carnitine palmitoyltransferase-1 (CPT-1) activity, was depressed (P < 0.05) by approximately 50% with obesity (6.8 +/- 2.2 vs. 13.7 +/- 1.4 nmole CO(2).g(-1).h(-1)). The CPT-1-independent event of palmitoyl carnitine oxidation was also depressed (P < 0.01) by approximately 45%. There were significant negative relationships (P < 0.05) for adiposity with palmitate (r = -0.76) and palmitoyl carnitine (r = -0.82) oxidation. Muscle CPT-1 and citrate synthase activity, an index of mitochondrial content, were also significantly (P < 0.05) reduced ( approximately 35%) with obesity. CPT-1 (r = -0.48) and citrate synthase (r = -0.65) activities were significantly (P < 0.05) related to adiposity. These data suggest that lesions at CPT-1 and post-CPT-1 events, such as mitochondrial content, contribute to the reduced reliance on fat oxidation evident in human skeletal muscle with obesity.     

Activity of human dihydrolipoamide dehydrogenase is reduced by mutation at threonine-44 of FAD-binding region to valine

Dihydrolipoamide dehydrogenase (E3) is a member of the pyridine nucleotide-disulfide oxidoreductase family. Thr residues are highly conserved. They are at the active site disulfide-bond regions of most E3s and other oxidoreductases. The crystal structure of Azotobacter vinelandii E3 suggests that the hydroxyl group of Thr that are involved in the FAD binding interact with the adenosine phosphate of FAD. However, several prokaryotic E3s have Val instead of Thr. To investigate the meaning and importance of the Thr conservation in many E3s, the corresponding residue, Thr-44, in human E3 was substituted to Val by site-directed mutagenesis. The mutant's E3 activity showed about a 2.2-fold decrease. Its UV-visible and fluorescence spectra indicated that the mutant might have a slightly different microenvironment at the FAD-binding region.     

Surface antigens of gonococci: correlation with virulence and serum resistance

Encapsulated and non-encapsulated variants of one strain of gonococcus were compared for their capacity to produce infection in chambers implanted subcutaneously in mice, for their reactions with specific antibody and for their precipitation with wheat germ agglutinin. Only the encapsulated variant could infect implanted chambers. Specific rabbit antiserum raised against the non-encapsulated variant killed both variants. Saline extracts and lipopolysaccharide preparations of the encapsulated variant differed from those of the non-encapsulated one in their reactions with wheat germ agglutinin and antibody in diffusion and electrophoresis tests. Preparations from infective encapsulated gonococci reacted with wheat germ agglutinin while those from the non-encapsulated variant did not. Immunodiffusion tests showed that lipopolysaccharides from both variants share a common antigenic determinant, but saline extracts and lipopolysaccharides from encapsulated gonococci possess an additional determinant. The significance of these findings is discussed.     

Effect of neuraminidase treatment on serum reactivity to autologous leukemic blast cells

Summary The sera of 35 patients with acute lymphoblastic leukemia (ALL) and acute non-lymphoblastic leukemia (ANLL) were tested for reactivity against cell surface antigens of autologous leukemic blast cells by protein A assay (PA), immune adherence assay (IA), and anti-C3 mixed hemadsorption assay (C3-MHA). Autologous serum reactivity was detectable by PA in four cases and by LA and C3-MHA in about half the patients. Autologous serum reactivity occurred more often in ALL than in ANLL. Absorption studies revealed that in one patient only the autologous reactivity was directed against a restricted antigen, which could be detected only on the individual T-ALL blast cells. All other autologous antibodies detected unspecific antigens. Neuraminidase treatment had two effects: first, it increased antibody attachment to antigens which are also present on untreated cells; secondly, after neuraminidase treatment an antigen was detectable on the cell surface which could also be demonstrated on neuraminidase-treated non-leukemic cells (e.g., erythrocytes). Neither of these two effects of neuraminidase treatment seems to be tumor-specific. Possible therapeutic effects of neuraminidase are probably caused by unspecific adjuvant effects of the enzyme.     

Lipopolysaccharide alteration is associated with induced resistance of Neisseria gonorrhoeae to killing by human serum

On SDS-PAGE, solubilized and proteinase K treated preparations of Neisseria gonorrhoeae strain BS4 (agar) showed differences in silver stained lipopolysaccharide (LPS) patterns, before and after induction to resistance to serum killing by incubation for 3 h at 37 degrees C with low Mr fractions from lysates of guinea pig red blood cells. Preparations from the original serum susceptible gonococci and LPS purified from such bacteria showed two components, but the preparations from the serum resistant gonococci were deficient in the higher Mr component. Furthermore, on immunoblotting with fresh human serum (FHS), the two LPS components of the susceptible gonococci reacted strongly with IgM. With preparations from the serum resistant gonococci there was no reaction in the area corresponding to the higher Mr component and a weaker reaction with the component of low Mr. Purified LPS from the susceptible gonococci neutralized the bactericidal activity of FHS against N. gonorrhoeae strain BS4 (agar) probably by reacting with the relevant antibody, since heated FHS was no longer bactericidal when mixed with a source of complement (human placental serum) after prior reaction with the LPS. These neutralization tests coupled with the results of immunoblotting strongly suggest that increased serum resistance is due to the lack of the high Mr LPS moiety.     

A mitochondria-targeted nitroxide is reduced to its hydroxylamine by ubiquinol in mitochondria

Piperidine nitroxides such as TEMPOL act as antioxidants in vivo due to their interconversion among nitroxide, hydroxylamine, and oxoammonium derivatives, but the mechanistic details of these reactions are unclear. As mitochondria are a significant site of piperidine nitroxide metabolism and action, we synthesized a mitochondria-targeted nitroxide, MitoTEMPOL, by conjugating TEMPOL to the lipophilic triphenylphosphonium cation. MitoTEMPOL was accumulated several hundred-fold into energized mitochondria where it was reduced to the hydroxylamine by direct reaction with ubiquinol. This reaction occurred by transfer of H() from ubiquinol to the nitroxide, with the ubisemiquinone radical product predominantly dismutating to ubiquinone and ubiquinol, together with a small amount reacting with oxygen to form superoxide. The piperidine nitroxides TEMPOL, TEMPO, and butylTEMPOL reacted similarly with ubiquinol in organic solvents but in mitochondrial membranes the rates varied in the order: MitoTEMPOL > butylTEMPOL > TEMPO > TEMPOL, which correlated with the extent of access of the nitroxide moiety to ubiquinol within the membrane. These findings suggest ways of using mitochondria-targeted compounds to modulate the coenzyme Q pool within mitochondria in vivo, and indicate that the antioxidant effects of mitochondria-targeted piperidine nitroxides can be ascribed to their corresponding hydroxylamines.