Rhythm analysis of clipped data: Examples using circadian data

Summary It has previously been proved that, in rhythm analysis, it makes little difference to the outcome if the data are analyzed in clipped form (that is, reduced to zero crossings). Loss of amplitude information has little effect on analyses for periodicity, a fact which can be used to advantage both for data collection and for data analysis.Rhythm analyses are common in studies which seek to characterize circadian oscillations. I have subjected eight time series of real biological data to time series analysis, then reduced the data to the clipped form and re-analyzed it with the same techniques. I did this using three techniques for rhythm analysis: autocorrelation, power spectral analysis, and Enright's periodogram. The data consisted of perchhopping activity records from sparrows exhibiting entrained or freerunning circadian rhythms. For all three techniques, results with real data conformed with the mathematical theorem for analysis of clipped data.I Thank K. Adler, E. Kluth, G. Wyche, Mr. Killan, C. Cromack, J. Alvis, and the Notre Dame Computer Center. Support was provided by postdoctoral fellowship 1 FO2-HD-52858 to S. Binkley; grants to K. Adler (NSF GB-30547 and NTH FR-07033-05). Sparrow data used in this report were gathered while S. Binkley was a graduate student at the U. of Texas in Austin (NIH traineeship 5T01 GM-00836-08) and with funds from an NIH program project grant (HD-03803-02) to M. Menaker.     

Pineal N-acetyltransferase in chickens: Rhythm persists in constant darkness

Summary The daily rhythm in an enzyme, pineal serotonin N-acetyltransferase activity was studied in chickens kept in conditions of constant dark. The rhythm persisted and had a period length of approximately 24 hours which are characteristics of truly circadian rhythms. The detailed documentation of the rhythm (Fig. 1) shows the shape of the oscillation and clear anticipation of the time of lights on. Both the rise and fall of the rhythm occur without stimulation by light or dark.We thank Robert Weiner, William Finlay, Jay Freedman, Kathy Hall, Sylvia Goddes, Dr. S. K. Roberts, and the Temple University Computer Activity. Supported by NSF grant GB-43215, NSF Institutional Award, and Temple Grant-in-Aid to S. Binkley.     

AII amacrine neurons of the rat retina show diurnal and circadian rhythms of parvalbumin immunoreactivity

We investigated parvalbumin immunoreactivity (PA-IR) in the retinas of rats maintained on a 12:12 h light:dark cycle, or after being placed in constant darkness for 24–72 h. Retinas were harvested at zeitgeber and circadian times 02:00, 06:00, 10:00, 14:00, 18:00 and 22:00 h. PA-IR was found primarily in retinal amacrine cells of the AII subtype. In a light/dark cycle, PA-IR showed a clear rhythm, with a low near zeitgeber time (ZT) 10:00 h and a peak near ZT 18:00 h. The ratio of immunofluorescence intensities at these timepoints was >15-fold. When animals were kept in complete darkness for 1–3 days, the rhythm of PA-IR was still preserved, but was progressively reduced in amplitude. The rhythm of PA-IR inferred from immunohistochemical data was confirmed by Western blots. We conclude that PA-IR in the rat retina shows an underlying circadian rhythm that is enhanced by cyclic light. The regulation may involve translocation of the protein between cell compartments and/or new protein synthesis.This study was supported by an OTKA grant (T 34160), NIH grants NS 37919 (R.S.) and ET 03570, NSF grant IBN-96418886 (R.S.), and grants from the Helen Hoffritz Charitable Trust and Research to Prevent Blindness, Inc. R.G. was also in receipt of a János Bolyai fellowship     

The pineal body: Site of extraocular perception of celestial cues for orientation in the tiger salamander (Ambystoma tigrinum)

Summary Tiger salamanders (Ambystoma tigrinum) trained to orient in a particular compass direction under the sun fail to orient in the trained direction if they are (i) eyeless and simultaneously have the brain covered with opaque plastic or are (ii) eyeless and pinealectomized (Fig. 1–2, Table 1). Salamanders with either the eyes or the pineal intact and unobstructed continue to orient in the trained direction. These data strongly support the hypothesis that the pineal body is an effective extraocular photoreceptor (EOP) for compass orientation in tiger salamanders.We thank M.P. Farrell for developing computer programs for data analysis. B. Bailey and R. Walton helped conduct tests. Financial support was provided for separate phases of this research by a Biomedical Science Support Grant (NIH FR 07033-05) and NSFgrants GB-30647 and BMS 75-18693 to K. Adler and an Indiana Academy of Science Research Grant, a postdoctoral fellowship (NSF GU-2058), a Miami University Research Grant and an NSF grant (GB-41102) to D.H. Taylor.     

A radioimmunoassay for quantifying carbonic anhydrase isozymes in crude lysates

A radioimmunoassay was developed for quantifying each of the two genetically distinct forms of primate carbonic anhydrase, carbonic anhydrases I and II, in unpurified lysates. Under the given experimental conditions, the assay is capable of detecting a minimum of 0.025 g of carbonic anhydrase I and 0.005 g of carbonic anhydrase II. There is approximately 98% reproducibility upon repeated assays of a given hemolysate.Supported by NIH training grant 5-TO1-GM-71-11 and NIH research grant 1-PO1-GM-15419-02.NIH postdoctoral trainee.     

Protein staining of ribboned epon sections for light microscopy

Summary Procedures are described for obtaining and handling ribboned epon sections 0.3–2 thick for light microscopy, and for the cytological application of two intense acid dyes, Aniline Blue Black and Coomassie Brilliant Blue R 250. The technique allows precise localization of proteins and some other materials, and, because the sections are ribboned, facilitates three-dimensional visualization of the structures involved. The dyes may be used in combination with the periodic acid-Schiff reaction and with autoradiography.This work was supported in part by a Public Health Service fellowship 5-F2-GM-22, 031-02 to the author and in part by NSF grant GB 3460 to Dr. W. A. Jensen.     

Histochemical studies of pollen: storage pockets in the endoplasmic reticulum (ER)

Summary The pollen grain of cotton (Gossypium hirsutum) was examined histochemically at the light and electron microscope level. The cytoplasm of the pollen contains an unusual storage unit which consists of a pocket of endoplasmic reticulum (ER) containing lipid droplets and dictyosome vesicles. The ER pockets are large enough to be seen with the light microscope if thin enough sections are used (0.3–1.5). The results of the histochemical analyses show that the dictysome vesicles are rich in carbohydrate and contain protein and lipid as well. The ER contains large amounts of protein which may be arginine rich. Some carbohydrates may also be present in the ER. The ER is covered with ribosomes so that the pockets are unusually rich storage units containing abundant protein, carbohydrate, lipid and RNA. The light microscope localization of carbohydrates was confirmed by the periodic acid-silver method. Other storage units in the cytoplasm were also studied. A new method for the embedding of plant tissue for thin sectioning for light microscopy is presented.This work was supported by a Public Health Service fellowship 5-F2-GM-22, 031-02 from the National Institute of General Medical Sciences, by NSF grant GB 3460, by NIH grant 5-RO1-CA 0356-10 and by the Miller Institute for Basic Science.     

Retinal and extraretinal photoreceptors mediate entrainment of the circadian locomotor rhythm in lizards

Summary The circadian locomotor activity rhythms of 7 species of lizards can readily be entrained (synchronized) toLD12: 12 (30–50 lux: 0) fluorescent light cycles after complete surgical removal of both eyes. Removal of the parietal eye and pineal organ does not prevent entrainment of blinded lizards. Appropriate control experiments established that lightper se, and not low amplitude temperature cycles or other obvious environmental variables, was the entraining stimulus for blinded lizards. In some cases, blocking the penetration of light to the brains of blinded lizards caused them to free-run (express their endogenous circadian rhythm) in the presence of a dim green light cycle, to which they had previously entrained, suggesting that the brain is the site of the extraretinal photoreceptor(s) mediating entrainment. The extraretinal photoreceptor(s) is capable of intensity discrimination since changing the intensity of aLD 12: 12 fluorescent light cycle caused a change in the phase-relationship between the entrained activity rhythm and the light cycle in a blinded gekko. The lateral eyes are also involved in mediating entrainment since removal of the lateral eyes of thoseSceloporus olivaceus which previously entrained to a dim green light cycle [LD 12: 12 (0.05 lux: 0)] caused them to free-run. Also, blinding had noticeable effects on the entrained activity patterns of some species of lizards.I thank Michael Menaker, Jeffrey Elliott, Sue Binkley, Joseph Silver, Ed Kluth, George Wyche, Bruee Rouse, Nancy Leshikar, Lili Mostafavi, Janet Alvis, Celeste Cromack, A. L. Mackey and Jean Rogers for their suggestions and technical assistance. Support for this work was provided by NIH grant HD-03803-02 (to M. Menaker); NSP grant GB-8138 (to M. Menaker); NSF traineeship GZ-1336 (to H. Underwood); and MH traineeship 5T01GM00836-09 (to H. Underwood).     

Preferential recovery of uniparental streptomycin resistant mutants from diploid Chlamydomonas reinhardtii

Summary Analysis of the streptomycin resistant mutants recovered from control and N-methyl-N-nitro-N-nitrosoguanadine (MNNG) treated haploid cultures of C. reinhardtii reveal that approximately 60% of the mutants are of the sr-1 type known to show Mendelian inheritance while 40% are of the sr-2 and sm-3 types known to be inherited in a uniparental (UP) manner. In contrast, most if not all streptomycin mutants recovered from similarly treated diploid cultures of C. reinhardtii are of the UP variety. Failure to recover sr-1 mutants from the diploid stock is explained by our findings that diploids heterozygous for Mendelian streptomycin resistance (sr-1/sr-1 ⁺) are both stable and sensitive to streptomycin. Efficient recovery of UP streptomycin resistant mutants from diploids can be explained by the observations of Gillham (1963a, 1969) which demonstrate that diploids heterozygous for an sr-2 mutation (sr-2/sr-2 ⁺) segregate resistant and sensitive progeny during mitotic cell division.The utility of diploids for isolating new UP mutant genotypes, for establishing the cellular localization of the UP genome(s), and for characterizing the rules governing UP gene segregation is discussed.Supported by NIH postdoctoral fellowship GM 52359 to R.W.L., NIH predoctoral traineeship GM 02007 to K.P.V., and NSF grant GB-22769 to N.W.G. and J.E.B.     

A Cytophotometric analysis of the DNA in the nucleus of the giant cell,R-2, in Aplysia

DNA was cytophotometrically measured in Feulgen stained nuclei of R-2, the giant neuron of the abdominal ganglion in Aplysia. The data indicate that the nucleus hag a volume of more than 7 X 10^{6 3} in large animals, and contains as much as 75000 times the haploid amount of DNA. To our knowledge, this is the most highly polyploid nucleus yet described. Furthermore, the amount of DNA increases with growth, going from approximately 2000 times the haploid amount in small animals to over 75000 times in large animals. The data suggest that the increase in DNA occurs in increments, each increment having approximately twice the DNA as the one before. Thus we suggest that the increase in DNA in the nucleus of R-2 results from the entire genome replicating without accompanying cell division.This study was supported by USPHS Grants no. 5R01NS07711 and 5R01-NS08109 and NSF grant no. GB7284. Richard E. Coggeshall is a recipient of Career Development Award 5-K3-GM-31754 and Frank J. Swartz is a recipient of a travelling fellowship GM05079.     

Growth of crustacean muscles and muscle fibers

Summary The superficial flexor (SF) muscle of the crayfish (Procambarus clarkii) abdomen increases in volume in direct proportion to increases in total body weight during ontogeny. This increase in SF muscle mass occurs solely (Figs. 1, 2) by an increase in the width and length of SF muscle fibers (i.e., the number of SF muscle fibers remains constant). Unlike vertebrate muscle fibers, these crustacean muscle fibers increase in length by increases in sarcomere length (Fig. 3). This increase in sarcomere length during ontogeny must occur via a continuous lengthening of actin and myosin filaments since the relative lengths of the A and I bands remain essentially unchanged as these fibers lengthen. Similar results are reported for the opener muscle of the cheliped (Figs. 5–7).We suggest that fiber number is specified for many crayfish muscle masses since for a given species of crayfish, certain muscle masses contain a set number of fibers within rather narrow limits, and the number of fibers is often significantly different in homologous muscle masses of the same species or in the same muscle mass of different species. Finally, it would seem that similar processes are operating both during embryonic growth and during regeneration in crayfish and in some other crustaceans, since fiber number is not significantly different in opener muscles from normal and regenerated limbs in crayfish and in the crabGecarcinus lateralis.We would like to thank Mr. Martis Ballinger and Mr. Mark R. Meyer for their aid and histology and photography, Mr. Michael Bouton for his assistance in several of the experiments, and Drs. Alan Templeton and Laurence Fox for their help in the statistical analysis of the data. This research was supported by NSF grant No. GB-30199 and NIH grant No. NS-08609.     

Electron microscope studies on collagen

Summary The present paper describes the effect of intensive tryptic digestion of native tropocollagen (TC) macromolecules in solution. Contrary to earlier investigations it has been found that the trypsin treatment results in a fragmentation of the TC molecules. The addition of ATP to solutions exposed to the enzyme yields SLS fragments, 2250 Å in length. Comparison between these and normal SLS type aggregates shows that the scission occurs in a well-defined locus adjacent to the -1/2-line seen after positive staining. The significance of this finding is discussed.This study was supported by grant NB-02215-04 from the National Institute of Neurological Diseases and Blindness, Public Health Service, U.S.A. and a Student research fellowship from the Norwegian Research Council for Science and the Humanities. This aid is gratefully acknowledged.I am indebted to Mrs. J. Line Vaaland and Mr. B. V. Johansen for technical assistance.     

The adrenal: A new target organ of the calciotropic hormone 1,25-dihydroxyvitamin D3

Summary Target cells for 1,25-dihydroxyvitamin D₃ were demonstrated in the adrenal medulla by frozen-section autoradiography. The appearance of these target cells was age-dependent in neonatal mice. Immunocytochemical staining for phenylethanolamine-N-methyltransferase revealed that both epinephrine and non-epinephrine cells concentrate 1,25-dihydroxyvitamin D₃ in their nuclei. In contrast, immunocytochemical staining for vitamin D-dependent calcium-binding protein (D-CaBP) demonstrated that D-CaBP immunoreactivity is localized in only a small percentage of adrenomedullary cells, in mice and rats. Comparison of PNMT and D-CaBP immunoreactivities in sequential sections showed that epinephrine-producing cells do not contain D-CaBP. These results indicate that adrenal medullary cells have receptors for 1,25-dihydroxyvitamin D₃ and that 1,25-dihydroxyvitamin D₃ may directly affect certain functions of these endocrine cells.Supported by US PHS grant PCM8200569 and postdoctoral training grant AM07240-02     

Polysaccharidases and the control of cell wall elongation

Summary Avena coleoptile sections were treated with a fraction of a fungal filtrate containing a potent cellulase. Elongation rate was not affected although turgor pressure remained constant and wall extensibility was increased. These data show that the simple weakening of cell walls is not sufficient to promote growth and suggest that endogenous polysaccharidases are not the means by which the growth rate of the coleoptile is regulated.This work was supported in part by a predoctoral and a postdoctoral fellowship from NSF     

Nuclear and cytoplasmic DNA synthesis in cotton embryos: a correlated light and electron microscope autoradiographic study

Summary To investigate the possibility, implied by an earlier report, that large amounts of degradable DNA are probably present in the cytoplasm of young cotton embryos, an investigation was undertaken to establish the distribution, amount and metabolic stability of DNA in cotton embryos. Several sensitive cytochemical tests failed to detect any but small amounts of extranuclear DNA. Quantitative determination of the nucleic acid content of embryos during embryogenesis showed that the amounts of DNA and RNA remained fairly constant during embryogenesis, with a ratio of RNA to DNA of about 3.5 to 1. Quantitative autoradiography at both the light and electron microscope levels of sections from embryos pulse-labeled with ³H-thymidine showed that the grain density over the nucleus and cytoplasm did not change during a seven-hour period after labeling, nor did the distribution of label in the cytoplasm. Virtually all incorporation was eliminated by the inclusion of iododeoxy-uridine in the medium. Almost all of the nuclear label and at least 90% of the cytoplasmic label after ³H-thymidine incorporation was eliminated by deoxyribonuclease. It was concluded that there are no unusual features related to DNA distribution or metabolism in cotton embryo; i.e., that only small amounts of DNA are present in the cytoplasm and that all of the DNA is metabolically stable.Approximately 40% of the cytoplasmic grains after ³H-thymidine labeling were not associated with either plastids or mitochondria (i.e., were more than 0.1 micron distant). No fully satisfactory explanation for such an apparently high figure could be given.This work was supported by a Public Health Service fellowship 5-F2-GM-22,031-02 from the National Institute of General Medical Sciences, by NSF grant GB 3460, by NIH grant 5-R01-Ca0356-10 and by Miller Institute for Basic Science.     

The ultrastructure of the oyster brown cell,a cell with a fenestrated plasma membrane

Summary The oyster brown cell, a connective tissue cell of uncertain function and affinity, was characterized in the electron microscope by (1) the presence of large cytoplasmic granules, (2) fenestrations of the plasma membrane, and (3) an extensive tubular network originating in, or emptying into, the plasma membrane fenestrations. The brown cell did not appear to be a cell involved in glycogen storage or in the manufacture of exportable protein. The extensive tubular network and the membrane slits suggested that the brown cell may have been involved in the processing of biological fluids.This work was supported in part by Public Health Service Contract No. 5 To 1 ES00038-02, Health Sciences Advancement Award No. RR06138, and Tumor Biology Training Grant, NIH CA 05245.We wish to thank Miss Grete Nilsen for her expert technical assistance and Mr. Bob Munn for his help in the use of the electron microscope and for proof reading our MS. Our appreciation is also extended to Dr. J. Luft, Dr. A. K. Sparks, Miss P. Phelps, Mr. M. DeVault, and to the personnel of the Johnson Oyster Company, Inverness.     

Molecular characterization and cell-specific expression of an ion transport peptide in the tobacco hornworm, <Emphasis Type="Italic">Manduca sexta</Emphasis>

The crustacean hyperglycemic hormone (CHH) peptides regulate diverse physiological processes from reproduction to metabolism and molting in arthropods. In insects, the ion transport peptides (ITP), also members of the CHH family, have only been implicated in ion transport. In this study, we sequenced a nucleotide fragment spanning the conserved A1/A2 region of the putative CHH/ITP gene. This fragment was amplified from larval cDNA of the tobacco hornworm, Manduca sexta and showed a high degree of sequence conservation with the same region from other insects and, to a lesser degree, with that of crustacean species, suggesting the presence of a Manduca-specific CHH/ITP mRNA (MasITP mRNA). CHH-like immunocytochemical analyses with two crustacean antisera (from Carcinus maenas and Cancer pagurus) identified the presence of CHH-like immunoreactivity in nervous tissue of all developmental stages, but not in the gut of M. sexta. Specifically, CHH-like peptides localized to paired type IA₂ neurosecretory cells of the pars lateralis of the brain (projecting ipsilaterallly to the corpora cardiaca-allata complex) and to neurosecretory cells and transverse nerves of the ventral nerve cord in larvae, pupae, and adults. The distribution of the putative MasITP peptide shifted during development in a manner consistent with metamorphic reorganization. A comparison of hemolymph equivalents of CHH detected by enzyme-linked immunosorbent assay with CHH-like immunoreactivity in transverse nerves provided evidence for the release of MasITP from the transverse nerves into the hemolymph at insect ecdysis. These data suggest the presence of an insect ITP in M. sexta and a role for this hormone during ecdysis. This research was funded by the National Institutes of Health (MBRS SCORE Program-NIGMS) to M.F. (grant no. 2S06 GM52588-09), by the National Center on Minority Health and Health Disparities (grant no. 5P20-MD000262), an NIH RISE graduate fellowship to A.L.D. (5 R25 GM59298), an NIH PREP fellowship to C.C.H. and M.A.U. (5 R25 GM64078), an NSF CSU LS-AMP fellowship to C.C.H. (HRD-9802113), and by NIH MBRS-MARC to M.D.P. (T34 GM08574) and NIH MA/MS-PhD Bridge Scholarship to A.L.D. and C.C.H. (5R25 GM48972).     

Gradual increase in the electrical excitability of crayfish slow muscle fibers produced by anoxia or uncouplers of oxidative phosphorylation

Summary The electrical properties of crayfish (Procambarus clarkii) tonic flexor muscle fibers have been studied after exposure to anoxia or treatment with uncouplers of oxidative phosphorylation. These fibers do not normally generate action potentials; after anoxia, or treatment with any of three uncouplers (dinitrophenol, dicoumarol, CCCP), they gradually develop the ability to generate action potentials over the course of several hours. This change in excitability is not accompanied by any change in fiber resting potential or input resistance. The time course of uncoupler action is strongly temperature-dependent; it is speeded in fibers treated with cyanide, and slowed in fibers treated with iodoacetate. The possibility that the increase in excitability is caused by a decrease in the internal pH of the fibers is discussed.Abbreviations CCCP carbonyl cyanide-m-chlorophenylhydra-zone - DNP 2,4-dinitrophenol The majority of this work was carried out in the laboratory of Dr. Donald Kennedy at Stanford, supported by an NSF Predoctor-al Fellowship and NIH grant NS-02944; it is a pleasure to acknowledge Dr. Kennedy's continuing advice and encouragement throughout these experiments. The anoxia experiments were done in the laboratory of Dr. S. Hagiwara at UCLA, where the author is a postdoctoral fellow of the Helen Hay Whitney Foundation.     

Molecular characterization of meiotic recombination within the major histocompatibility complex of the mouse: Mapping of crossover sites within theI region

The molecular analysis of crossing-over within the mouse major histocompatibility complex provides a useful approach for the study of the structural characteristics of meiotic recombination. In this study five intra-I-region recombinants, each derived fromI ^k/I ^b heterozygotes, were characterized for restriction-fragment length polymorphisms (RFLPs) characteristic of theI region of the two parental strains. Southern blot analysis of intra-I recombinant strains A.TBR2, A.TBR3, A.TBR5, A.TBR13, and A.TBR17 using sixI-region DNA probes revealed that the point of crossing-over in all five recombinants occurred within a 6.2-kbKpnI-EcoRI segment located within theE gene. The segments of DNA containing the crossover point from each of the recombinant chromosomes were cloned by screening partial genomic libraries constructed in gt7 bacteriophage. Construction of partial restriction maps of the cloned segments from the parental and recombinant chromosomes permitted the boundaries of the area containing the crossover site to be narrowed to a 4.0-kb segment located almost entirely within an intron of theE gene. The recognition that the points of crossing-over in all five recombinants studied are clustered in a relatively small area of theI region provides further evidence for a hot spot of recombination associated with theE _ß gene.This work was supported by Grants AI14424 and AI20317 from the National Institutes of Health. J. Kobori was supported by a postdoctoral fellowship from the Arthritis Foundation. E. Zimmerer was supported by a postdoctoral fellowship from the Charles and Johanna Busch Fund of the Bureau of Biological Research. D. Spinella was supported by a predoctoral fellowship from the Charles and Johanna Busch Fund.