Cytoplasmic recycling of 60S preribosomal factors depends on the AAA protein Drg1

Allelic forms of DRG1/AFG2 confer resistance to the drug diazaborine, an inhibitor of ribosome biogenesis in Saccharomyces cerevisiae. Our results show that the AAA-ATPase Drg1 is essential for 60S maturation and associates with 60S precursor particles in the cytoplasm. Functional inactivation of Drg1 leads to an increased cytoplasmic localization of shuttling pre-60S maturation factors like Rlp24, Arx1, and Tif6. Surprisingly, Nog1, a nuclear pre-60S factor, was also relocalized to the cytoplasm under these conditions, suggesting that it is a previously unsuspected shuttling preribosomal factor that is exported with the precursor particles and very rapidly reimported. Proteins that became cytoplasmic under drg1 mutant conditions were blocked on pre-60S particles at a step that precedes the association of Rei1, a later-acting preribosomal factor. A similar cytoplasmic accumulation of Nog1 and Rlp24 in pre-60S-bound form could be seen after overexpression of a dominant-negative Drg1 variant mutated in the D2 ATPase domain. We conclude that the ATPase activity of Drg1 is required for the release of shuttling proteins from the pre-60S particles shortly after their nuclear export. This early cytoplasmic release reaction defines a novel step in eukaryotic ribosome maturation.     

Implementation of Tuberculosis Intensive Case Finding,Isoniazid Preventive Therapy,and Infection Control ("Three I's") and HIV-Tuberculosis Service Integration in Lower Income Countries

SettingWorld Health Organization advocates for integration of HIV-tuberculosis (TB) services and recommends intensive case finding (ICF), isoniazid preventive therapy (IPT), and infection control (“Three I’s”) for TB prevention and control among persons living with HIV.ObjectiveTo assess the implementation of the “Three I’s” of TB-control at HIV treatment sites in lower income countries.DesignSurvey conducted between March-July, 2012 at 47 sites in 26 countries: 6 (13%) Asia Pacific, 7 (15%), Caribbean, Central and South America, 5 (10%) Central Africa, 8 (17%) East Africa, 14 (30%) Southern Africa, and 7 (15%) West Africa.ResultsICF using symptom-based screening was performed at 38% of sites; 45% of sites used symptom-screening plus additional diagnostics. IPT at enrollment or ART initiation was implemented in only 17% of sites, with 9% of sites providing IPT to tuberculin-skin-test positive patients. Infection control measures varied: 62% of sites separated smear-positive patients, and healthcare workers used masks at 57% of sites. Only 12 (26%) sites integrated HIV-TB services. Integration was not associated with implementation of TB prevention measures except for IPT provision at enrollment (42% integrated vs. 9% non-integrated; p = 0.03).ConclusionsImplementation of TB screening, IPT provision, and infection control measures was low and variable across regional HIV treatment sites, regardless of integration status.     

The ultrastructural organization of the mature spermatozoon of Luidia clathrata (Say) (Echinodermata: Asteroidea)

The sperm of Luidia clathrata are morphologically typical of asteroid sperm. The head is spherical and contains the nucleus and acrosomal complex. The nucleus has an anterior indentation in which rests the acrosomal complex. There is no evidence of a centriolar fossa along the posterior border of the nucleus. The acrosome is a cup-shaped structure containing a less electron dense central region. The periacrosomal material is homogeneous in nature, and the subacrosomal specialization of the periacrosomal materials appear as bands of varying electron density. The middle piece is an annular band of mitochondria which surrounds the proximal and distal centrioles. The centrioles exhibit the typical nine triplet arrangement. Both the centrioles and the axoneme project to one side of the middle piece region. Associated with the distal centriole is an elaborate pericentriolar process.     

Predicted Delays in the Activation of the Contractile System

The capacitance C'(e), presumed to be located across the walls of the transverse tubules of twitch fibers, was identified in earlier impedance measurements by virtue of having a resistance in series with it. When the voltage V(m) across the surface membrane is made to vary, the voltage V(c) across C'(e) will be delayed with respect to V(m), the extent of the delay depending on the location of the series resistance. Model 1 assumes that the resistivity of the lumen of the tubules is negligible; model 2 assumes that the series resistance arises entirely in the tubular lumen; model 3 assumes that the resistivity of the tubular lumen is small, but not negligible and that the bulk of the resistance arises in a structure directly in series with C'(e) and having a similar geometric distribution. If V(m) varies sinusoidally, the relative value of V(c(max)) will fall with increasingly higher powers of the frequency at the center of the fiber if model 2 is applicable, whereas models 1 and 3 predict that V(c(max)) will fall at high frequency only in proportion to the frequency everywhere in the cross-section of the fiber. Equations have been derived for the voltage change V(c) in response to a step change of V(m) and during an action potential. On the assumption that contraction is initiated when V(c) reaches mechanical threshold, the delay between the activation of myofibrils on the axis of the fiber and at the surface would amount to 2.6 msec in model 2 and 0.25 msec in model 3 for frog fibers of about 100 mum diameter during a twitch.     

Dihydroxyphenylalanine and Dopamine Are Released from Portal Vein Together with Noradrenaline and Dihydroxyphenylglycol During Nerve Stimulation

The overflows of 3,4-dihydroxyphenylalanine, dopamine, noradrenaline, and 3,4-dihydroxyphenylglycol in canine portal vein superfused in vitro were studied before, during, and after depolarization of sympathetic nerve endings. The four compounds were separated from superfusate and from tissue on Sep-Pak C-18 cartridges and quantified by HPLC with electrochemical detection. Physiological and biochemical methods were used to show that the compound released was most probably 3,4-dihydroxyphenylalanine; the identity of the other endogenous compounds has been established previously. Release of 3,4-dihydroxyphenylalanine was calcium and frequency dependent, inhibited by a-m-L-p-tyrosine (an inhibitor of tyrosine hydroxylase) and augmented by 3-hydroxybenzylhydrazine (an inhibitor of aromatic amino acid decarboxylase). The overflows of dopamine, noradrenaline, and 3,4-dihydroxyphenylglycol from the vein were calcium and frequency dependent. It was estimated that under control conditions, approximately 80% of the total 3,4-dihydroxyphenylalanine that was synthesized was directed to catecholamine biosynthesis, approximately 8% overflowed from the vein, and approximately 14% remained unchanged within the tissue. It is concluded that 3,4-dihydroxyphenylalanine and dopamine are released together with noradrenaline and 3,4-dihydroxyphenylglycol from portal vein upon nerve depolarization.     

Precursors and Metabolites of Norepinephrine in Sympathetic Ganglia of the Dog

3,4-Dihydroxyphenylalanine, dopamine, epinephrine, 3,4-dihydroxyphenylglycol, and 3,4-dihydroxyphenylacetic acid as well as norepinephrine were measured in dog lumbar sympathetic ganglia. The responses of these compounds to several classes of stimuli were investigated using an isolated time-resolved superfusion system. Nonselective (i.e., amphetamine and high K+) and receptor-mediated selective (oxotremorine) stimuli were used to evoke releases. The overflows of all compounds were measured by HPLC with electrochemical detection. The efficiency of each stimulus was estimated by normalizing the amount of evoked release to the total neurotransmitter pool when the stimulus was applied; i.e., fractional release was calculated. Overflows of all compounds except 3,4-dihydroxyphenylalanine were enhanced by a 10-min 100 microM amphetamine stimulus, and each of the catecholamine pools (dopamine, norepinephrine, and epinephrine) was affected to the same degree. By contrast, the 3,4-dihydroxyphenylalanine and dopamine pools were more readily releasable than the norepinephrine pool with a 10-min 80 mM K+ stimulus, and these releases were Ca2+ dependent. Epinephrine was released in preference to norepinephrine by a 10-min 1 mM oxotremorine stimulus. The data suggest the existence of at least three types of neurons in dog lumbar ganglia and are consistent with previous histological observations.     

EFFECT OF THE ADMINISTRATION OF L-5-HYDROXYTRYPTOPHAN AND A MONOAMINE OXIDASE INHIBITOR ON GLUCOSE METABOLISM IN RAT BRAIN

The effects of the presence of large amounts of 5-HT and of its precursor 5-HTP in brain on cerebral utilization of glucose were studied. [U-¹⁴C]Glucose was injected to fed rats that had previously been treated with L-5-HTP, L-5-HTP and an inhibitor—N-[β-(2-chlorophenoxy)-ethyl]-cyclopropylamine hydrochloride (Lilly-51641)-of MAO, or Lilly-51641 alone. Such treatment increased the concentrations of 5-HTP and 5-HT in the brain. After treatment with 5-HTP and Lilly-51641, and to a lesser extent with Lilly-51641 alone, the concentration of glucose in plasma was increased. However, the uptake of glucose by the brain did not appear to be proportionately increased, and this suggested an impairment in this mechanism. After the administration of Lilly-51641 alone and more especially of Lilly-51641 plus 5-HTP, the concentration of glucose in the brain was increased. This increase was thought to be due to an impairment of glucose utilization, because the flux of ¹⁴C from glucose to amino acids in the brain was reduced. The concentrations of most major amino acids in the brain were not greatly affected by these treatments. GABA and alanine concentrations in the brain were modestly increased after treatment with 5-HTP alone or in combination with Lilly-51641. The present results suggest that the metabolism of glucose to amino acids in the brain is altered when the concentration of 5-HTP, or more especially that of 5-HT, in the brain is increased.