Does in vitro activation of postsynaptic alpha 2-adrenoceptor utilize intracellular Ca2+ for contraction in dog saphenous vein?

Dog saphenous vein spiral strips were employed to determine whether an intracellular source of Ca2+ is used for contraction upon activation of the alpha 2-adrenoceptor by B-HT 920 in Ca2+-free Krebs solution containing 50 microM EGTA. The studies were carried out in parallel with the activation of the alpha 1-adrenoceptor by phenylephrine (Phe) under the condition that B-HT 920 (10(-5) M) and Phe (2 x 10(-6) M) gave rise to a similar level of responses in Ca2+-containing Krebs solution. A similar level of responses to these agonists at equieffective concentrations in Ca2+-free medium were also observed. Such responses to Phe and B-HT 920 were inhibited by 10(-7) M rauwolscine and 10(-7) M prazosin, respectively, and were not affected by 10(-7) M nifedipine or 5 mM Mn2+. The responses to B-HT 920 (10(-5) M) and submaximal concentration of Phe (2 x 10(-6) M) in Ca2+-free medium were additive. However, if the vascular strips were first contracted maximally with 10(-4) M Phe in Ca2+-free medium to deplete the intracellular Ca store, subsequent addition of B-HT 920 failed to induce additional response. Our results strongly suggest that activation of alpha 2-adrenoceptor in dog saphenous vein in Ca2+-free medium indeed utilizes intracellular Ca2+ for contraction. We also found that the failure of earlier studies to demonstrate the contractile effects of B-HT 920 in dog saphenous vein was due to experimental artifacts derived from the use of high concentration of EGTA and artificial pH-buffering reagent.     

Extrachromosomal circular DNAs in Drosophila melanogaster: Comparison between embryos and Kc0% cells

We established the size distribution of extrachromosomal covalently closed circular DNA molecules from embryos of various Drosophila melanogaster strains and from Kc0% tissue culture cells. In embryos, more than 80% of the circular DNA molecules are smaller than 2.5 kb and all the distributions show a peak of molecules of between 200 and 400 bp. The Kc0% cell distribution differs mainly from that of embryos in that 48% of the molecules have a size between 4 and 8 kb. Correlating with this, circular molecules homologous to copia, 412 and 297 were detected only in Kc0% cells. The three tandemly repeated families containing the 5S genes, the histone genes and the 240 bp repeat of the ribosomal DNA intergenic spacer, which had previously been identified in circular DNAs from embryos, were also found in cultured cells. A fourth tandemly repeated family corresponding to the 1.688 g/cm³ satellite DNA was detected, both in embryos and Kc0% cells. It consists of circular multimeric molecules containing multiple copies of the 359 bp repeated unit. No circular DNA molecules homologous to the actin genes, the type I ribosomal DNA insertion, or the F and I transposable elements were found in embryos or Kc0% cells. Thus it appears that the extrachromosomal circular DNA molecules from embryos and from tissue culture cells differ mainly in the presence of circular copies of the copia-like transposable elements.     

Characterization of intracellular ice formation in Drosophila melanogaster embryos

Cryomicroscopy and differential scanning calorimetry (DSC) were used to characterize the incidence of intracellular ice formation (IIF) in 12- to 13-hr-old embryos of Drosophila melanogaster (Oregon-R strain P2) as influenced by the state of the eggcase (untreated, dechorionated, or permeabilized), the composition of the suspending medium (with and without cryoprotectants), and the cooling rate. Untreated eggs underwent IIF over a very narrow temperature range when cooled at 4 or 16 degrees C/min with a median temperature of intracellular ice formation (TIIF50) of -28 degrees C. The freezable water volume of untreated eggs was approximately 5.4 nl as determined by DSC. IIF in dechorionated eggs occurred over a much broader temperature range (-13 to -31 degrees C), but the incidence of IIF increased sharply below -24 degrees C, and the cumulative incidence of IIF at -24 degrees C decreased with cooling rate. In permeabilized eggs without cryoprotectants (CPAs), IIF occurred at much warmer temperatures and over a much wider temperature range than in untreated eggs, and the TIIF50 was cooling rate dependent. At low cooling rates (1 to 2 degrees C/min), TIIF50 increased with cooling rate; at intermediate cooling rates (2 to 16 degrees C/min), TIIF50 decreased with cooling rate. The total incidence of IIF in permeabilized eggs was 54% at 1 degree C/min, and volumetric contraction almost always occurred during cooling. Decreasing the cooling rate to 0.5 degree C/min reduced the incidence of IIF to 43%. At a cooling rate of 4 degrees C/min, ethylene glycol reduced the TIIF50 by about 12 degrees C for each unit increase in molarity of CPA (up to 2.0 M) in the suspending medium. The TIIF50 was cooling rate dependent when embryos were preequilibrated with 1.0 M propylene glycol or ethylene glycol, but was not so in 1.0 M DMSO. For embryos equilibrated in 1.5 M ethylene glycol and then held at -5 degrees C for 1 min before further cooling at 1 degree C/min, the incidence of IIF was decreased to 31%. Increasing the duration of the isothermal hold to 10 min reduced the incidence of IIF to 22% and reduced the volume of freezable water in embryos when intracellular ice formation occurred. If the isothermal hold temperature was -7.5 or -10 degrees C, a 10- to 30-min holding time was required to achieve a comparable reduction in the incidence of IIF.     

TGF-beta 1-induced inhibition of mouse mammary ductal growth: developmental specificity and characterization

TGF-beta 1, implanted into growing mouse mammary glands, was previously shown to inhibit ductal growth in an apparently normal and fully reversible manner. In this report we extend these findings to show that TGF-beta 1 inhibition is highly specific. In pregnant or hormone-treated mice, doses of TGF-beta 1 that were capable of fully inhibiting ductal elongation had little effect on the proliferation of lobuloalveolar structures. Additionally, the inhibitory action of TGF-beta 1 on ducts is epithelium-specific, resulting in cessation of DNA synthesis in the rapidly proliferating epithelium of mammary end buds, but does not inhibit DNA synthesis in the stroma surrounding the end buds. At the cellular level, transplant studies showed that TGF-beta 1 inhibited the regeneration of mammary ductal cells when implanted into mammary gland-free fat pads by suppressing the formation of new end buds, without inhibiting maintenance DNA synthesis in ductal lumenal epithelium; this observation indicates the potential of TGF-beta 1 to maintain patterning by suppressing adventitious lateral branching. The time-course of TGF-beta 1 inhibition of end buds was rapid, with cessation of DNA synthesis by 12 hr, followed by loss of the stem cell (cap cell) layer. The question of glandular exposure to TGF-beta 1 administered in EVAc implants was also investigated. Incorporation of TGF-beta 1 into EVAc was found not to degrade the hormone, while the release kinetics of the ligand from implants, its retention in the gland, and the demonstrable zone of exposure were consistent with observed inhibitory effects. These results support the hypothesis that TGF-beta 1 is a natural regulator of mammary ductal growth.     

Expression and characterization of the tau subunit of phosphorylase kinase

A cDNA encoding the entire tau subunit of rabbit skeletal muscle phosphorylase kinase was reconstructed and inserted into a plasmid containing the Escherichia coli ptac promoter and a constructed plasmid containing the ptac promoter and bacterial chloramphenicol acetyl transferase (CAT) gene, respectively. A significant phosphorylase kinase activity was found, in the first case. In the second case, a fused protein containing 73 amino acids from the CAT protein was obtained. After renaturation, the CAT-tau subunit protein shows enzymatic activity similar to the HPLC-purified and renatured tau subunit.     

Cytochalasin D inhibits basal body migration and ciliary elongation in quail oviduct epithelium

Summary The effects of cytochalasin D (CD) were studied by scanning (SEM) and transmission (TEM) electron-microscopic examination at different stages of ciliary differentiation in epithelial cells of quail oviduct. Immature quails were prestimulated by estradiol benzoate injections to induce ciliogenesis in the undifferentiated oviduct. After 24 h of CD culture, SEM study revealed inhibition of ciliogenesis and dilation of the apex of non-ciliated cells. TEM study showed that 2 h of CD treatment produced dilation of lateral intercellular spaces, after 6 h of treatment, this resulted in intracellular macrovacuolation. Vacuoles were surrounded by aggregates of dense felt-like material. CD also induced the disappearance of microvilli, and rounding of the apical surface of undifferentiated cells and those blocked in ciliogenesis. Centriologenesis was not inhibited by CD; basal bodies assembled in generative complexes in the supranuclear region after 24 h of treatment. However, the migration of mature basal bodies towards the apical surface was impaired. Instead, they anchored onto the membrane of intracellular vacuoles; growth of cilia was induced in the vacuole lumen. Cilium elongation was disturbed, giving abnormally short cilia with a dilated tip; microtubules failed to organize correctly.     

Patterns of [13N]ammonium uptake and assimilation by Frankia HFPArl3

Nitrogen-starved cells of Frankia strain HFPArl3 incorporated [¹³N]-labeled ammonium into glutamine serine (glutamate, alanine, aspartate), after five-minute radioisotope exposures. High initial endogenous pools of glutamate were reduced, while total glutamine increased, during short term NH _inf4 ^sup+ incubation. Preincubation of cells in methionine sulfoximine (MSX) resulted in [¹³N]glutamine reduced by more than 80%, while [¹³N]glutamate and [¹³N]alanine levels increased. The results suggest that glutamine synthetase is the primary enzyme of ammonium assimilation, and that glutamate dehydrogenase and alanine dehydrogenase may also function in ammonium assimilation at low levels. Efflux of [¹³N]serine and lesser amounts of [¹³N]glutamine was detected from the Frankia cells. The identity of both Ser and Gln in the extracellular compartment was confirmed with gas chromatography/mass spectrometry. Serine efflux may be of significance in nitrogen transfer in Frankia.Abbreviations Pthr phosphothreonine - Aad -amino-adipate - MSX methionine sulfoximine     

Circadian locomotor rhythms in the desert iguana I. The role of the eyes and the pineal

Summary The pineal and the eyes are known to be important components in the circadian system of some species of lizards; their effects may be mediated by the hormone melatonin. We examined the role played by these structures in the desert iguana (Dipsosaurus dorsalis). Surgical removal of the pineal had no effect on circadian locomotor rhythms, even though this procedure abolished the circadian rhythm of melatonin in the blood. Furthermore, when the isolated pineal of Dipsosaurus was studied in organ culture, it showed no circadian rhythm of melatonin secretion, as do pineals of some other lizard species, although it did produce large quantities of this hormone. Bilateral ocular enucleation had only small effects on the freerunning period of locomotor rhythms, without affecting melatonin levels in the blood. Behavioral circadian rhythms persisted in desert iguanas subjected to both enucleation and pinealectomy. These data suggest that neither the pineal nor the eyes are central components of the circadian pacemaking system in Dipsosaurus, nor is melatonin critically involved in maintaining its organization.Abbreviations CT circadian time - ZT zeitgeber time - LL constant light - LD light-dark cycle - DD constant darkness - freerunning circadian period     

Actigraphy: A Means of Assessing Circadian Patterns in Human Activity

-Twenty-three diurnally active (0705-2333), healthy persons between 22 and 54 yrs of age and without history of sleep abnormality were monitored continuously for 120 consecutive hr (five days) by wrist actigraphy. Circadian rhythms of high amplitude were detected by cosinor analysis for each participant and for the groups of 10 males and 13 females with the average span of heightened activity timed between ∼1330 and 1605. The circadian peak-trough difference in wrist movement was marked, equalling aproximately 75% of the 24-hr mean level. In 19 of 23 participants, the 24-hr mean of wrist activity varied between 140-180 movements/min, with four persons exhibiting lesser means of 110-140 movements/min. With respect to the daytime span of activity, the mean wrist movement of individual participants ranged from 155-265 movements/min, with the majority (20/23) varying between 185-245 movements/min. During nocturnal sleep the mean wrist activity level was quite low, varying between individuals from 5 to 25 movements/min for 21 of 23 persons. Wrist actigraphy proved to be well-accepted and was a most reliable means of monitoring aspects of body movement during activity and sleep in ambulatory persons adhering to usual life habits and pursuits.