Unilateral lesions of the hamster suprachiasmatic nuclei: evidence for redundant control of circadian rhythms

Summary Several properties of vertebrate circadian rhythms can be attributed to the behavior of an underlying pacemaker system which is composed of two separate but mutually interacting circadian oscillators. As originally formulated, the model for such a pacemaker system proposed that two oscillators or populations of oscillators have different properties, specifically in their responses to light (Pittendrigh 1974; Pittendrigh and Daan 1976b). We have tested the proposition that the right and left suprachiasmatic nuclei (SCN) of the golden hamster contribute in different ways to the regulation of circadian rhythmicity by measuring the wheel-running activity rhythms of hamsters with lesions to either the right or left SCN. Although effects of unilateral or other partial SCN lesions on pacemaker properties were observed, these effects were not different in hamsters receiving right- or left-side lesions. More specifically: (1) free-running period () in constant light was shorter in lesioned hamsters irrespective of the side lesioned (Fig. 3a), and the total amount of SCN destruction was found to correlate with (Fig. 4). (2) Phase-angle difference () of some lesioned hamsters (both right- and left-side) during entrainment to LD, 1410 was significantly more positive than that of controls (Fig. 3b). (3) The rate of phase-shift following a shift of the light/dark cycle was not different in hamsters with right- or left-side lesions (Fig. 3c). And (4) the simultaneous expression of different circadian periods, similar to splitting, was observed in hamsters with unilateral lesions (Fig. 5). It is concluded that the right and left SCN are similar in their contributions to the control of circadian rhythmicity and that there is as yet no evidence for the permanent loss of multioscillator properties resulting from the destruction of only one of the two SCN.Abbreviations SCN suprachiasmatic nuclei or nucleus - LD light/dark cycle - LL constant light - DD constant dark - circadian period - activity time - rest time - phase angle - phase-angle difference - SD standard deviation - SE standard error - ANOVA analysis of variance     

Purification and properties of microsomal UDP-glucuronosyltransferase from rat liver.

p-Nitrophenol conjugating activity associated with liver microsomal UDP-glucuronosyltransferase (EC 2.4.1.17) was purified 150- to 200-fold from cell-free homogenates. The purification scheme included solubilization with the nonionic detergent Lubrol WX, anion exchange chromatography at pH 6.0 and 7.5, and affinity chromatography with UDP-hexanolamine Sepharose 4B. The enzyme purified as a phospholipid-protein complex and was shown to consist of a single polypeptide chain of molecular weight 59,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Amino acid analysis indicated approximately 531 mol of amino acids/59,000 g of enzyme and a molar ratio of nonpolar to polar residues of 1.08. During fractionation, the enzyme displayed instability with such steps as gel filtration, dialysis, or ultrafiltration of dilute samples; however, upon adsorption to ion exchange resins or storage in concentrated form, the enzyme was reasonably stable. The active lipoprotein complex showed both size and charge heterogeneity as judged by gel filtration and electrofocusing. Three forms of the enzyme resolved by isoelectric focusing had isoelectric points which averaged pH 6.68, 6.56, and 6.31. Polypeptide compositions of these electrophoretically distinct phospholipid protein complexes were indistinguishable on the basis of sodium dodecyl sulfate-polyacryl-amide gel electrophoresis, suggesting that the charge heterogeneity may be the result of differences in the phospholipid content of the lipoprotein complex.     

Basolateral plasma membrane localization of ouabain-sensitive sodium transport sites in the secretory epithelium of the avian salt gland

The distribution of Na+ pump sites (Na+-K+-ATPase) in the secretory epithelium of the avian salt gland was demonstrated by freeze-dry autoradiographic analysis of [(3)H] ouabain binding sites. Kinetic studies indicated that near saturation of tissue binding sites occurred when slices of salt glands from salt-stressed ducks were exposed to 2.2 μM ouabain (containing 5 μCi/ml [(3)H]ouabain) for 90 min. Washing with label-free Ringer's solution for 90 min extracted only 10% of the inhibitor, an amount which corresponded to ouabain present in the tissue spaces labeled by [(14)C]insulin. Increasing the KCl concentration of the incubation medium reduced the rate of ouabain binding but not the maximal amount bound. In contrast to the low level of ouabain binding to salt glands of ducks maintained on a freshwater regimen, exposure to a salt water diet led to a more than threefold increase in binding within 9-11 days. This increase paralleled the similar increment in Na+-K+-ATPase activity described previously. [(3)H]ouabain binding sites were localized autoradiographically to the folded basolateral plasma membrane of the principal secretory cells. The luminal surfaces of these cells were unlabeled. Mitotically active peripheral cells were also unlabeled. The cell-specific pattern of [(3)H]ouabain binding to principal secretory cells and the membrane-specific localization of binding sites to the nonluminal surfaces of these cells were identical to the distribution of Na+-K+-ATPase as reflected by the cytochemical localization of ouabain-sensitive and K+-dependent nitrophenyl phosphatase activity. The relationship between the nonluminal localization of Na+-K+-ATPase and the possible role of the enzyme n NaCl secretion is considered in the light of physiological data on electrolyte transport in salt glands and other secretory epithelia.     

Cloning of multiple copies of immunoglobulin variable kappa genes in cosmid vectors

The possibility of cloning large segments of DNA in cosmid vectors offers distinct advantages, in particular for the study of multigene families. Large size fragments of mouse embryo DNA were successfully cloned in the cosmid pHC 79. Twelve recombinants hybridizing specifically to an immunoglobulin kappa chain variable region probe were identified. In 9 of these recombinants, the size of the insert ranges from 30 to 43 kilobases. Factors affecting the cloning efficiency of a complex mammalian genome in cosmids were studied. The stability of these recombinant cosmids and the preparation of recombinant cosmid DNA are also discussed.     

Antibodies directed toward human erythrocyte Ca2+-ATPase: effect on enzyme function and immunoreactivity of Ca2+-ATPases from other sources

Antibodies directed against purified human erythrocyte Ca²⁺-ATPase (purified according to a procedure modified from V. Niggli, J. T. Penniston, and E. Carafoli, 1979, J. Biol. Chem., 254, 9955–9958) were raised in rabbits. In competitive radioimmunoassay tests of immunological cross-reactivity, human erythrocyte Ca²⁺-ATPase shows a consistent pattern of immunological similarity to the Ca²⁺-ATPases derived from cell surface fractions of other species, such as rat and dog erythrocyte ghosts, rat corpus luteum plasma membranes, and rat brain synaptic plasma membranes. On the other hand, a purified Ca²⁺-ATPase preparation from rabbit skeletal muscle sarcoplasmic reticulum failed to show any immunological similarity to the human enzyme. The amount of Ca²⁺-ATPase protein in the erythrocyte ghosts was estimated to be about 0.6 μg/mg ghost protein, which was not too different from the calculated value of 1.2 ± 0.2 μg/mg ghost protein (mean ± SD, n = 6) based on the calmodulin binding studies of the erythrocyte ghosts. Anti-Ca²⁺-ATPase immunoglobulin G inhibited enzyme activity and calcium transport, showing that at least one subpopulation of antibodies can block the active site of the enzyme. The antibodies had no effect on the binding of calmodulin to erythrocyte membranes.     

A molecular model of RGD ligands. Antibody D gene segments that direct specificity for the integrin alpha IIb beta 3.

Following an ill-defined activation event, the Arg-Gly-Asp (RGD) recognition site of the platelet integrin, glycoprotein IIb-IIIa (alpha IIb beta 3), can bind to fluid-phase, RGD-containing protein ligands, such as fibrinogen, or to the murine monoclonal IgM, PAC-1, which contains the sequence Arg-Tyr-Asp (RYD) within the third complementarity-determining region of its heavy chain (H3). PAC-1 has thus become a widely exploited marker of platelet alpha IIb beta 3 activation. In this report, we compare PAC-1 with two murine IgG, OP-G2 (IgG1 kappa) and LJ-CP3 (IgG1 kappa), that also contain the sequence RYD in H3 but bind to alpha IIb beta 3 without prior activation. Each antibody can inhibit the binding of the other two to intact platelets or to purified IIb-IIIa, the binding of each antibody is completely inhibited by peptides containing RGD, and H3 of each antibody uses the germline D-gene DSP 2.10 (CTATAGGTACGAC) which includes the sequence RYD. Two other murine IgG, HP20 and PCG1-1, cloned and sequenced by other laboratories, also utilize the DSP 2.10 sequence, but neither antibody binds to alpha IIb beta 3. From a comparison of the H3 sequences of these antibodies, we have developed a molecular model of the H3 loop region which can explain these differences in specificity. This model predicts that both the ability to bind to alpha IIb beta 3 and the activation dependence of that binding are a function of the orientation and, therefore, accessibility of the RYD sequence. This model and refinements thereof can be exploited to study the molecular basis for specificity and affinity of RGD-containing ligands for integrins.     

Muscle triglyceride metabolism during exercise.

Skeletal muscle cell contains a considerable amount of triglycerides. The amount stored depends on the animal species as well as on muscle fiber composition. It is well documented that triglycerides in the fast-twitch red muscle and to a lesser extent in the slow-twitch muscle, but not those in the fast-twitch white muscle, are mobilized during prolonged exercise. Yet, little is known about the regulation of the metabolism of muscle triglycerides either at rest or during exercise. This is well reflected by the fact that an enzyme responsible for the hydrolysis of muscle triglycerides has not been identified. Mobilization of muscle triglycerides during exercise seems to be under both adrenergic and noradrenergic control. Accumulation of lactic acid and reduction in muscle pH are likely to be strong inhibitors of muscle triglyceride lipolysis. Reduction of carbohydrate availability accelerates mobilization of muscle triglycerides during exercise. The relationship between the plasma free fatty acids and muscle triglyceride metabolism seems to be complex. It has been proposed that most free fatty acids entering the muscle cell is esterified before being oxidized, but this is arguable for contracting skeletal muscles. It is suggested that most free fatty acids entering contracting high oxidative myocytes are transported directly to the mitochondria. A much lesser portion is likely esterified. It is proposed that triglycerides stored in the contracting muscle cell are mobilized when the delivery of the blood-borne-free fatty acids to the mitochondria is insufficient.     

Localization of DNA sequences to a region within Xp11.21 between incontinentia pigmenti (IP1) X-chromosomal translocation breakpoints.

Incontinentia pigmenti (IP) is an X-linked dominant disorder characterized by developmental anomalies of the tissues and organs derived from embryonic ectoderm and neuroectoderm. An IP locus, designated IP1, probably resides in Xp11.21, since five unrelated patients with nonfamilial IP have been identified who possess constitutional de novo reciprocal X;autosome translocations involving Xp11.21. We have used a series of somatic cell hybrids containing the rearranged chromosomes derived from three of the five IP1 patients, along with other hybrid cell lines, to map probes in the vicinity of the IP1 locus. Five anonymous DNA loci--DXS422, DXS14, DXS343, DXS429, and DXS370--have been mapped to a region within Xp11.21, between two IP1 X-chromosomal translocation breakpoints; the IP1 t(X;17) breakpoint is proximal (centromeric) to this region, and the IP1 t(X;13) and t(X;9) X-chromosomal breakpoints lie distal to it. While no IP1 translocation breakpoint has yet been identified by pulsed-field gel electrophoretic (PFGE) analysis, an overlap between three probes--p58-1, 7PSH3.5, and cpX210--has been detected, placing these probes within 125 kb. Four probes--p58-1, 7PSH3.5, cpX210, and 30CE2.8--have been helpful in constructing a 1,250-kb PFGE map of the region between the breakpoints; these results suggest that the IP1 X-chromosomal translocation breakpoints are separated by at least this distance. The combined somatic cell hybrid and PFGE analyses we report here favor the probe order DXS323-(IP1 t(X;13), IP1, t(X;9]-(DXS422, DXS14, DXS343, DXS429, DXS370)-(IP1 t(X;17), DXZ1). These sequences provide a starting point for identifying overlapping genomic sequences that span the IP1 translocation breakpoints; the availability of IP1 translocation breakpoints should now assist the cloning of this locus.     

Effects of neonatal septal lesions on reproductive behavior of male and female rats

The purpose of this study was to examine the effects of neonatally placed septal lesions (SL) in male, female, and androgenized female rats on reproductive behavior. Animals were castrated as adults and tested for both feminine and masculine sexual behavior. After treatment with estradiol benzoate (EB) alone (2 μg daily for 3 days), only the females with SL which had not been given testosterone propionate (TP) neonatally showed a facilitation of lordosis behavior. Following EB (2 μg for 3 days) plus 0.5 mg progesterone (P), both the lesioned and the sham-operated female groups showed an increase in the display of lordosis in either hormonal condition. All animals were given a pretest for masculine sexual behavior and tested on Days 4, 7, 11, and 15 of daily TP treatment (150 μg/day). There was no effect of the neonatally placed SL on masculine sexual behavior in female rats or in female rats androgenized with 30 μg TP. However, lesioned females treated neonatally with 1 mg TP showed a marginal enhancement of masculine sexual behavior. Male rats given SL neonatally showed a marked enhancement of masculine sexual behavior compared to that of controls. These results suggest that, depending on the neonatal hormone environment, SL selectively increase behavioral sensitivity to hormones. Although neonatally lesioned females show behavioral responses similar to females given SL as adults, male rats given SL neonatally are unique in that they show enhanced masculine sexual behavior whereas males lesioned as adults do not.     

Cell-free synthesis of a large translation product of prolactin messenger RNA.

RNA prepared from rat anterior pituitaries or from prolactin-secreting pituitary tumors has been shown to direct the synthesis of a large form of prolactin in a cell-free system derived from wheat germ. Immunoprecipitation of cell-free reactions demonstrated the synthesis of a product which was recognized by a specific antiprolactin antisera. Analysis of the immunoprecipitate on sodium dodecyl sulfate containing polyacrylamide gels suggested that the cell-free product has a molecular weight of approximately 28,000 compared to 22,500 for prolactin. RNA prepared by completely different techniques from rat pituitary and a pituitary tumor resulted in identical large translation products. Translation of tumor RNA in a cell-free system from Krebs ascites cells also resulted in a similar large product. The identity of the cell-free product as prolactin was confirmed by comparing peptides derived from the cell-free product and prolactin. The results of these studies suggest that prolactin messenger RNA directs the cell-free synthesis of a product which contains the amino acid sequence of prolactin but which has an addition at one or both ends of the molecule.