The Lipid Peroxidation Product 4-Hydroxynonenal is Formed by - and is able to Attract - Rat Neutrophils in vivo

4-Hydroxynonenal (HNE), a major aidchydic product of lipid peroxidation, is a chemoattractant for neutrophilic polymorphonuclear granulocytes in vitro. The question was studied, whether HNE is formed during the ingress of neutrophils in the Sephadex model of inflammation. The polydextrane Sephadex G-200, which causes an acute aseptic traumatic inflammation, was injected subcutaneously into rats. The implants were excised 6-36 hours later, and the neutrophils separated from the exsudate by centrifugation. After extraction with dichloromethane HNE was identified in the exsudate by non-derivative reversed phase HPLC in combination with on-line uv-spectroscopy. The concentration of HNE in the inflammatory focus did not correlate with the number of neutrophils present. While the peak of HNE coincided with the time point of the highest turnover rate of neutrophils (0.13 μM at 6 hrs after implantation), the highest number of neutrophils (about 100 million cells) occurred not earlier than 18 hrs later (24 hrs after onset of inflammation).

When neutrophils were isolated from the inflammatory focus and stimulated with Zymosan, they were able to produce HNE in vitro depending on the time of isolation. The highest production of HNE (0.17 μM) by phagocyting neutrophils was observed at the shortest inflammation time studied (3 hrs). In order to compare these results with the oxidative burst of neutrophils the formation of superoxide was also measured by the cytochrome c reduction assay in vitro. The maximum of the production rate of superoxide anion was observed at the same inflammation time (6 hrs), when the HNE maximum occurred. Cells which ingressed earliest (at 3 hrs) showed the highest production rate of superoxide per cell (307 × 10^-18 moles per cell and 30min).

The ability of HNE to attract neutrophils in vivo was studied by adding synthetic HNE to the Sephadex gel and measuring the ingression of neutrophils afterwards. The application of 1 μM HNE in the focus did not change the number of neutrophils but 10 μM HNE increased the cell number by a factor of 3.

The results indicate that HNE is not only a chemoattractant for rat neutrophils in vitro but also in vivo. It is suggested that HNE is produced by selfdestruction of neutrophils during a traumatic inflammation and its production seems to be tightly connected to the oxidative burst of neutrophils. The idea of HNE as part of an autocatalytic cycle is supported whereby neutrophils which immigrate into an inflammatory focus produce HNE which stimulates the ingress of new neutrophils.     

Plasma Corticosterone, Motor Activity and Metabolic Circadian Patterns in Streptozotocin-Induced Diabetic Rats

Experiments were conducted in male rats to study the effects of streptozotocin-induced diabetes on circadian rhythms of (a) plasma corticosterone concentrations; (b) motor activity; and (c) metabolic patterns. Animals were entrained to LD cycles of 12: 12 hr and fed ad libitum.

A daily rhythm of plasma corticosterone concentrations was found in controls animals with peak levels at 2400 hr and low values during the remaining hours. This rhythm was statistically confirmed by the cosinor method and had an amplitude of 3.37μg/100 ml and the acrophase at 100 hr. A loss of the normal circadian variation was observed in diabetic animals, with a nadir at the onset of light period and high values throughout the remaining hours; cosinor analysis of these data showed no circadian rhythm, delete and a higher mean level than controls.

As expected, normal rats presented most of their motor activity during the dark period with 80+ of total daily activity; the cosinor method demonstrated a circadian rhythm with an amplitude of 60+ of the mean level and the acrophase at 0852 hr. Both diabetic and control rats showed a similar activity during the light phase, but diabetic animals had less activity than controls during the night and their percentage of total daily activity was similar in both phases of the LD cycle (50+ for each one). With the cosinor method we were able to show the persistence of a circadian rhythm in the motor activity of diabetic rats, but with a mesor and amplitude lower than in controls (amplitude rested at 60+ of the mean level) and its acrophase advanced to 0148 hr.

The metabolic activity pattern of diabetic rats also changed: whereas controls showed a greater metabolic activity during the night (70+ food; 82+ water; 54+ urine; 67+ faeces), diabetics did not show differences between both phases of the LD cycle. Water ingested and urine excreted by the diabetic group were higher than normal during light and dark periods; food consumed and faeces excreted were higher than controls only in the light phase.

These data suggest that alterations in circadian rhythms of plasma corticosterone and motor activity are consecutive to the loss of the feeding circadian pattern, due to polyphagia and polydipsia showed by these animals, which need to extend intakes during the light and dark phases.     

Lipid Peroxidation in the Cortex and Medulla of Rabbit Kidneys Subjected to Cold Ischaemia and the Value of Protective Agents

The storage of rabbit kidneys for 24hr at 0 C in isotonic saline resulted in significantly increased rates of lipid peroxidation, as measured by the formation of thiobarbituric acid-reactive material and Schiff bases during in vitro incubation of homogenates prepared from the cortex and medulla. In addition, the content of thiobarbituric acid-reactive material in the medulla was also significantly elevated as a result of cold storage for 24 hr.

The effects of antioxidants (vitamin E), iron-chelation (desferoxamine) and inhibitors of arachidonic acid oxidation (indomethacin and dazmegrell on the rate of lipid peroxidation in homogenates prepared from ischaemic kidneys were studied. This demonstrated that lipid peroxidation in the cortex was predominantly non-specific and iron-catalysed whereas in the medulla approximately 50% of the TBA-reactive material was formed enzymically from arachidonic acid by cyclooxygenase.     

A Bioreactor-Crystallizer for L-Malic Acid Production

A bioreactor with associated crystallizer for the accumulation of a highly concentrated slurry product has been developed and investigated. The transformation of Ca-fumarate to Ca-L-malate by the action of the fumarase of immobilized Brevibacterium flavum cells focussed on the performance of this newly-devised bioreactor-crystallizer system.

The following results were obtained

(1) The fumarase reaction in the bioreactor proceeded at a rate that was first-order in apparent substrate concentration.

(2) The reaction rate increased with the addition of Na₂-fumarate to the substrate solution.

(3) The reaction rate was independent of the substrate circulation rate and the initial substrate concentration in the crystallizer.

(4) Fumarase activity of immobilized B. flavum cells was stable after 10 repeated uses over a period of 10 days.

(5) Maximum concentration of the product, final conversion ratio of the substrate and the productivity of the bioreactor-crystallizer system were much higher than those for a conventional bioreactor using solubilized Ca-fumarate as a substrate.     

Superoxide Radical Generation by Amadori Compounds

Several D-sugars were incubated with L-lysine or with L-arginine for 10 days. The resulting compounds are able to reduce nitrobluetetrazolium (NBT). This is prevented by superoxide dismutase (SOD), indicating that the superoxide radical is generated by the resulting Amadori compounds.

The formation of superoxide radical in vivo, as a result of nonenzymatic glycosylation of proteins, may be considered to be a contributory factor to the appearance of chronic complications of diabetes.     

The Effect of Substrate Hydrophobicity on the Kinetic Behaviour of Immobilized Candida rugosa Lipase

Candida rugosa lipase (EC 3.1.1.3.) was immobilized in a hydrophilic polyurethane foam and used in the hydrolysis of olive oil, in H-hexane. The results obtained were compared with those from a previous study, in which the same lipase preparation was used in the esterification of ethanol with butyric acid.

The initial rate of hydrolysis increased exponentially with increasing olive oil concentration. In contrast, for the esterification reaction, Michaelis-Menten kinetics with inhibition by both substrates, had been observed.

The effect of medium viscosity, stirring conditions and size of immobilization particles could not explain the observed kinetics of the hydrolytic reaction. However, a direct relationship was observed between the log P values of the reaction medium and the initial rate of hydrolysis, i.e., activation of the immobilized Candida rugosa lipase appears to be promoted by a high hydrophobicity of the reaction medium.

In the case of the esterification reaction, no similar correlation was found.     

Inhibition of oxidative DNA damage in vitro by extracts of brussels sprouts

Cruciferous vegetables have cancer preventive effects which may be due to reduction of oxidative DNA damage. We investigated the effect of an aqueous extract of cooked Brussels sprouts on formation of 7-hydro-8-oxo-2'-deoxyguanosine (8-oxodG) in calf thymus DNA in vitro. Damage was induced by a Fenton reaction, UVC (254 nm), UVA (365 nm), sunlamp light, and methylene blue with visible light.

The extract inhibited 8-oxodG formation in all systems except visible light with methylene blue. The IC₅₀ values were 6-20 μg/ml corresponding to the extract of 5-20 g of Brussels sprouts distributed in a volume of 50 L. The protective effect in the Fenton reaction was unaffected by addition of EDTA. After HPLC separation fractions were identified with similar DNA protective effects. Sinigrin, a glucosinolate abundant in Brussels sprouts, co-eluted with the most effective fraction and had DNA protective effects. In comparison with other antioxidants the patterns of effect of the extract in the five damage systems were more similar to that of sodium azide than to those of dimethylsulfoxide and vitamin C.

Constituents of Brussels sprouts can protect DNA by direct scavenging, e.g. hydroxyl radical and other oxidants, without prooxidant effects at concentrations potentially achievable by modest intake of the vegetable.     

On the Role of Energy Metabolism in Neurospora Circadian Clock Function

Neurospora crassa (bdA) mycelia were kept in liquid culture. Without rhythmic conidiation the levels of adenine nucleotides undergo circadian changes in constant darkness. Maxima occur 12-17 hr and 33-35 hr after initiation of the rhythm, i.e., at CT 0-6 hr. Pulses of metabolic inhibitors such as vanadate (Na₃Vo₄), molybdate (Na₂MoO₄: 2 H₂O), N-ethylmaleimide (NEM), azide (NaN₃), cyanide (NaCN) and oligomycin phase shift the circadian conidiation rhythm of Neurospora crassa. Maximal advance phase shifts are observed at about CT 6 with all inhibitors.

Pulses of N,N'dicyclohexylcarbodiimide (DCCD) and light phase shift the conidiation rhythm following a phase response curve different from those of the other agents (maximal advance at about CT 18-24). The phase shifts with DCCD and light are significantly larger in the wild type compared to the mitochrondrial mutant poky. Such differences are not found in PRCs of the protein synthesis inhibitor cycloheximide.

[³¹P] NMR spectra of wild type Neurospora crassa and the clock mutants frq 1 and frq 7 which differ in their circadian period lengths did not reveal differences in the concentrations of adenine nucleotides, pyridine nucleotides or sugar phosphates. Starvation causes drastic changes of the levels of adenine nucleotides, phosphate and mobile polyphosphate without effecting phase or period length of the circadian rhythm.     

Studies on the Preparation and Recoloration of Fuchsin Sulfurous Acid

Some of the factors affecting the recoloration of Schiff's Reagent (fuchsin sulfurous acid or FSA) by formaldehyde have been studied spectrophotometrically to determine the optimal conditions for the reaction of this reagent with aldehydes.

Of the various reducing agents utilized in the preparation of the leuco dye from basic fuchsin, sodium sulfite and bisulfite proved to be the most satisfactory for obtaining in the reagent maximal sensitivity to recoloration with minimal quantitative variation of results.

The relative proportions of reducing agent and basic fuchsin present in die leuco dye determine its sensitivity to recoloration. Under the conditions of the present experiments, greatest reagent recoloration was obtained when the leuco dye contained 0.01 mole of sodium bisulfite and 0.001 mole of basic fuchsin per 100 ml., a ratio of 10/1.

The recoloration of a given amount of FSA is related to the amount of aldehyde and the temperature of the reaction.

The present experiments indicate the desirability of standardizing the composition of FSA and the conditions under which it is used, if the results of different investigators are to be readily reproduced or compared.     

Circadian and Seasonal Variations of Plasma Insulin and Cortisol Concentrations in the Syrian Hamster, Mesocricetus Auratus

Circadian rhythms of plasma insulin, Cortisol, and glucose concentrations were examined in scotosensitive (reproductively sensitive to inhibitory effects of short daylengths) and scotorefractory male and female Syrian hamsters (Mesocricetus auratus) maintained on short (LD 10:14) and long (LD 14:10) daylengths. The baseline concentration (mean of all values obtained every 4 hr six times of day) of insulin was much greater in female than in male scotosensitive hamsters kept on short daylengths. These differences in insulin concentration may account for the observed heavy fat stores in female and low fat stores in male scotosensitive hamsters kept on short daylengths. The baseline concentrations of Cortisol were approximately equal in both scotosensitive and scotorefractory males held on short and long daylengths, but were relatively low in females held on short daylengths and especially high in scotorefractory females held on long daylengths.

The plasma concentrations of both cortisol and insulin varied throughout the day in many of the groups tested. However, the variations were not equivalent. The circadian variations of cortisol were similar irrespective of sex, seasonal condition and daylength. Peak concentrations generally occurred about 12 hr after light onset. In contrast, the circadian variations of insulin differed markedly. For example in male hamsters, robust daily variations were found in scotosensitive hamsters held on short daylengths but not on long daylengths and in scotorefractory hamsters held on long daylengths but not on short daylengths. Furthermore, the daily peak occurred during the light in the scotosensitive hamsters and during the dark in the scotorefractory animals. Neither the daily feeding pattern (about 60% consumed during dark) nor the daily variations of glucose concentration varied appreciably with seasonal condition or daylength. They do not appear to determine nor directly reflect the variations in cortisol and glucose concentrations. It is postulated that the daily rhythms of cortisol and insulin are regulated by different neural pacemaker systems and that changes in the phase relations of circadian systems account in part for seasonal changes in body fat stores.     

Oxidative Effects of Iron on Erythrocytes

In this work we have investigated the effects of iron-induced free radical formation in normal human erythrocytes in vitro, as a model system for studying iron damage, and in erythrocytes from patients with β-thalassaemia major. The resulting oxidative effects were measured in terms of methaemoglobin formation and reduced glutathione loss. The effects of desferrioxamine, an iron-chelating agent, were also investigated.

The results show that the increased methaemoglobin formation after iron-induced oxidative stress is consistent with a decline in the intracellular glutathione levels and that this process is inhibited by desferrioxamine. Similar treatment of red cell haemolysates produces less methaemoglobin. This suggests that, on exposure of intact erythrocytes to iron-induced free radical effects, the red cell membrane exacerbates the breakdown of the antioxidant defences of the cell and the oxidation of haemoglobin.     

Commentary: The Role of Iron-Sulfur Clusters in in vivo Hydroxyl Radical Production

The in vivo production of HO- requires iron ions, H₂O₂ and O₂- or other oxidants but probably does not occur through the Haber-Weiss reaction. Instead oxidants, such as O₂-, increase free iron by releasing Fe(II) from the iron-sulfur clusters of dehydratases and by interfering with the iron-sulfur clusters reassembly. Fe(II) then reduces H₂O₂, and in turn Fe(III) and the oxidized cluster are re-reduced by cellular reductants such as NADPH and glutathione. In this way, SOD cooperates with cellular reductants in keeping the iron-sulfur clusters intact and the rate of HO- production to a minimum.

O₂- and other oxidants can release iron from Fe(II)-containing enzymes as well as copper from thionein. The released Fe(III) and Cu(II) are then reduced to Fe(II) and Cu(I) and can then participate in the Fenton reaction.

In mammalian cells oxidants are able to convert cytosolic aconitase into active IRE-BP, which increases the “free” iron concentration intracellularly both by decreasing the biosynthesis of ferritin and increasing biosynthesis of transferrin receptors.

The biological role of the soxRS regulon of Escherichia coli, which is involved in the adaptation toward oxidative stress, is presumably to counteract the oxidative inactivation of the iron clusters and the subsequent release of iron with consequent increased rate of production of HO.     

Influence of Igepal reverse micellar and micellar systems on activity and stability of heme peroxidases

The activities of horseradish peroxidase (HRP) and lactoperoxidase (LPO) entrapped in reverse micelles of Igepal CO-520 in cyclohexane were studied. When the molar ratio of water to surfactant, w ₀ was ≥13, the activity of HRP encapsulated in the water pool of the reverse micelle was comparable with that measured in buffer. For LPO, however, lower activity was observed after its incorporation into the same system.

The activity of the investigated peroxidases was also measured in an aqueous solution of Igepal CO-720 or after incubation with this surfactant. The enzymes became inactivated in an aqueous micellar solution of Igepal CO-720, although this process was reversible.

The stability of HRP and LPO at 37 or 50°C was lower in the micellar systems than in buffer with the exception for HRP in reverse micelles at 50°C.     

Rapid reset of the corticosterone rhythm by food presentation in rats under acircadian restricted feeding schedule

Corticosterone levels were determined in the 7-week-old male rat maintained under different feeding and lighting schedules. At 4 weeks of age, the animals were kept either under a natural photoperiod (LD) or were subjected to continuous illumination (LL). Access to food was either ad libitum or restricted to an 8 hr span per 24 hr (circadian) or 32 hr (acircadian).

The food signal seemed able to synchronize the corticosterone rhythm to its own circadian periodicity, irrespective of the lighting regimen. No synchronization was observed in serially sampled LL or LD rats under an acircadian feeding schedule. Instead, the group acrophase appeared 24 hr subsequent to food presentation. Regarding individual patterns, many rats showed an acrophase or a peak also at that time. We speculate that an endogenous circadian mechanism was reset by the food signal, whenever it appeared.     

The internal synchronization of five circadian functions of the rabbit

Five different physiological functions of the rabbit (hard faeces and urine excretion, food and water intake and locomotor activity) were registered during LD 12:12 and during continuous light conditions (LL).

(1) In LD 12:12 a strong synchronization of the five parameters existed. The minima of all functions consistently occurred during the hours of light. The nocturnal percentage of overall 24-hr events was increased significantly in 'hard faeces excretion' (66±8 (S.D.) %), 'water intake' (64±15 (S.D.) %) and 'urine excretion' (58±10 (S.D.) %). The nocturnal percentage of locomotor activity was significantly increased during the dark-hours in 9 out of 14 animals. In the other five individuals prominent peaks were present even during the photoperiod. On the average of all 14 animals 5S±13 (S.D.) % of the 24 hr events of locomotor activity occurred during the night. Despite a trough during the cessation of hard faeces excretion the events of food intake were not elevated significantly during the dark hours.

(2) During LL the synchronization of the five functions within each animal persisted during the complete 90-day LL period. A free-running circadian rhythm with-: = 24.8±0.5 (S.D.) hr was present in the four rabbits kept in LL conditions within 5-16 days after the withdrawal of the zeitgeber.

(3) In addition to the circadian period the power spectrum analysis of data obtained during LD 12:12 revealed significant ultradian periods of an average period length of 11,6 hr (hard faeces and urine excretion), 8 hr (food and water intake, locomotor activity) and 4 hr (food intake, locomotor activity). During the free-run ultradian periods of 8 and 3.2-4.2 hr were significant in almost all parameters.

(4) During LL the level of locomotor activity was reduced for 13±16 (S.D.) %, the events of food intake were increased for 17±12 (S.D.) %.

(5) The reinserted LD 12:12 zeitgeber re-entrained the circadian rhythms within 25-45 days.

(6) These results provided evidence of a predominant nocturnality of the rabbits under investigation.     

Oxidation of Low Density Lipoprotein Upon Sequential Exposure to Copper Ions

Copper-induced LDL oxidation is characterized by an 'induction phase' (lag phase) during which the endogenous antioxidants are consumed, followed by a 'propagation phase' in which the LDL-associated polyunsaturated fatty acids are oxidized. Oxidation products may play an important role in the propagation of the oxidative process in the arterial intima as they increase the permeability of the damaged endothelium to various plasma components, including LDL. We therefore found it of interest to investigate the kinetics of LDL oxidation in vitro under conditions where LDL is sequentially exposed to Cu²⁺-induced oxidation.

The results of our studies demonstrate that when native LDL is exposed to copper oxidation in a medium containing oxidized LDL, oxidation of the added LDL may be almost instantaneous. Furthermore, even when native LDL is added to 'oxidizing LDL' towards the end of the lag phase or during the propagation phase it becomes oxidized after a very short lag. This oxidation process, occurring in spite of the possible protective effect of the antioxidants present in the newly added LDL, indicates that although antioxidants prolong the latency period by preventing the formation of active free radicals, when such radicals are present in the system, oxidation propagates. These results lend strong support to the generally accepted paradigm regarding the mechanism of propagation of lipid oxidation.

In view of the effect of oxidation products on the permeability of the endothelium, the observed shortening of the lag period may result in a vicious cycle, independent of the LDL-associated antioxidants, leading to continuing oxidation and foam cell formation.     

Maturity and fertility of rhesus monkey oocytes collected at different intervals after an ovulatory stimulus (human chorionic gonadotropin) in in vitro fertilization cycles

In rhesus monkeys undergoing ovarian stimulation for in vitro fertilization (IVF), a midcycle injection of human chorionic gonadotropin (hCG) substitutes for the LH surge and induces preovulatory oocyte maturation. The time interval between injection and oocyte collection, ideally, allows for the completion of oocyte maturation without ovulation, which would reduce the number of oocytes available for harvest. To evaluate the influence of this time interval on oocyte parameters following hCG administration, we conducted a series of gonadotropin treatment protocols in 51 animals in which the interval from hCG administration to follicular aspiration was systematically varied from 27 to 36 hr. Follicle number and size, evaluated prior to hCG administration by sonography, did not vary significantly or consistently with preovulatory maturation time. Oocytes were harvested by laparotomy or laparoscopy, and scored for maturity before insemination. The percentage of mature, metaphase II (MII) oocytes at recovery increased significantly with increasing preovulatory time and was inversely proportional to that of metaphase I (MI) oocytes. However, oocyte yield tended toward a progressive decrease with increasing preovulatory maturation times from a high of 27 oocytes at 27 hr to a low of 17 oocytes/animal at the 36 hr time interval. Fertilization levels declined significantly from a high of 50% at 27 hr to a low of 30% at 36 hr. Thus, although higher percentages of mature oocytes were recovered at the longer time intervals, optimal oocyte/embryo harvests were realized after the shorter time intervals (27 and 32 hr) and are most compatible with the goal of achieving high yields of fertile oocytes and embryos following gonadotropin stimulation in rhesus monkeys. © 1996 Wiley-Liss, Inc.     

Technical Procedures for the Study of the Nucleolonema

In a recent paper¹ the authors have shown filamentous structures within the nucleolus of all cells. This structure was named the nucleolonema. In the present paper the technical procedures which prove their existence are described in detail.

There are two groups: a) Silver impregnation procedures and iron-pyrogallic stain for fixed material; b) phase contrast microscopy, dark-field illumination and oblique transillumination for fresh material.

The suitable objects are mentioned in every case in order to obtain the best results.     

α-Chymotrypsin Immobilized on Chitin. Relationships Between the Enzyme Kinetic Constant and Support Structure

-Chymotrypsin was immobilized on chitin from squills, lobsters and prawns by means of glutaraldehyde. Hydrolase and peptide synthetase activities were determined in aqueous and homogeneous aqueous-organic media, respectively.

The results show -chymotrypsin immobilized on chitin from prawn to be the most active immobilized derivative based on its synthetase activity (90% yield of Bz-Tyr-Leu-NH₂ in carbonate buffer, p^H 9 containing 70% 1,4- butanediol).

The relationship between the kinetic constant of hydrolysis and chitin structure was also studied. -Chymotrypsin immobilized on prawn chitin was found to be the best derivative in kinetic terms.

The stability of the three derivatives was studied at 37C.     

Olencira praegustator (Crustacea, Isopoda, Cymothoidae) parasitic on Brevoortia species (Pisces, Clupeidae) from the southeastern coasts of North America: review and re-description

The cymothoid isopod Olencira praegustator (Latrobe, 1802), the sole species of the genus Olencira Leach, 1818, has been the subject of several previous studies, but has remained incompletely described. This species is re-described and figured from the Atlantic menhaden Brevoortia tyrannus (Latrobe, 1802), the type host from the estuarine systems of the South Carolina coast, USA. The general morphology, mouthparts and appendages of the female, male and other stages of its lifecycle are described.

The geographical distribution and hosts recorded for O. praegustator are reviewed. Biological data are summarized and discussed. Areas that need additional studies include the infection process and the effect of the parasite on its host.