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 Stability Of So-Called Axonal Acid Phosphatase As Determined By Experiments In Its “Stainability”

Since the histochemical method for exhibiting acid phosphatase in bodily tissues is said to depend upon the enzyme acting on suitable substrates, it is possible to test its stability by various tests. It has been found that the background element or elements, whatever they may b e, concerned with the “staining” properties of the reaction are very stable and somewhat resistant to destruction. So-called acid phosphatase in spinal cord axons has not been inactivated by subjecting it to various fixing solutions, changes in temperature and pH, relatively prolonged post-mortem autolysis nor by well known enzyme inactivators. It is believed that the “staining” reaction may be dependent fundamentally on other factors than enzymatic activity.     

Fusion with enucleated fibroblasts corrects “I-cell” defect

Fusions have been carried out between fibroblasts from patients with “I-cell” disease and enucleated human fibroblasts with a single lysosomal enzyme deficiency derived from patients with G_M1-gangliosidosis, Sandhoff disease and mannosidosis. Pure cytoplasts were obtained using cytochalasin B treatment followed by fluorescence activated cell sorting. After fusion with whole “I-cells”, the cybrid populations showed a restoration of deficient lysosomal enzyme activity and also the abnormal electrophoretic pattern characteristic for the residual hexosaminidase activity in “I-cells” was found to be corrected. The results described in this paper indicate that the defective post-translational modification, which is responsible for the multiple lysosomal enzyme deficiency, can be corrected by a factor that is stable for at least three days in enucleated cells. During this period the cytoplasmic factor can act without the need of de novo synthesis but the absence of correction in in vitro experiments shows that cellular integrity is required.     

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.     

Immunofluorescent and ultrastructural studies of “sarcomeric” units in stress fibers of cultured non-muscle cells

Immunofluorescence and electron microscopy were used to study the organization of actin, myosin and α-actinin in the “sarcomeric” units of the stress fibers of a selected non-muscle cell type. The results of indirect immunofluorescence confirm that myosin and α-actinin are periodically distributed in discrete units along stress fibers and demonstrate that they are alternately spaced. This relationship is required by a sarcomere model of stress fiber construction. A comparison between immunofluorescent and EM images of stress fibers confirms that α-actinin is confined to Z line-like dense bodies, myosin to the spaces in between. The most intriguing result is that by immunofluorescence a periodic distribution of actin can be detected in some fibers. This may indicate that even actin is periodically distributed in non-muscle “sarcomeres”.     

Sphenophyllum miravallis Vetter and Bowmanites cupulatus sp.n. from the “Illinger Flözzone” (“Heusweiler Schichten”, Lower Stephanian, Saar Basin, German Federal Republic)

This paper deals with a detailed study of Sphenophyllum miravallis Vetter, a member of the “Sphenophyllum thonii group”. New material from the Reisbach colliery, working the “Illinger Flözzone” of the “Heusweiler Schichten” (Lower Stephanian, Saar Basin, German Federal Republic), is described morphologically and anatomically, and the species is discussed. The new material enlarges the known range of variability of the normal aspect of the foliage, i.e. the foliage of the thinner branches. Thicker stems with their aberrant polymorphous foliage, and cellular details, are described for the first time. An emended diagnosis is given. Comparisons with other species are made.

The new species Bowmanites cupulatus is introduced to accommodate fructufications most probably belonging to Sphenophyllum miravallis.

S. crenulatum Knight ex Wagner is considered to be a heterotypic synonym of S. miravallis, the latter name having priority.     

Notes on Technic: “Clearing” Steel Knife Epon Sections in a Polystyrene Film Sandwich

Serial sectioning epoxy embedments by steel knife permits rapid light microscope survey of large tissue volumes, and preselection of areas of interest for electron microscopy. Acetate film (Hollander 1970) and Turtox plastic slides (West 1972) have been suggested as substrates upon which the sections may be “cleared” with an added layer of cured epoxy. In our experience, these substrates are excessively adherent to Epon, and “cleared” sections thinner than 40-50 μm cannot be released from them reliably. The following method is suitable for processing Epon sections 10 or more microns thick.     

Aromatase immunoreactivity in the rat brain: Gonadectomy-sensitive hypothalamic neurons and an unresponsive “limbic ring” of the lateral septum-bed nucleus-amygdala complex

The aromatase (estrogen synthetase) enzyme catalyzes the conversion of androgens to estrogens in peripheral tissues, as well as in the brain. Our study aimed at comparing the brain distribution of aromatase-immunoreactive neurons in male and female, normal and gonadectomized rats. Light microscopic immunostaining was employed using a purified polyclonal antiserum raised against human placental aromatase. Two anatomically separate aromatase-immunoreactive neuronal systems were detected in the rat brain: A “limbic telencephalic” aromatase system was composed by a large population of labeled neurons in the lateral septal area, and by a continuous “ring” of neurons of the laterodorsal division of the bed nucleus of stria terminalis, central amygdaloid nucleus, stria terminalis, and the substantia inominata-ventral pallidum-fundus striati region. The other, “hypothalamic” aromatase system consisted of neurons scattered in a dorsolateral hypothalamic area including the paraventricular, lateral and dorsomedial hypothalamic nuclei, the subincertal nucleus as well as the zona incerta. In addition, a few axon-like processes (unresponsive to gonadectomy) were present in the preoptic-anterior hypothalamic complex, the ventral striatum, and midline thalamic regions. No sexual dimorphism was observed in the distribution or intensity of aromatase-immunostaining. However, 3 days, 2, 3, 8, 16, or 32 weeks after gonadectomy, aromatase-immunoreactive neurons disappeared from the hypothalamus, whereas they were still present in the limbic areas of both sexes. The results indicate the existence of two distinct estrogen-producing neuron systems in the rat brain: (1) a “limbic ring” of aromatase-labeled neurons of the lateral septum-bed nucleus-amygdala complex unresponsive to gonadectomy; and (2) a sex hormone-sensitive “hypothalamic” aromatase neuron system.     

The development of a “microtitre” fluctuation test for the detection of indirect mutagens, and its use in the evaluation of mixed enzyme induction of the liver

The “Microtitre” Fluctuation test recently introduced for the detection of direct mutagens has been adapted for the detection of indirect mutagens through the incorporation of an “S9-mix” metabolic system. It compares favourably with Greens' original method for the detection of a range of chemical mutagens.

The technique has been employed in the evaluation of mixed enzyme induction using phenobarbitone and β-naphthoflavone (benzoflavone). as a safe substitute for Aroclor-1254. The post-mitochondrial preparations from rats induced with the combined inducers had a similar “metabolic competence” to those derived from Aroclor induced animals. Such a combination would therefore provide a useful alternative to Aroclor-1254 for routine screening.

It was found that the level of “S9” present in the metabolic system greatly affected the quantitative mutagenic response. This varied considerably from chemical to chemical and underlined the need for such preliminary investigations in routine screening.     

Basophilic “Dark Cells:” Optimal Conditions for Toluidine Blue Staining

Differential staining of basophilic “dark cells” of epithelia in thick epoxy sections with toluidine blue is critically affected by fixation and embedding conditions. Optimal conditions are described.     

Membrane alterations associated with “transformation” by BUdR in BUdR-dependent cells : Fluorescence polarization studies with a lipid probe

Steady state and nanosecond fluorescence polarization studies were carried out on membranes of a “bromodeoxyuridine (BUdR) dependent” cell line (B4) derived from a malignant Syrian hamster melanoma line. When grown in the presence of BUdR B4 cells resemble transformed cells (in terms of several biological characteristics), while B4 cells grown in the absence of BUdR resemble untransformed cells. B4 cells were labelled with the lipid probe 1,6-diphenyl-1,3,5-hexatriene, which had been used previously to show that fluorescence polarization values of membrane lipids of virally transformed cells are higher than fluorescence polarization values of membrane lipids of untransformed cells. The steady state fluorescence polarization values of membrane lipids of B4 cells in BUdR were found to be larger than those of cells in the absence of BUdR, and the change in fluorescence polarization values was found to be fully reversible. Nsec rotational correlation time experiments confirmed and extended the steady state results. The results of the fluorescence polarization studies suggest that the membranes of B4 cells grown in the presence of BUdR resemble those of virally transformed cells while membranes of B4 cells grown in the absence of BUdR resemble those of untransformed cells.     

Flexibility versus “rigidity” of the functional architecture of AChE active center

Functional architecture of the AChE active center appears to be characterized by both structural “rigidity”, necessary to stabilize the catalytic triad as well as by flexibility in accommodating the different, high affinity AChE ligands. These seemingly conflicting structural properties of the active center are demonstrated through combination of structural methods with kinetic studies of the enzyme and its mutant derivatives with plethora of structurally diverse ligands and in particular with series of stereoselective covalent and noncovalent AChE ligands. Thus, steric perturbation of the acyl pocket precipitates in a pronounced stereoselectivity toward methylphosphonates by disrupting the stabilizing environment of the catalytic histidine rather than through steric exclusion demonstrating the functional importance of the “rigid” environment of the catalytic machinery. The acyl pocket, the cation-binding subsite (Trp86) and the peripheral anionic subsite were also found to be directly involved in HuAChE stereoselectivity toward charged chiral phosphonates, operating through differential positioning of the ligand cationic moiety within the active center. Residue Trp86 is also a part of the “hydrophobic patch” which seems flexible enough to accommodate the structurally diverse ligands like tacrine, galanthamine and the two diastereomers of huperzine A. Also, we have recently discovered further aspects of the role of both the unique structure and the flexibility of the “hydrophobic patch” in determining the reactivity and stereoselectivity of HuAChE toward certain carbamates including analogs of physostigmine. In these cases the ligands are accommodated mostly through hydrophobic interactions and their stereoselectivity delineates precisely the steric limits of the pocket. Hence, the HuAChE stereoselectivity provides a sensitive tool in the in depth exploration of the functional architecture of the active center. These studies suggest that the combination of “rigidity” and flexibility within the HuAChE gorge are an essential element of its molecular design.     

Rotational relaxation of 1,6-diphenylhexatriene in membrane lipids of cells acclimated to high and low growth temperatures.

Measurement of the time-resolved fluorescence depolarization of 1,6-diphenylhexatriene (DPH) in artificial bilayers of microsomal membrane lipids from Tetrahymena gives detailed information concerning the molecular motion of this probe and fluid properties of the membrane lipids which are obscured with steady-state methods. The rotational motion of DPH in these lipids from cells acclimated to 15 and 39.5 degrees C growth temperatures was anisotropic, which agrees with recent time-resolved studies of this probe in synthetic phospholipid systems. Evaluation of DPH polarization data obtained from these lipid fractions at their respective growth temperatures showed differences in physical properties which suggest that "viscosity", per se, of the microsomal lipids is not a strictly regulated as it is in prokaryotic systems. Rotational relaxation of DPH in 39.5 degrees C microsomal lipids measured at 15 degrees C is more complex than that of either lipid fraction measured at its actual growth temperature, suggesting that the probe has partitioned into two dissimilar environments within the bilayer. Similar effects are observed in the microsomes of 39.5 degrees C cells by freeze-fracture electron microscopy following rapid cooling to 15 degrees C. Under these conditions, two distinct regions are observed on the fracture faces, suggesting a correlation between lipid phase changes and alterations in membrane structure.     

Characterization of the plasmids comprising the “R Factor” R5 and their relationships to other R plasmids

The “R factor” R5 confers resistance to tetracycline (Tc), streptomycin (Sm) and spectinomycin (Sp), chloramphenicol (Cm), sulfonamides (Su), kanamycin (Km), and mercuric ion (Mer). This phenotype is mediated by the presence of two R plasmids: pMH1 and pMH2, having approximate weights of 18.5 and 62 megadaltons (Mdal), respectively. pMH1 encodes Sm, Su, Cm, Mer, and Km resistance, and is nonconjugative. pMH2 confers Sm, Su, Cm, Mer, and Tc resistance, is conjugative, and belongs to the FII incompatibility group. NR79 is a 63-Mdal R plasmid encoding the same resistances as “R5,” and was derived from the same geographical source. It belongs to the FII incompatibility group and is conjugative. Analysis of restriction endonuclease digestion patterns and polynucleotide sequence homologies indicate that pMH1, pMH2, and NR79 are closely related. In addition, pMH2 and NR79 exhibit nearly complete homology to R100. Restriction endonuclease maps and resistance gene locations for pMH1, pMH2, and NR79 have been derived and a model for the evolutionary relationships of these plasmids is presented.     

Bound Water and Cryptobiosis:Thermodynamic Properties of Water at Biopolymer Surfaces

The molecular mechanisms underlying the adaptations to water loss developed in several tardigrade species remain poorly understood. It seems, however, that the binding of the disaccharide trehalose to membranes and other cellular components at low water contents is important for the tolerance to extreme drought. Trehalose is thus thought to replace interfacial- or “bound” water and enhance the conformational stability of labile macromolecules. To gain further insight into this we investigate here thermodynamic properties of water bound to the protein lysozyme at low water content (<100 water molecules pr. protein). It appears that this surface water has a higher enthalpy and higher entropy than the bulk liquid. These observations call for re-evaluation of the term “bound water” since “bound” carries the connotation of a low-energy, ordered (i.e. low-entropy) state.

To rationalize these observations it is suggested that — in addition to the self-evident energetic contribution from biopolymer-water contacts — the properties of interfacial water are dominated by two effects. These are i) the ability of water to facilitate fast movements of individual parts of biopolymers and ii) the high molecular cohesion in the aqueous bulk. Thus, the hydration of a surface leads to enhanced flexibility in the biopolymer and breakage in the network of hydrogen bonding in the liquid bulk, and these effects collectively increase the enthalpy and entropy of the system. As a result, the thermodynamic parameters of hydration of lysozyme carry the thermodynamic hallmarks of an order → disorder process, even for the first hundred (i.e. most strongly associated) water molecules. We discuss these data for protein hydration together with some recent, very similar observations for the hydration of lipid bilayer membranes.     

“Survival in air” of the blue mussel Mytilus edulis L. as a sensitive response to pollution-induced environmental stress

Mussels, Mytilus edulis, were exposed for periods of 6 weeks at various locations in Dutch coastal waters during 1989 and 1990. “Survival in air” showed to be a sensitive response parameter for indicating pollution induced environmental stress in transplanted mussels sampled from eight field sites. Increased tissue contaminant levels, especially PCBs and PAHs, correlated with a reduced survival time during aerial exposure. Three weeks exposure of mussels in the laboratory to 1 μg · 1⁻¹ PCBs affected the aerial survival time negatively. Laboratory experiments did not indicate that lowered salinity influences the “Survival in air” response after sufficient acclimation (15 days), facilitating the use of this response parameter in both marine and estuarine waters.     

Influence of sex, slaughter weight and carcass weight on “non-carcass” and carcass quality in segureña lambs

The effects of sex, slaughter weight and carcass weight on carcass characteristics and meat quality traits were evaluated using 100 Segureña lambs. The management of all lambs was similar prior to slaughter at 19–25 kg. Slaughtered animals with a hot carcass weight below 20 kg were assigned to class B, and those greater than 22 kg to class C. Carcass weight had a significant influence on “non-carcass” components, dressing percentage, subjective carcass conformation, fat deposits, carcass fatness, bone and most carcass measurements. Sex had a significant effect on age at slaughter, “non-carcass” components, rib measurements, dressing percentage, fat deposits, and neck and shoulder percentage. As the weight increased, the carcass measurements also increased. Concurrently, while improving the conformation indices of the carcass, leg and dressing percentages, neither the commercial cuts of the animal nor tissue composition was significantly affected. Sex primarily affected the quantity of all types of fat deposits.     

A biochemical approach toward the systematics of the Leptothorax “muscorum”Group in North America (Hymenoptera: Formicidae)

Enzyme patterns have been used to distinguish among a number of morphologically very similar ant species belonging to the Leptothorax “muscoru” group in North America. Not counting the already described species L. retractus, L. sphagnicolus and L. crassipilis, the complex apparently consists of at least three or four more different taxa.     

Detection of Radio-Elements in Histological Slides by Coating with Stripping Emulsion—The “Strip-Coating” Technic

The “strip-coating” technic offers some improvement of the published methods for the autographic detection of Tadio-elements in sections by stripping films. Reproducible results are easily obtained with Ilford Half Tone Stripping Plate Emulsion. The method does not lend itself readily to the preparation of the large number of slides usually required for biological research.     

Membrane disruption, fusion and resealing in the course of exocytosis in Paramecium cells

Ca²⁺-ionophore-mediated trichocyst exocytosis was followed by scanning electron microscopy, freeze-cleaving and ultrathin sectioning after surface labelling in vivo with negatively charged hemepeptides. The apical trichocyst membrane and the superposed cell membrane portion (encircled by ˜300 nm large “rings” of membrane-intercalated particles) undergo fragmentation, while both membranes involved fuse with each other within the “rings”. Subsequently cell membrane materials spread centropetally to the region within the “rings” allowing the cell membrane to become resealed and the trichocyst membrane to become detached. Exocytosis does not result in any remarkable integration of trichocyst membrane materials into the cell membrane.