Potential Toxicological Problems Associated with Antioxidants in Plastics and Rubber Consumables

Antioxidants and stabilisers are of critical importance in the manufacture and use of polymer artifacts. Most conventional additives are readily leached from plastics or rubbers by foodstuffs or body fluids with consequent danger associated with the potential toxicity in the body. Strategy for the development of biologically “safe” stabilisation systems for polymers are discussed and it is concluded that the attachment of the appropriate reactive functional group to the polymer backbone by “reactive processing” provides the best means of immobilising the functional group to leaching fluids without sacrificing its protective action.     

Interim Report of the Committee on Histologic Mounting Media: Resinous Media

The Fisher “Permount” naphthalene polymer, the Hartman Leddon “H.S.R.” terpene polymer resin, a Monsanto polystyrene P-1, the Will Corporation “Diaphane” and “Green Diaphane”, and the du Pont “Lucite” methyl methacrylate polymer were examined, and the possibility of use of some other plastics was also explored. The first 5 mentioned were tested for color preservation of a variety of stains in comparison with Canada balsam and Clarite X. From this point of view polystyrene, the Hartman Leddon “H.S.R.” and the Fisher “Permount” resins were the most satisfactory, then the “Diaphanes”. Both “Permount” and “H.S.R.” show some yellowing. The H.S.R. with a melting point of 115°C, the Permount with 150°C. melting point, and the Polystyrene with a thermal denaturation point above 220°C. all excell Canada balsam in heat resistance. Trimethylbenzene, cymene and monoamylbenzene appear to be the best solvents for polystyrene. Mounts made in a solution of 20 g. polystyrene in 100 ml. trimethylbenzene can be packed flat slide to slide in 24 hours after mounting without sticking together.

This report is not intended to deprecate the use of other resinous mounting media which have not as yet been tested or compared with those mentioned herein.     

A comparison of eating habits between retired or semi-retired aged subjects and younger subjects in full-time work

An analysis of eating habits in older retired subjects (“No Work group”) and younger subjects employed in full-time work (“Work group”) has been carried out. It used a questionnaire that assessed why individuals chose to eat or not to eat meals during the course of the day, and subjective responses to the meals. The questionnaire was answered every three hours over a “typical week” which, for the Work group, entailed working during the weekdays and resting at the weekend. For the “No Work” group, breakfast was the most frequently taken meal of the day whereas, for the “Work” group, this meal was often missed. Patterns of meal intake were not significantly different between the weekdays and weekend for the “No Work” group, but the “Work” group ate more hot food at the weekend. The reasons cited for eating/not eating a meal and for choosing the type of meal eaten were dominated by hunger/lack of hunger in both groups. In addition, whereas habits were also important for the “No Work” group, it was time availability or the lack of it that was of major importance to the Work group, though this was significantly less important at the weekend. Meals which required more time for preparation and cooking were appreciated significantly more (appetite before the meal, enjoyment during it, and satiety afterwards) than meals such as snacks and cold food, which could be prepared more quickly. Some implications of these results, with regard to regular meals acting as a social zeitgeber in older subjects and the additional constraints imposed upon night workers, are considered.     

Comparison of the Antimicrobial Adhesion Potential of Human Body Fluid Glycoconjugates Using Fucose-Binding Lectin (PA-IIL) of Pseudomonas aeruginosa and Ulex europaeus Lectin (UEA-I)

Pseudomonas aeruginosa produces a fucose-binding lectin (PA-IIL) which strongly binds to human cells. This lectin was shown to be highly sensitive to inhibition by fucose-bearing human milk glycoproteins. Since the glycans of these glycoproteins mimic human cell receptors, they may function as decoys in blocking lectin-dependent pathogen adhesion to the host cells. Human saliva and seminal fluid also contain such compounds, and body fluids of individuals who are “secretors” express additional fucosylated (alpha 1,2) residues. The latter are selectively detected by Ulex europaeus lectin UEA-I. The aim of the present research was to compare the PA-IIL and UEA-I interactions with human salivas and seminal fluids of “secretors” and “nonsecretors” with those obtained with the respective milks. Using hemagglutination inhibition and Western blot analyses, we showed that PA-IIL interactions with the saliva and seminal fluid glycoproteins were somewhat weaker than those obtained with the milk and that “nonsecretor” body fluids were not less efficient than those of “secretors” in PA-IIL blocking. UEA-I, which interacted only with the “secretors” glycoproteins, was most sensitive to those of the seminal fluids.     

Method for Treating and Processing Whole Chick Embryos for Autoradiography, Immunocytochemistry and other Techniques

A method is presented for In situ treatment of whole chick embryos with drugs and immunocytochemical and fixative reagents that resembles conditions “in ovo.” The chick embryo is placed in a “shell-less” culture system where it is contained by an agar ring allowing for treatment in vivo. The conceptus (embryo + membranes) is then mounted on a microporous membrane and inserted into a filter device connected to a three-way stopcock that permits fluids to be changed using syringes. The embryo is then processed in toto or after embedding and sectioning for light or electron microscopy. The proposed handling system decreases technical artifacts and changes in the topographic microanatomy produced by conventional manipulation of chick embryos. This method is useful also for directly observing and recording changes in the embryo during drug treatments and allows processing with dangerous reagents without their direct contact with the operator. It is simple, inexpensive and requires only minimal technical training.     

The Pragmatic Approach to Masking

This paper advocates the use of a pragmatic approach to the problem of masking in real-life situations involving an abrupt change in the timing of sleep, i.e. shiftwork and “jet-lag” situations. Although “pure” chronobiological research has pointed to the importance of taking masking effects into account, the techniques that it has provided for doing so are extremely difficult to apply in real-life situations. The approach advocated here is based on Wever's pioneering work, and involves estimating the normative endogenous and exogenous components of the circadian rhythm in body temperature. These estimates are then used to: (a) simulate the results of shiftwork studies; and (b) to “remove” the exogenous component in “jet-lag” studies to allow analysis of the estimated endogenous component. The simulated curves obtained cross-correlated extremely highly with published night-shift temperature curves, while the “removal” of the exogenous component resulted in very similar findings to those obtained in temporal isolation studies. It is concluded that this pragmatic approach to masking may prove extremely useful in interpreting the results of field studies of shiftwork and “jet-lag”.     

"Live memory" of the cell, the other hereditary memory of living systems

We propose to designate by the term “live memory” of the cell, the cytoplasmic memory. This phenomenon consists of non-genetic memory, but nevertheless includes transmission function, which may be “hereditary” via the ovum, from mother cell to daughter cell, or simply within the same cell from instant t to instant t + 1. To understand this notion of “live memory”, its role and interactions with DNA must be resituated; indeed, operational information belongs as much to the cell body and to its cytoplasmic regulatory protein components and other endogenous or exogenous ligands as it does to the DNA database. We will see in Section 2, using examples from recent experiments in biology, the principal roles of “live memory” in relation to the four aspects of cellular identity, memory of form, hereditary transmission and also working memory.     

Mitochondrial damage and intralysosomal degradation in cellular aging

Normal mitochondrial respiration is associated with a continuous production of superoxide and hydrogen peroxide, inevitably resulting in minor macromolecular damage. Damaged cellular components are not completely turned over by autophagy and other cellular repair systems, leading to a progressive age-related accumulation of biological “garbage” material, such as defective mitochondria, cytoplasmic protein aggregates and an intralysosomal undegradable material, lipofuscin. These changes primarily affect neurons, cardiac myocytes and other long-lived postmitotic cells that neither dilute this “garbage” by mitotic activity, nor are replaced by newly differentiated cells. Defective mitochondria are insufficient in ATP production and often generate increased amounts of reactive oxygen species, further enhancing oxidative stress. Lipofuscin-loaded lysosomes, in turn, poorly turn over mitochondria that gradually leads to the overload of long-lived postmitotic cells with “garbage” material, decreased adaptability and eventual cell death.     

Temperature profiles, and the effect of sleep on them, in relation to morningness-eveningness in healthy female subjects

There were 15 healthy female subjects, differing in their position on the “morningness-eveningness” scale, studied for 7 consecutive days, first while living a sedentary lifestyle and sleeping between midnight and 08:00 and then while undergoing a “constant routine.” Rectal temperature was measured at regular intervals throughout this time, and the results were subjected to cosinor analysis both before and after “purification” for the effects of physical activity. Results showed that there was a phase difference in the circadian rhythm of core temperature that was associated with the morningness score, with calculations that “morning types” would be phased earlier than “evening types” by up to about 3h. This difference in phase (which was also statistically significant when the group was divided by a median split into a “morning group” and an “evening group”) could not be attributed to effects of waking activity and existed in spite of the subjects keeping the same sleep-wake schedule. Moreover, it persisted when the subjects' data had been purified and when the data were obtained from the constant routine. That is, there was an endogenous component to this difference in phase of the core temperature. The morning group also showed a greater fall of core temperature during sleep; this was assessed in two ways, the main one being a comparison of constant routine and nychthemeral data sets after correction for any effects of activity. Even though the morning group was sleeping at a later phase of their circadian temperature rhythm than was the evening group, neither group showed a fall of temperature due to sleep that varied with time elapsed since the temperature acrophase. It is concluded that another factor that differs between morning and evening types is responsible for this difference. (Chronobiology International, 18(2), 227-247, 2001)     

Substitutes for Ethyl Alcohol

Three substitute solvents were investigated as substitutes for ethanol in nine commonly used staining fluids. Anhydrous methanol (“Bioloid” grade, Will Corporation) was found an entirely satisfactory substitute in these fluids, and isopropanol only slightly inferior.     

Plasma Concentration of Nine Hormones and Neurotransmitters During Usual Activities or Constant Bed Rest for 34 H

Thyroid-stimulating hormone (TSH), Cortisol, melatonin, prolactin, luteinizing hormone (LH), delta-sleep-inducing peptide (DSIP), its phosphorylated form (P-DSIP), heart rate, and body temperature were measured every half hour during two 24-h periods in five normal men. τ-Amino-butyric acid (GABA) and 3-methoxy-4-hydroxyphenylglycol (MHPG) were measured less frequently. The first period, the “activity” condition, included usual daily activities. The second period, or “rest” condition, consisted of fasting, constant bed rest during 34 h, and partial light deprivation. Compared with the “rest” condition, the “activity” condition increased heart rate, temperature, LH, and TSH in most subjects, and Cortisol in two of five subjects. It retarded the onset of nocturnal Cortisol and melatonin secretion. The temporal pattern and the absolute values of the concentrations of DSIP, P-DSIP, MHPG, GABA, and prolactin showed no or minimal changes during the two conditions. In spite of the influence of the “activity” versus “rest” condition on several hormones, the mean concentrations as well as the temporal organization of their secretion into plasma were quite stable within each subject, whereas they varied much more between individuals. TSH, Cortisol, and melatonin values were also stable within an 8-month period in one subject who was studied on four occasions. The results illustrate that the patterns of hormones rhythms and their reactivity to changes in the environment are, to a large extent, specific to each subject.     

Sleep and Circadian Rhythms of Temperature and Urinary Excretion on a 22.8 hr “Day”

Two groups of subjects (total N = 6) were studied in an isolation chamber for a period of 3 weeks whilst living on a 22.8 hr “day”. Regular samples of urine were taken when the subjects were awake, deep body temperature was recorded continuously and polygraphic EEG recordings were made of alternate sleeps. The excretion in the urine of potassium, sodium, phosphate, calcium and a metabolite of melatonin were estimated.

Measurements of the quantity and quality of sleep were made together with assessments of the temperature profiles associated with sleep. In addition, cosinor analysis of circadian rhythmicity in urinary variables and temperature was performed.

The 22.8 hr “days” affected variables and subjects differently. These differences were interpreted as indicating that the endogenous component of half the subjects adjusted to the 22.8 hr “days” but that, for the other three, adjustment did not occur. When the behaviour of different variables was considered then some (including urinary potassium and melatonin, sleep length and REM sleep) appeared to possess a larger endogenous component than others (for example, urinary sodium, phosphate and calcium), with rectal temperature behaving in an intermediate manner. In addition, a comparison between different rhythms in any subject enabled inferences to be drawn regarding any links (or lack of them) that might exist between the rhythms. In this respect also, there was a considerable range in the results and no links between any of the rhythms appeared to exist in the group of subjects as a whole.

Two further groups (total N=8) were treated similarly except that the chamber clock ran at the correct rate. In these subjects, circadian rhythms of urinary excretion and deep body temperature (sleep stages and urinary melatonin were not measured) gave no evidence for deterioration. We conclude, therefore, that the results on the 22.8 hr “day” were directly due to the abnormal “day” length rather than to a prolonged stay in the isolation chamber.     

Regulation of estrogen receptor protein and messenger ribonucleic acid by estradiol and progesterone in rat uterus

The objective of this study was to examine the mechanisms of estrogen receptor (ER) processing and replenishment in the uterus of ovariectomized rats after estradiol and progesterone treatment. Uterine ER binding activity, ER protein and ER mRNA were measured by receptor binding exchange assay, Western blot and slot blot, respectively. The regulation of ER levels in rat uterus by estradiol and progesterone was very dramatic. Changes in ER protein were faithfully reflected by changes in binding activity. Estradiol caused receptor “processing” within 4 h of administration followed by recovery or “replenishment” of ER levels to the initial level by 20 h. The term “processing” has previously been used to describe the loss of ER binding activity in the early phase of estradiol-action, but it was never clear whether the ligand binding site was inactivated by processing or if the receptor molecule actually disappeared. This study shows that receptor “processing” constitutes disappearance of receptor protein and the later “replenishment” phase represents new ER protein rather than recycling of “processed” receptor. Progesterone-action, on the other hand, influenced only the “replenishment” phase by blocking recovery of ER protein. ER mRNA was suppressed by estradiol at 8 h, after the receptor was “processed” and “replenishment” already initiated. Progesterone, on the other hand, did not alter the steady state level of the message. Other mechanisms, such as regulation of translation rate of existing mRNA and changes in the rate of degradation of ER proteins are more likely involved in acute regulation of ER by these ovarian steroid hormones.     

The pet dogs ability for learning from a human demonstrator in a detour task is independent from the breed and age

There are many indications and much practical knowledge about the different tasks which various breeds of dogs are selected for. Correspondingly these different breeds are known to possess different physical and mental abilities. We hypothesized that commonly kept breeds will show differences in their problem solving ability in a detour task around a V-shaped fence, and also, that breed differences will affect their learning ability from a human demonstrator, who demonstrates a detour around the fence. Subjects were recruited in Hungarian pet dog schools. We compared the results of the 10 most common breeds in our sample when they were tested in the detour task without human demonstration. There was no significant difference between the latencies of detour, however, there was a trend that German Shepherd dogs were the quickest and Giant Schnauzers were the slowest in this test. For testing the social learning ability of dogs we formed three breed groups (“utility”, “shepherd” and “hunting”). There were no significant differences between these, all the breed groups learned equally well from the human demonstrator. However, we found that dogs belonging to the “shepherd” group looked back more frequently to their owner than the dogs in the “hunting” group. Further, we have found that the age of pet dogs did not affect their social learning ability in the detour task. Our results showed that the pet status of a dog has probably a stronger effect on its cognitive performance and human related behaviour than its age or breed. These results emphasize that socialization and common activities with the dog might overcome the possible breed differences, if we give the dogs common problem solving, or social learning tasks.     

Time-Qualified Reference Limits (Chronodesms) for 24-Hour Blood Pressure Values: Classical and Bayesian

Background - This paper is a concrete example of the problems raised by the need of constructing the time-qualified reference limits (chronodesms) for blood pressure (BP), in order to clinically estimate the hemodynamic parameter in its intrinsic nychtohemeral variability. Methods - Assuming that the noninvasive ambulatory BP monitoring (ABPM) is the eligible technique for this need, it must be realized that the BP chronodems may be of two types, depending on the sample being used for their calculation. The first type may be regarded as “ a priori ” because of the fact that they are derived by a sample of normotensive subjects who are unavoidably recruited via “ causal ” sphygmomanometric measurements and reclassified as normotensive by comparing their ABPM to the fixed reference limits (monodesms) given by WHO (monodiagnosis). Therefore, the “ a priori ” BP chonodesms are by principle derived by subjects who could not be correctly classified as normotensive, their ABPM being not tested versus the time-varying physiological limits. The second type may regarded as “ a posteriori ” in virtue of the fact that they may be constructed on a sample which contemplates the previous subjects who result to be true normotensive via the reassessment of their ABPM versus the “ a priori ” BP chronodesms (chronodiagnosis). The “ a posteriori ” chronodesms may be regarded as biometrically reliable, whether the sample for their construction is additionally constituted by those subjects of the local population who have been erroneously monodiagnosed as hypertensive, while they result to be true normotensive via the chronodiagnostic comparison of their ABPM versus the “ a priori ” BP chronodesms. Results - The biometric reliability of the “ a posteriori ” BP chronodems is demonstrated by the fact that their upper limits are statistically significantly less pronounced due to the fact that they are provided by a sample which has been depured by the falsely monodiagnosed normotensives. Conclusions - The “ a posteriori ” BP upper chronodesms are the time-qualified reference limits which should be used in clinical practice for the chronodiagnosis of hypertension.     

Some Introductory Comments on Silver Staining

Silver staining has become a versatile method for the visualization of specific cell structures and products. The similarity of the impregnation “nuclei” of reduced silver staining to the silver “specks” or “nuclei” of the latent image in photography is noted. “Physical” development (reduction of ionic silver in solution) in silver staining as compared to “chemical” development (reduction of ionic silver remaining in a silver halide crystal) in photographic procedures is briefly discussed.     

Nitric oxide and peroxynitrite. The ugly, the uglier and the not so good: a personal view of recent controversies

Nitric oxide, a gaseous free radical, is poorly reactive with most biomolecules but highly reactive with other free radicals. Its ability to scavenge peroxyl and other damaging radicals may make it an important antioxidant in vivo, particular in the cardiovascular system, although this ability has been somewhat eclipsed in the literature by a focus on the toxicity of peroxynitrite, generated by reaction of O^·-₂ with NO^· (or of NO^- with O₂). On balance, experimental and theoretical data support the view that ONOO^- can lead to hydroxyl radical (OH^·) generation at pH 7.4, but it seems unlikely that OH^· contributes much to the cytotoxicity of ONOO^-. The cytotoxicity of ONOO^- may have been over-emphasized: its formation and rapid reaction with antioxidants may provide a mechanism of using NO^· to dispose of excess O^·-₂, or even of using O^·-₂ to dispose of excess NO^·, in order to maintain the correct balance between these radicals in vivo. Injection or instillation of “bolus” ONOO^- into animals has produced tissue injury, however, although more experiments generating ONOO^- at steady rates in vivo are required. The presence of 3-nitrotyrosine in tissues is still frequently taken as evidence of ONOO^- generation in vivo, but abundant evidence now exists to support the view that it is a biomarker of several “reactive nitrogen species”. Another under-addressed problem is the reliability of assays used to detect and measure 3-nitrotyrosine in tissues and body fluids: immunostaining results vary between laboratories and simple HPLC methods are susceptible to artefacts. Exposure of biological material to low pH (e.g. during acidic hydrolysis to liberate nitrotyrosine from proteins) or to H₂O₂ might cause artefactual generation of nitrotyrosine from NO^-₂ in the samples. This may be the origin of some of the very large values for tissue nitrotyrosine levels quoted in the literature. Nitrous acid causes not only tyrosine nitration but also DNA base deamination at low pH: these events are relevant to the human stomach since saliva and many foods are rich in nitrite. Several plant phenolics inhibit nitration and deamination in vitro, an effect that could conceivably contribute to their protective effects against gastric cancer development.     

Enhancement of Enzyme-Catalyzed Production of Reactive Oxygen Species by Suspensions of “Crocidolite” Asbestos Fibres

Ethylene release from methylthio-ketobutyric acid is an indicator for activated oxygen species of the OH '-radical type. Xanthine oxidase plus xanthine or diaphorase in the presence of NADH and j uglone produce OH'-type oxy-radicals. The production of reactive oxygen species in these enzymatic systems is enhanced by “crocidolite” asbestos fibres.     

Therapeutic applications of ultrasound

Therapeutic applications of ultrasound predate its use in imaging. A range of biological effects can be induced by ultrasound, depending on the exposure levels used. At low levels, beneficial, reversible cellular effects may be produced, whereas at high intensities instantaneous cell death is sought. Therapy ultrasound can therefore be broadly divided into “low power” and “high power” applications. The “low power” group includes physiotherapy, fracture repair, sonophoresis, sonoporation and gene therapy, whereas the most common use of “high power” ultrasound in medicine is probably now high intensity focused ultrasound. Therapeutic effect through the intensity spectrum is obtained by both thermal and non-thermal interaction mechanisms. At low intensities, acoustic streaming is likely to be significant, but at higher levels, heating and acoustic cavitation will predominate. While useful therapeutic effects are now being demonstrated clinically, the mechanisms by which they occur are often not well understood.     

Principles in the selection of inorganic elements by organisms — Application to molybdenum enzymes

Some basic principles were delineated with regard to the selection of inorganic elements by organisms; they are “basic fitness rule”, “abundance rule”, “efficiency rule” and “evolutionary pressure”. The basic fitness rule was then applied to the case of molybdenum, to show how inherently fit molybdenum is to the biological functions carried out by those enzymes dependent on it, including xanthine oxidase, sulfite oxidase, nitrate reductase and nitrogenase.