Assessment of counting efficiencies for labeled protein and other macromolecules in filter disk assays

A several-fold greater counting efficiency is observed for protein labeled with [³H]leucine than for free [³H]leucine using a conventional filter disk assay. A similar, though less marked, effect is noted for ¹⁴C-labeled molecules. These results are comparable to those reported by others for counting efficiencies of labeled DNA and deoxynucleotides and illustrate the generality of this effect with regard to macromolecules and their low-molecular weight precursors. This phenomenon, presumably due to differences in the distribution of large and small molecules within filters, gives rise to errors in the quantitation of macromolecule synthesis if a counting efficiency identical to that of the precursor is assumed to apply. A convenient method for determining counting efficiencies of various molecules bound to filters is presented which eliminates this problem.     

Response of poly(adenylic acid) polymerase in rat liver nuclei and mitochondria to stravation and re-feeding with amino acids.

Poly(adenylic acid) polymerase was extracted from liver nuclei and mitochondria of rats either fed ad libitum, starved overnight or starved and then re-fed with a complete amino acid mixture for 1-3 h. The enzymes were partially purified and assayed by using exogenous primers. Starvation resulted in an 80% decrease in the total activity of the purified nuclear enzyme, and the mitochondrial enzyme activity diminished to almost zero after overnight starvation. Measurements of the protein content of whole nuclei or mitochondria and of the enzyme extracts from these organelles indicated that the decrease in enzyme activity on starvation was not caused by incomplete extraction of the enzyme from the starved animals. Re-feeding the animals with the complete amino acid mixture increased the total activity of poly(A) polymerase from the nuclei and mitochondria by 1.9-fold and 63-fold respectively. Under these conditions, the total protein content of the nuclei and mitochondria increased by only 13 and 32% respectively. These data indicate that poly(A) polymerase is one of the cellular proteins specifically regulated by amino acid supply.     

Intracellular localization of GDP-l-fucose: Glycoprotein and CMP-sialic acid: Apolipoprotein glycosyltransferases in rat and pork livers

A GDP-l-fucose:glycoprotein fucosyltransferase which transfers l-fucose to terminal β-N-acetyl-d-glucosaminyl residues of sialidase-, β-galactosidase-treated α₁-acid glycoprotein and a CMP-sialic acid:glycoprotein sialyltransferase acting on sialidase-treated apolipoprotein-Ala₁ from human very low density lipoprotein have been shown to be concentrated in rat liver Golgi apparatus preparations at enrichments of 40- and 45-fold, respectively, and in pork liver Golgi-rich fractions at enrichments of 35- and 20-fold, respectively. A second fucosyltransferase acting on sialidase-treated α₁-acid glycopretein was absent from rat liver and was enriched only 13-fold in a pork liver Golgi-rich fraction. The smooth-surfaced microsome fraction was the only other rat liver subcellular fraction with appreciable levels of the GDP-l-fucose: β-N-acetyl-d-glucosaminide fucosyltransferase and the lipoprotein sialyltransferase (enrichments of 2.6- and 5.2-fold, respectivley). This enrichment could not be attributed to the plasma membrane content of the smooth microsome fraction since plasma membrane fractions from rat liver were shown to have relatively low concentrations of these two transferases (enrichments of 0.3 or less). Rat liver plasma membrane was also shown to have similarly low relative specific activities for three other glycosyltransferases (sialyl-, galactosyl-, and N-acetylglucosaminyl-). The accurate determination of the glycosyltransferase activities of the plasma membrane fraction required the use of relatively low concentrations of plasma membrane and relatively high concentrations of nucleotide-sugars in order to avoid interference by the high nucleotide-sugar pyrophosphatase and hydrolase activities of this fraction.     

PARTICIPATION OF DOPAMINE- AND SEROTONIN-RECEPTORS IN THE DISAGGREGATION OF BRAIN POLYSOMES BY l-DOPA AND l-5-HTP

Abstract— It has previously been shown that the disaggregation of brain polysomes and suppression of brain protein synthesis observed in rats given the amino acids l -dopa or l -5-HTP is mediated by the decarboxylation products dopamine and serotonin. Present studies demonstrate that the poly-some disaggregation is caused by the interactions of the monoamines with specific receptor sites. Thus, dopa-induced disaggregation is blocked if rats are pretreated with haloperidol or pimozide (but not methysergide or cyproheptadine), while 5-HTP-induced disaggregation is blocked by methysergide or cyproheptadine (but not by haloperidol or pimozide).
Pretreatment of rats with MK-486, a drug that inhibits dopa decarboxylase in blood vessels and peripheral tissues but not brain, does not block dopa-induced brain polysome disaggregation; hence this disaggregation depends on the interaction of dopamine with receptors in the brain parenchyma. Brain polysomes are not disaggregated in rats given intraperitoneal apomorphine (or intracisternal dopamine). The disaggregation caused by dopa is not reduced in animals pretreated with sufficient intracisternal 6-hydroxydopamine to cause major damage to catecholaminergic nerve terminals.     

House mice bred for many generations in two environments

Wild house mice, Mus musculus L., were trapped, and their descendants reared, in permanently mated pairs, for a number of generations in two laboratory environments, at about 21°C (controls) and -3°C, respectively. All mice had sawdust and cottonwool for bedding; but the nests of those at -3°C were colder than those in the warm, and fluctuated greatly in temperature.
Reproductive performance was inferior in the cold environment: more pairs were barren, and the fecund pairs reared fewer young than the controls. Yet litters at birth were usually larger in the cold, and the young at three weeks were always heavier. Over ten generations nestling mortality declined at -3°C.
From generation 1 on, adult mice at -3°C were heavier than the controls, but there was no corresponding increase in body length. Tails were much shorter relative to body length in the first generations in the cold, but returned to control proportions by generation 10. Most of the structural changes in the cold accord with the "rules" of Bergmann and Allen.
The incidence of abnormal sixth lumbar vertebrae was low in all generations at both temperatures.
After nine generations, some mice were transferred from the cold to the warm environment, and bred for a further three generations. There they outstripped the controls both in reproductive performance and in growth. They also had more fat, and a heavier and longer small intestine; but the heart, stomach and kidneys were lighter than those of the controls. Adrenal weights at 21°C declined over the generations, but those of the mice at -3°C did not.
The secular changes observed, especially those in the cold environment, are attributed principally to differential selection of genotypes, not to inbreeding; but maternal effects may also have been involved.     

Plasma immunoreactive melanotrophic hormones in patients on maintenance haemodialysis.

Circulating levels of melanotrophic horomones and ACTH were determined in patients treated by maintenance dialysis for chronic renal failure. Plasma melanotrophic hormone levels were greatly increased in all patients studied (125-1100 ng/1 as compared with 12-36 ng/1 in normal adults) and were correlated with the duration of treatment. Skin pigmentation, especially in exposed areas, was notably increased, particularly in those patients with the highest plasma melanotrophic concentrations. Plasma ACTH levels were normal or only slightly raised and circulating corticosteroid concentrations, as determined by a fluorimetric method, all lay within the physiological range. The dissociation between ACTH and melanotrophic hormone levels in these patients may have been the result of a slower metabolic clearance of the latter.     

Clobetasone butyrate,a new topical corticosteroid: clinical activity and effects on pituitary-adrenal axis function and model of epidermal atrophy.

Clobetasone butyrate is a new corticosteroid, selected for study because of its combination of good activity in the vasoconstriction test and low systemic activity in animals. Formulated as an 0.05% ointment and cream (Molivate) it was clinically effective in patients with eczema, its activity being significantly greater than that of hydrocortisone 1% or fluocortolone 0.2% (Ultradil). Under conditions that predispose to maximal percutaneous absorption clobetasone butyrate ointment had minimal effect on hypothalamic-pituitary-adrenal function. In an animal model of cutaneous atrophy it caused less thinning of the epidermis than steroids other than hydrocortisone. Clobetasone butyrate 0.05% ointment and cream gave every indication of offering clinically effective topical anti-inflammatory activity with a wide margin of safety.     

Abdominal surgical emergencies in the puerperium.

Since surgical emergencies in the puerperium are rare, surgeons and obstetricians amass little experience of them. There may be considerable delay in making a diagnosis, firstly, because the abdominal symptoms are thought to be related to the pregnancy, and, secondly, because the signs are rarely convincing in contrast to the symptoms. Any of the common surgical emergencies may occur in pregnancy and the puerperium and there are some others that appear to be a particullar problem of the puerperium.