Histone-like protein fractions of Kluyveromyces fragilis and their relation to the cell cycle.

Protein fractions were obtained from Kluyveromyces fragilis by pH titration, a technique used for histone extraction, following the inclusion of a distilled water extraction stage, and the fractions partially characterized. The inclusion of a distilled water step resulted in a tenfold purification of the fraction obtained at pH 2.20 as compared with pH titration alone. In synchronous cultures induced with 2'-deoxyadenosine or prepared by selection this fraction displayed a stepwise accumulation, and doubled in quantity at a point about one third of a cycle after cell division. The fraction obtained at pH 1.35 resembled calf thymus f2a2 histone in its extraction properties and amino acid composition, and also showed a possible stepwise accumulation. Other fractions appear to accumulate exponentially.     

Phagocytosis of mast cell granules results in decreased macrophage superoxide production

The mechanism by which phagocytosed mast cell granules (MCGs) inhibit macrophage superoxide production has not been defined. In this study, rat peritoneal macrophages were co-incubated with either isolated intact MCGs or MCG-sonicate, and their respiratory burst capacity and morphology were studied. Co-incubation of macrophages with either intact MCGs or MCG-sonicate resulted in a dose-dependent inhibition of superoxide- mediated cytochrome c reduction. This inhibitory effect was evident within 5 min of incubation and with MCG-sonicate was completely reversed when macrophages were washed prior to activation with PMA. In the case of intact MCGs, the inhibitory effect was only partially reversed by washing after a prolonged co-incubation time. Electron microscopic analyses revealed that MCGs were rapidly phagocytosed by macrophages and were subsequently disintegrated within the phagolysosomes. Assay of MCGs for superoxide dismutase (SOD) revealed the presence of significant activity of this enzyme. A comparison of normal macrophages and those containing phagocytosed MCGs did not reveal a significant difference in total SOD activity. It is speculated that, although there was no significant increase in total SOD activity in macrophages containing phagocytosed MCGs, the phagocytosed MCGs might cause a transient increase in SOD activity within the phagolysosomes. This transient rise in SOD results in scavenging of the newly generated superoxide. Alternatively, MCG inhibition of NADPH oxidase would explain the reported observations.     

Hyperoxia alters effect of calcium on rat alveolar macrophage superoxide production

The effect of hyperoxia on the Ca2+ dependence of stimulated superoxide anion radical (O2-.) production (the respiratory burst) of rat alveolar macrophages was investigated. Enhancement of the concanavalin A (con A)-stimulated respiratory burst by extracellular Ca2+ was suppressed by O2 exposure. Similarly, the inhibitory effect of verapamil on the con A-stimulated respiratory burst was reduced by O2 exposure. O2 exposure also inhibited con A stimulation that was independent of Ca2+ entry. Exposure to O2 also caused a decline in O2-. production stimulated by either A23187 or phorbol myristate acetate (PMA). With A23187 stimulation, extracellular Ca2+ was essential for either air-exposed (control) or O2-exposed cells. With PMA, stimulation was independent of extracellular Ca2+ for either air or O2-exposed macrophages and verapamil did not inhibit. Free intracellular Ca2+ concentration ([Ca2+]i) was measured in control and O2-exposed alveolar macrophages. Hyperoxic exposure did not alter [Ca2+]i in unstimulated cells. In controls, con A stimulated an immediate increase in [Ca2+]i followed by a rapid decrease and a second rise and fall. The second elevation was suppressed by verapamil or ethyleneglycol-bis (beta-aminoethylether)-N,N'-tetraacetic acid or O2 exposure. The results of both the respiratory burst assays and measurement of con A-stimulated changes in [Ca2+]i suggest that Ca2+ entry involved in stimulus-response coupling is suppressed in cellular O2 toxicity.     

The mechanism of superoxide production by the antimycin-inhibited mitochondrial Q-cycle

Superoxide production from antimycin-inhibited complex III in isolated mitochondria first increased to a maximum then decreased as substrate supply was modulated in three different ways. In each case, superoxide production had a similar bell-shaped relationship to the reduction state of cytochrome b(566), suggesting that superoxide production peaks at intermediate Q-reduction state because it comes from a semiquinone in the outer quinone-binding site in complex III (Q(o)). Imposition of a membrane potential changed the relationships between superoxide production and b(566) reduction and between b(562) and b(566) redox states, suggesting that b(562) reduction also affects semiquinone concentration and superoxide production. To assess whether this behavior was consistent with the Q-cycle mechanism of complex III, we generated a kinetic model of the antimycin-inhibited Q(o) site. Using published rate constants (determined without antimycin), with unknown rate constants allowed to vary, the model failed to fit the data. However, when we allowed the rate constant for quinol oxidation to decrease 1000-fold and the rate constant for semiquinone oxidation by b(566) to depend on the b(562) redox state, the model fit the energized and de-energized data well. In such fits, quinol oxidation was much slower than literature values and slowed further when b(566) was reduced, and reduction of b(562) stabilized the semiquinone when b(566) was oxidized. Thus, superoxide production at Q(o) depends on the reduction states of b(566) and b(562) and fits the Q-cycle only if particular rate constants are altered when b oxidation is prevented by antimycin. These mechanisms limit superoxide production and short circuiting of the Q-cycle when electron transfer slows.     

Gonadotrophic cells of the rat hypophysis and their relation to hormone production

Summary The results of these experiments clearly indicate that the PAS-red and PAS-purple gonadotrophs of the rat anterior hypophysis are functionally as well as tinctorially distinct cell types. The PAS-red cells located peripherally in control animals (peripheral gonadotrophs of Purves and Griesbach) produce LH. Following castration they begin to appear in greater numbers in the central areas and by 45 days following castration they are predominant in both central and peripheral portions of the glands. At this time after castration the gonadotrophic content of the pituitary gland is predominantly luteinizing in character.The PAS-purple cells which are found in the central portions of control glands (central gonadotrophs of Purves and Griesbach) produce FSH. They appear peripherally following castration and are the predominant type of gonadotrophic cell in the glands of short term (10-day) castrates. The gonadotrophic content of such glands is chiefly FSH as shown by bioassay.By 45 days after castration the LH producing peripheral gonadotrophs and the FSH producing central gonadotrophs have lost their characteristic distribution patterns. Obviously, then, these two gonadotrophic types cannot be accurately followed on the basis of a restricted regional location in the pituitary but must be differentiated on the basis of specific cytological features and staining characteristics.This study was supported by USPHS Grant RG 4723 and by contract between Office of Naval Research, Department of the Navy, and University of Texas.     

Available soil nitrogen in relation to fractions of soil nitrogen and other soil properties

The available N of 27 soils from England and Wales was assessed from the amounts of N taken up over a 6-month period by perennial ryegrass grown in pots under uniform environmental conditions. Relationships between availability and the distribution of soil N amongst various fractions were then examined using multiple regression. The relationship: available soil N (mg kg^–1 dry soil)=(N_min×0.672)+(N_inc×0.840)+(N_mom×0.227)–5.12 was found to account for 91% of the variance in available soil N, where N_min=mineral N, N_inc=N mineralized on incubation and N_mom=N in macro-organic matter. The N mineralized on incubation appeared to be derived largely from sources other than the macro-organic matter because these two fractions were poorly correlated. When availability was expressed in terms of available organic N as % of soil organic N (N_ao) the closest relationship with other soil characteristics was: N_ao=[N_inc×(1.395–0.0347×CN_mom]+[N_mom×0.1416], where CN_mom=CN ratio of the macro-organic matter. This relationship accounted for 81% of the variance in the availability of the soil organic N.The conclusion that the macro-organic matter may contribute substantially to the available N was confirmed by a subsidiary experiment in which the macro-organic fraction was separated from about 20 kg of a grassland soil. The uptake of N by ryegrass was then assessed on two subsamples of this soil, one without the macro-organic matter and the other with this fraction returned: uptake was appreciably increased by the macro-organic matter.     

Cytosolic calcium, calcium fluxes, and regulation of alveolar macrophage superoxide anion production

The recently available compound quin-2, which acts as a high affinity fluorescent indicator for calcium in the cytosol, was used to examine the role of calcium mobilization in the alveolar macrophage during the stimulation of 0-2 production by the tripeptide N-formyl norleucyl leucyl phenylalanine (FNLLP). After preloading with quin-2, the production of 0-2 was measured in conjunction with the transfer of 45Ca+2 and changes in quin-2 fluorescence upon stimulation with FNLLP. When cells were maintained in low (10 microM) extracellular calcium medium the presence of 1.5 mM quin-2 in the cytosolic space partially inhibited the rate of 0-2 production upon stimulation by FNLLP. Addition of 1 mM Ca+2 to the medium prior to stimulation rapidly restored the cell's capability to produce 0-2 upon stimulation at rates equal to control and extended the duration of stimulated 0-2 production as well. Quin-2 fluorescence measurements indicated an increase in cytosolic Ca+2 upon stimulation with FNLLP. This increase was lowest under conditions in which 0-2 production was inhibited. The addition of 1 mM Ca+2 to the medium caused by itself a rapid but transient increase in cytosolic Ca+2 as measured with quin-2 without stimulating 0-2 production. This intracellularly redistributed calcium was determined to be the source of the greater increase in cytosolic calcium during stimulation in the presence of high extracellular calcium. Measurements of 45Ca+2 transfer demonstrated a buffering of cytosolic Ca+2 changes by quin-2, which in low calcium medium could deplete calcium stores. It is suggested that this effect, prior to stimulation, was responsible for the mitigated 0-2 response for those cells maintained in low calcium medium, wherein calcium stores could not be replenished. These results suggested that the cell's mechanism for regulating cytosolic and bound calcium concentrations may also play an integral role in its normal mechanism for stimulated 0-2 production. They further support the postulate that the commonly observed rise in the concentration of calcium in the cytosol upon formyl peptide stimulation is a concomitant but nonregulatory event only.     

Biochemical changes associated with the mechanism controlling superoxide radical formation in the aging rotifer

Levels of the superoxide radical (SOR) and lipid peroxides were measured and found to increase during aging in the short-lived rotifer, Asplanchna brightwelli. Life-span was altered by changes in environmental temperature, absence of light, diet restriction, exposure to ultraviolet radiation, and addition of vitamin E to the diet. Each of the conditions that lengthened life-span decreased SOR and lipid peroxide levels, and each condition that shortened life-span increased levels of SOR and lipid peroxides. Additional experiments indicated that on the third day of age, there was a significant increase in Ca2+ uptake and phospholipase A2 activity in membrane samples and an elevation in superoxide dismutase and catalase activity in rotifer homogenates. In addition, SOR concentration was inhibited by the addition of bromophenacyl bromide and indomethacin to membrane samples. By day 5 there was also a significant increase in the lysosomal enzyme, alpha-mannosidase. The results of this study indicate that levels of the SOR and lipid peroxides are coupled to rotifer life-span and that activation of phospholipase A2 may contribute to the elevation of these agents in older animals.     

Inhibition of neutrophil sulfhydryl groups by choloromethyl ketones. A mechanism for their inhibition of superoxide production

The inhibition of O2- production by serine protease inhibitors such as chloromethyl ketone derivatives, has been used as evidence to indicate that protease activity is essential for the production of O2- by neutrophils. However, chloromethyl ketones are potent inhibitors of sulfhydryl groups. This study demonstrates that chloromethyl ketones inhibited non-protein sulfhydryl groups as well as O2- production by human neutrophils stimulated with phorbol myristate acetate (PMA). Their inhibition of O2- production could be prevented by reduced glutathione. The results suggest that inhibition of O2- production by chloromethyl ketones is largely due to their inhibition of sulfhydryl groups.     

Changes in the superoxide production and other macrophage functions could be related to the mortality of mice with endotoxin-induced oxidative stress

Free radicals and proinflammatory cytokines from phagocytes have been implicated in the pathogenesis of endotoxic shock, a disease with high mortality caused by Gram-negative bacterial endotoxin. In the present study, male BALB/c and Swiss mice received intraperitoneally lipopolysaccharide (LPS) at 100 mg/kg and 150 mg/kg, respectively, that led to a lethal endotoxic shock (100 % of mortality before 30 h). Swiss mice injected with 100 mg/kg, that did not show lethal endotoxic shock, were also studied. Peritoneal macrophages were obtained from animals at 2, 4, 12 or 24 h after injection of LPS or saline (control) solutions. Superoxide anion and tumor necrosis factor (TNFalpha) production were determined in these cells as well as other functions such as adherence capacity, chemotaxis and phagocytosis. The increase in superoxide anion production after endotoxin injection was higher in cells from mice with lethal shock than in those with non-lethal shock. However, the enhancement of TNFalpha production was similar in all cases, although in Swiss mice the highest levels of TNFalpha were observed at 1.5 h after endotoxin injection, while in BALB/c mice they occurred at 2 h after LPS injection. This oxidative stress was also revealed by the other functions analyzed, since adherence to substrate and phagocytosis were stimulated and chemotaxis was decreased after endotoxin injection as compared to controls, the differences being even more significant in animals with lethal shock. These data suggest that these changes, mainly the increased production of free radicals even more than the TNFalpha release, could be involved in mouse mortality caused by LPS.     

Protein kinase C has both stimulatory and suppressive effects on macrophage superoxide production.

Unlike resident peritoneal macrophages (RPM) or tumor necrosis factor alpha (TNF alpha)-primed bone marrow-derived macrophages (BMM), unprimed BMM do not generate superoxide in response to the protein kinase C (PKC) activator, phorbol myristate acetate (PMA). However, these cells do contain significant levels of PKC activity. In contrast to PMA, zymosan induces the generation of superoxide in unprimed BMM, as well as in TNF alpha-primed BMM and RPM. Staurosporine, a potent PKC inhibitor, failed to affect the zymosan-induced production of superoxide by unprimed and TNF alpha-primed BMM and RPM, in spite of substantial inhibition of PMA-induced superoxide production by the primed BMM and RPM. However, when PKC was depleted from unprimed BMM by prolonged (24 h) treatment with phorbol dibutyrate (PdBt) (10(-7) M) the ability of zymosan to induce the production of superoxide was greatly diminished. Such a result could be interpreted as suggesting a role for PKC in the zymosan-induced response, a conclusion which contrasts with the inhibitor data. However, PKC depletion, in this case, is achieved via the PdBt-induced activation of PKC. It is thus possible that it is the initial activation of PKC, rather than its depletion, that suppresses superoxide production. Consistent with this interpretation, the co-stimulation of unprimed BMM with both zymosan and PMA resulted in a reduced superoxide release compared to zymosan alone. The activation of PKC therefore appears to have a suppressive effect on the generation of superoxide by unprimed cells. We thus conclude that PKC is not required for zymosan-induced superoxide production by either primed or unprimed macrophages and suggest that PKC may be involved in regulatory mechanisms restricting superoxide production by macrophages. However, since PMA alone can initiate the release of superoxide from primed BMM and RPM, it would appear that PKC can mediate both stimulatory and suppressive signals for macrophage superoxide production.     

Ultrastructural cytochemical properties of elastinassociated microfibrils and their relation to fibronectin

Summary The characteristics of elastin-associated microfibrils were investigated in the tunica adventitia of mouse aortas at the ultrastructural cytochemical level. The high iron diamine-thiocarbohydrazide-silver proteinate (HID-TCH-SP) method specific for sulphate groups was used with and without prior treatment ofen bloc specimens with either monopersulphate or cupric sulphite reagent. Amorphous elastin formed a clearly identifiable central core with microfibrils located both peripherally and interstitially. Sequential oxidation with monopersulphate and HID-TCH-SP demonstrated a characteristic staining for oxytalan fibres and intensely stained the microfibrils, whereas amorphous elastin stained weakly. Sequential thiosulphation with cupric sulphite and HID-TCH-SP for the demonstration of disulphide linkages and sulphydryl groups intensely stained microfibrils and weakly to moderately stained the amorphous elastin. This reactivity of the microfibrils was not altered by digestion with chondroitinase ABC, performed prior to or after treatment with either monopersulphate or cupric sulphite. In the specimens not exposed to either monopersulphate or cupric sulphite there was no definite HID-TCH-SP staining of microfibrils and amorphous elastin. Further, immunostaining with rabbit antibody specific for mouse fibronectin localized fibronectin in the microfibrils but not in the amorphous, elastin. These results indicate that elastin-associated microfibrils in mouse aorta lack stainable sulphate complex carbohydrates but are enriched with either disulphide or sulphydryl groups, or both, and further demonstrate the close correlation between these glycoproteins and fibronectin.     

Biochemical features of microbial keratinases and their production and applications

Keratinases are exciting proteolytic enzymes that display the capability to degrade the insoluble protein keratin. These enzymes are produced by diverse microorganisms belonging to the Eucarya, Bacteria, and Archea domains. Keratinases display a great diversity in their biochemical and biophysical properties. Most keratinases are optimally active at neutral to alkaline pH and 40–60°C, but examples of microbial keratinolysis at alkalophilic and thermophilic conditions have been well documented. Several keratinases have been associated to the subtilisin family of serine-type proteases by analysis of their protein sequences. Studies with specific substrates and inhibitors indicated that keratinases are often serine or metalloproteases with preference for hydrophobic and aromatic residues at the P1 position. Keratinolytic enzymes have several current and potential applications in agroindustrial, pharmaceutical, and biomedical fields. Their use in biomass conversion into biofuels may address the increasing concern on energy conservation and recycling.     

The mechanism of mitochondrial superoxide production by the cytochrome bc1 complex

Production of reactive oxygen species (ROS) by the mitochondrial respiratory chain is considered to be one of the major causes of degenerative processes associated with oxidative stress. Mitochondrial ROS has also been shown to be involved in cellular signaling. It is generally assumed that ubisemiquinone formed at the ubiquinol oxidation center of the cytochrome bc(1) complex is one of two sources of electrons for superoxide formation in mitochondria. Here we show that superoxide formation at the ubiquinol oxidation center of the membrane-bound or purified cytochrome bc(1) complex is stimulated by the presence of oxidized ubiquinone indicating that in a reverse reaction the electron is transferred onto oxygen from reduced cytochrome b(L) via ubiquinone rather than during the forward ubiquinone cycle reaction. In fact, from mechanistic studies it seems unlikely that during normal catalysis the ubisemiquinone intermediate reaches significant occupancies at the ubiquinol oxidation site. We conclude that cytochrome bc(1) complex-linked ROS production is primarily promoted by a partially oxidized rather than by a fully reduced ubiquinone pool. The resulting mechanism of ROS production offers a straightforward explanation of how the redox state of the ubiquinone pool could play a central role in mitochondrial redox signaling.     

Mechanisms for sister chromatid exchanges and their relation to the production of chromosomal aberrations

By taking advantage of the fact that fluorescent light (FL) induces strand breaks only in bromodeoxyuridine(BrdU)-substituted DNA, and that those breaks eventually lead to the formation of sister chromatid exchanges (SCEs), the response of SCEs to FL was studied carefully in Chinese hamster chromosomes in which, out of four DNA strands, BrdU-substitution had occurred either in one or three strands. The FL-induced SCE frequency did not differ greatly between these two types of chromosomes. However, when they were submitted to caffeine treatment, a drastic increase in the frequency was detected in the trifilarly-substituted chromosomes while a significant decrease occurred in the unifilarly-substituted chromosomes. Based on these results, a working hypothesis was developed that the SCE can arise by at least two different mechanisms, one operating at replicating points probably utilizing the machinery of DNA replication, and the other acting only in the post-replicational DNA portion, probably in a similar fashion as assumed in a general model of crossing over in the eukaryote. These dual mechanisms may account for the discrepancy encountered in the explanations of the induction of SCEs by various exogenous agents as well as spontaneous SCEs. The present study also showed that some, but clearly not all, of chromatid deletions are the outcome of the failure to complete SCEs arising through these mechanisms.     

Biochemical properties of Shigella flexneri and their practical significance

As the result of the study of 921 S. flexneri strains 1-6 and 4 (IV: 7,8), isolated in 31 regions of the USSR in 1975-1984, their biochemical characterization by 33 tests was made. All the strains under study proved to be typical in most of their constant signs, only some of strains 2a showed deviations in mannitol and some of strains 4a, in acetate. In strains of serovar 6, circulating in the USSR, specific features with respect to dulcitol and xylose were noted. The possibility of the biochemical subserovar typing of S. flexneri 1-5, X- and Y-var., with respect to maltose, arabinose, sorbitol and rhamnose was confirmed.