A comparison of some different methods for purifying core temperature data from humans

Nine healthy females were studied about the time of the spring equinox while living in student accommodations and aware of the passage of solar time. After 7 control days, during which a conventional lifestyle was lived under a 24h “constant routine,” the subjects lived 17 × 27h “days” (9h sleep in the dark and 18h wake using domestic lighting, if required). Throughout the experiment, recordings of wrist activity and rectal (core) temperature were taken. The raw temperature data were assessed for phase and amplitude by cosinor analysis and another method, “crossover times,” which does not assume that the data set is sinusoidal. Two different purification methods were used in attempts to remove the masking effects of sleep and activity from the core temperature record and so to measure more closely the endogenous component of this rhythm; these two methods were “purification by categories” and “purification by intercepts.” The former method assumes that the endogenous component is a sinusoid, and that the masking effects can be estimated by putting activity into a number of bands or categories. The latter method assumes that a temperature that would correspond to complete inactivity can be estimated from measured temperatures by linear regression of these on activity and extrapolation to a temperature at zero activity. Three indices were calculated to assess the extent to which exogenous effects had been removed from the temperature data by these purification methods. These indices were the daily variation of phase about its median value; the ratio of this variation to the daily deviation of phase about midactivity; and the relationship between amplitude and the square of the deviation of phase from midactivity. In all cases, the index would decrease in size as the contribution of the exogenous component to a data set fell. The purification by categories approach was successful in proportion to the number of activity categories that was used, and as few as four categories produced a data set with significantly less masking than raw data. The method purification by intercepts was less successful unless the raw data had been “corrected” to reflect the direct effects of sleep that were independent of activity (a method to achieve this being produced). Use of this purification method with the corrected data then gave results that showed least exogenous influences. Both this method and the purification by categories method with 16 categories of activity gave evidence that the exogenous component no longer made a significant contribution to the purified data set. The results were not significantly influenced by assessing amplitude and phase of the circadian rhythm from crossover times rather than cosinor analysis. The relative merits of the different methods, as well as of other published methods, are compared briefly; it is concluded that several purification methods, of differing degrees of sophistication and ease of application to raw data, are of value in field studies and other circumstances in which constant routines are not possible or are ethically undesirable. It is also concluded that such methods are often somewhat limited insofar as they are based on pragmatic or biological, rather than mathematical, considerations, and so it is desirable to attempt to develop models based equally on mathematics and biology. (Chronobiology International, 17(4), 539-566, 2000)     

The Lower Row Monkey Cage: An Overlooked Variable in Biomedical Research

A survey of 96 primatological articles revealed that cage location of research monkeys is rarely mentioned, although the environment of upper and lower row-housed animals markedly differs in terms of light quality, light intensity, and living dimension. Not accounting for these uncontrolled variables may increase variability of data and, consequently, the number of experimental animals needed to obtain statistically acceptable results. This study concluded that single-tier housing would be an important refinement of research methodology. Such housing would (a) enable all animals of a room to use the “arboreal ”dimension of their enclosure and retreat to “safe ”vantage points above the human “predator, ”(b) offer all animals access to uniform light, and (c) provide more favorable conditions for professional animal care.     

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.     

Animal protection measures in Taiwan: Taiwanese attitudes toward the animal protection law and animal shelters

This study used a questionnaire, self-administered by 387 Taiwanese citizens, to assess Taiwanese perception and knowledge of the Animal Protection Law and perception and use of animal shelters. Most respondents (87%) knew that Taiwan has legal protection for animals. A -5 to 5 summary score measured knowledge of companion animal dog-related legal requirements. The median score was 3, indicating that almost all respondents had inadequate knowledge about these requirements. Although more dog caregivers had adequate knowledge of the law than did nondog caregivers (prevalence ratio = 1.9, 95% confidence interval: 1.1, 3.2), only 19% had adequate knowledge. Although most respondents (81%) knew Taiwan has animal shelters, only 19 (5%) reported visiting or participating in shelter activities. Only 6 (32%) of shelter visitors or users had a “good or better ”impression of services received. Despite low usage of animal shelters, most respondents thought shelters should accept (87%) and relocate (90%) unwanted animals, capture strays (64%), assist in finding lost pets (79%), and educate the public about responsible ownership (85%). More than half (52%) thought shelters should perform euthanasia.     

"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.     

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.     

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.     

The Difference Between Activity When in Bed and Out of Bed. II. Subjects on 27-Hour “Days”

Nine healthy subjects have been studied while exposed to the normal alternation of light and dark, but with their sleep and activity pattern adjusted to a 27-h “day” for 17 imposed “days.” Rectal temperature showed clearly the competing influences of 27-h and 24-h components, and these were separated by the method of “purification.” The method indicated that the endogenous component had a constant amplitude throughout the experiment and remained entrained to solar (24-h) time; by contrast, the exogenous component followed the imposed 27-h “day” and increased rectal temperature in proportion to the amount of subjects' activity. Wrist movement was used to assess activity while in bed (attempting sleep) and out of bed (when naps were forbidden). While these results confirmed adherence of the subjects to the imposed 27-h “days,” they also showed that the dichotomy between “out of bed” activity and “in bed” inactivity depended on the phase relationship between endogenous (24h) and exogenous (27h) components. Thus, the dichotomy was highest and was equal to that during control days (with a conventional 24-h life-style) when the two components were in phase and lowest when the solar and imposed day were in antiphase. This was due to changes in activity, both during time spent in bed and out of bed.

We confirm that this protocol can produce valuable information about the properties of the circadian system in humans and the value of the process of purification of temperature data. We have established also that the very simple and noninvasive measurement of wrist movement, coupled with its use to calculate dichotomy indices, provides valuable information that both confirms and extends the results obtained from the more conventional (butalso more invasive) measurement of rectal temperature.     

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”.     

Contribution of response surface design to the development of glycerolysis systems catalyzed by commercial immobilized lipases

Two commercial immobilized lipases (“Lipozyme® IM” and “Novozym® 435”) were tested as biocatalysts for the glycerolysis of olive residue oil in n-hexane aimed at the production of monoglycerides (MG) and diglycerides (DG). A central composite rotatable design (CCRD) was followed to model and optimize glycerolysis as a function of both the amount of biocatalyst (L) and of the molar ratio glycerol/triglycerides (Gly/TG). For both biocatalysts, the production of free fatty acids (FFA) was described by second order models. In terms of MG and DG production, as well as of TG conversion, the best fits were obtained with first-order models. The highest MG productions were in the range 43–45% (w/w, on the basis of total fat) for both biocatalysts tested at a (Gly/TG) ratio of one. In the case of “Novozym 435”, the lowest load used (12%, w/w) gave the best results, in contrast with “Lipozyme IM” with which a concentration of about 26% (w/w) was necessary to obtain the highest production. Under these conditions, the amount of FFA produced was about 2% and 10% (w/w), respectively, for “Novozym 435” and “Lipozyme IM” catalyzed systems. Considering both FFA production and lipase loading, “Novozym 435” was shown to be a better biocatalyst for the glycerolysis of olive residue oil in n-hexane, aimed at the production of MG, than “Lipozyme IM”.     

Population dynamics, overpopulation, and the welfare of companion animals: new insights on old and new data

Americans profess a great love for their companion animals, and, indeed, their expenditures on food and other products for their dogs and cats would seem to confirm this. However, each year, many millions of dogs and cats are brought to animal shelters, where the majority are euthanized. Our inability to provide reasonably valid statistics related to this concern makes it difficult to offer a credible presentation on the need for a concerted effort to deal with the issue, design initiatives to ameliorate the problem, or evaluate progress and performance of these efforts. In this article we review some of the past efforts to document the scale of the “pet overpopulation ”problem. We reexamine long-term shelter statistics from a single shelter system and present new data that reflect a recent cooperative effort to understand the origins and disposition of dogs and cats received by animal shelters.     

The effect of recombinant human erythropoietin on serum selenium levels in hemodialysis patients

Successful results in the treatment of anemia, one of the main complications of chronic renal failure, can be achieved by the use of recombinant human erythropoietin (RhEPO), which is available almost fifteen years in clinics. On the other hand, as both chronic renal failure and maintenance hemodialysis reduce the levels of trace elements, this study was designed to evaluate the interaction potential of RhEPO with serum concentrations of selenium (Se) during four months. Thirty one adult hemodialysis outpatients participated in the study. Ten of them, not on any drug therapy to interact with RhEPO, recruited as “Control Group”, and the remainder, on RhEPO therapy, as “RhEPO Group”. Blood was drawn from the Control Group at the beginning of the study, and from the RhEPO Group at every month for four months. Serum erythropoietin levels were measured by a radioimmunoassay method and Se status by a spectrofluorometric method. It was found that Se levels were not affected by RhEPO treatment during 3 months of therapy, while an increase was seen on the fourth month. The observation indicates that the increase in serum Se levels would be significant in longer than three-month RhEPO treatment.     

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.     

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.     

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.     

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)     

Enclosure design and space utilization by Indian leopards (Panthera pardus) in four zoos in Southern India

Enclosure design and the use of enclosure space influence the activity budget of captive leopards. The study laid out in grids all enclosures on the base map and segregated these grids into 4 zones. Every 5 min, the study recorded the proportion of time spent in these zones with the leopards' behavior. Captive leopards most frequently used the “edge ”zone. Almost all leopards used the edge zone for stereotypic pacing, the “back ”zone for resting, and the “other ”zone for activity. The study positively correlated the proportion of time spent in the “enriched ”zone with activity levels exhibited by leopards housed in some enclosures and with resting in others. Thus, the study segregated structural objects in the enriched zone into activity-related features (e.g., logs) and rest-related features (e.g., trees and sleeping platforms). Compared with individuals housed in barren enclosures, leopards housed in structurally enriched on-exhibit enclosures exhibited higher levels of activity. Enclosure design was found to be an important factor influencing the welfare of leopards in captivity.     

Paraphyly of the Pseudophyllidea (Platyhelminthes: Cestoda): circumscription of monophyletic clades based on phylogenetic analysis of ribosomal RNA

Phylogenetic relationships of cestodes of the order Pseudophyllidea (Platyhelminthes: Cestoda) were examined using sequences of complete small subunit and partial (D1-D3 region) large subunit nuclear rDNA of members of all pseudophyllidean families. The results provide evidence of paraphyly of the order as indicated by previous molecular phylogenetic analyses based on a much lower number of species sequenced. Pseudophyllidean tapeworms represent an artificial assemblage comprising two unrelated clades. “Bothriocephalidea” is formed by four families sensu Bray et al. (1994), namely Bothriocephalidae, Echinophallidae, Philobythiidae and Triaenophoridae, whereas two other families, Diphyllobothriidae and Cephalochlamydidae, give rise to the “Diphyllobothriidea”. The present results indicate that “Bothriocephalidea” forms the most derived clade of all difossate and tetrafossate/bothriate tapeworm lineages which are considered to be basal relative to the rest of tetrafossate/bothridiate and acetabulate cestodes. By contrast, “Diphyllobothriidea”, which includes medically important parasites (Diphyllobothrium and Spirometra), appeared more basal, without a clearly resolved position within other difossate tapeworm lineages.     

Manipulation of the amount of RNA in post-mitotic nuclei

Because all (or almost all) nuclear RNAs are liberated to the cytoplasm during mitosis and then return to the post-mitotic nuclei, we expected that if cytoplasm were amputated from mitotic cells the post-division nuclei would possess less than normal amounts of RNA. Experiments performed with amebae (A. proteus) show that this is in fact what happens. Furthermore, since the enucleate fragment cut from a mitotic cell possesses an “excess” of returnable nuclear RNAs, a normal interphase nucleus implanted into such mitotic cytoplasm might be expected to acquire above-normal amounts of RNA. Experiments reported here show that this expectation also is realized. Thus, the regulation of the normal nuclear concentration of these RNAs involves mechanisms other than a limited number of intranuclear “binding” sites and most likely is restricted by the rate of synthesis of these RNAs.The demonstration that nuclei can be depleted or enriched for RNAs, many of which are unique to nuclei, makes it possible to determine the consequences for cell metabolism of altered amounts of nuclear RNA. Hopefully, such studies will reveal the function(s) of these RNAs.     

Effect of 0.25 N sodium chloride treatment on DNA denaturation in situ in thymus lymphocytes

Extraction of cells with 0.25 N NaCl changes the profile of DNA denaturation in situ. The portion of DNA denaturing at lower temperatures (“thermosensitive” fraction) shows increased sensitivity to heat following salt extraction while the “thermoresistant” DNA fraction is further stabilized. The results suggest that proteins extractable with 0.25 N NaCl while providing local counterions for DNA phosphates of the “thermosensitive” DNA fraction also decrease the strength of DNA-histone interactions within the “thermoresistant” fraction.