RNA and protein synthesis in cultured human fibroblasts derived from donors of various ages

RNA synthesis in human fibroblasts from donors of various ages was studied in fibroblasts made permeable to nucleoside triphosphates with the nonionic detergent Nonidet P40. Cells from donors of 11 years and older showed a 30-40% decline in total RNA synthesis. The decrease in RNA synthesis was primarily due to a lowering of RNA polymerase II activity (alpha-amanitin sensitive). Studies on the incorporation of leucine into protein also showed a 30-40% decrease in cells from older donors.     

The in vivo synthesis of stable RNA within the vagus nerve following vagotomy

The in vivo synthesis of rRNA and tRNA within the proximal region of the rat vagus nerve, up to and including the crush site, rapidly increased following vagotomy. Synthesis was maximal at 2 days post-crush when a 15-fold increase in rRNA was observed. This increase was localized to the region 2–3 mm either side of the crush site. Apparent DNA synthesis also appeared substantially elevated at 2, 4 and 7 days post-crush. These results are consistent with a Schwann cell hypertrophy at the site of injury which facilitates removal of axonal debri and myelin and provides structures to receive new axonal sprouts.     

Characterization of a second protein associated with virions of tobacco mosaic virus

An average of about one molecule of a 26,500-dalton protein (termed H_t protein) has been found per tobacco mosaic virus (TMV) virion. H_t protein has been detected in five strains of TMV (U₁, K, C_c, GA, and trace amounts in U₂) prepared from tobacco by a variety of isolation methods and purification treatments, followed by incubation of virions with either 5% Triton X-100, 80 mM EDTA, 0.2 M phosphate or any of several proteolytic enzymes. H_t protein is associated with the insoluble RNA after disruption of virions with cold 66% acetic acid; it will also associate or co-precipitate in vitro with other added RNAs under the same conditions, apparently in a nonspecific manner. This protein is considered to be coded for by the host rather than the virus, based on two observations: (1) H_t protein from strain C_c TMV propagated in tobacco is smaller than the protein extracted from the same strain isolated from legumes; (2) peptide analysis after limited proteolysis under denaturing conditions showed that the H_t proteins isolated from two very different TMV strains (U₁ and C_c) propagated in a common host were identical. If viral RNAs had coded for the H_t proteins, their amino acid sequences would have been expected to be different because of the known very dissimilar nucleotide sequences of the RNAs of the two strains. By comparing different classes of virus rods in which the coat protein subunits had been partially stripped from one end of the molecule (5′ end), it was concluded that H_t protein is not associated with a specific site in the virion. It is not preferentially associated with or absent from that proportion of TMV virions which are resistant to alkali stripping. Its location within the virions is not known, but it may be either intercalated between the coat proteins subunits or, more probably, lodged in the hole in the center of the virion. Its role in viral replication and pathogenicity, if any, remains to be determined.     

Feline syncytium-forming virus proviral DNA Time of synthesis and relationship to the host cell genome

An infectious DNA assay has been used to study the time of synthesis of feline syncytium-forming virus (FSFV) proviral DNA, and also its relationship to the host cell genome. When extracted late in infection, infectious FSFV proviral DNA was associated with high molecular weight DNA with the same buoyant density as host cell DNA. Furthermore, the apparent size of this DNA could be reduced by shearing, suggesting that the fragments containing FSFV provirus also contained sequences not essential for viral replication. These data suggest that the DNA provirus of FSFV was integrated into chromosomal DNA late in infection. Under single-cycle growth conditions, infectious FSFV DNA was detected after 1 hr in the Hirt supernatant and after 2 hr in the Hirt pellet DNA. Progeny virus was not detected until at least 5 hr postinfection. It was concluded that the synthesis of free proviral DNA and its subsequent integration into the host cell genome may be essential events in the replication of foamy viruses.     

Cycloleucine-induced vacuolation of myelin is associated with inhibition of protein methylation

Chickens injected with cycloleucine developed vacuolation of myelin similar to that seen in humans with vitamin B12 deficiency. Cycloleucine, an inhibitor of the formation of S-adenosylmethionine, decreased the incorporation of methyl groups into methylarginine in myelin basic protein in vivo. The need for methylcobalamin for the conversion of homocysteine to methionine and the requirement that myelin basic protein may be methylated, offer a rational explanation for the myelin lesions observed in cases of vitamin B12 deficiency where there is increasing evidence that methyl-, rather than adenosylcobalamin is required to prevent dysmyelination.     

Early protein synthesis in activated human peripheral blood mononuclear cells

Though B-cell division and Ig synthesis in response to pokeweed mitogen (PWM) require interaction with T-cells and monocytes, it is not clear which earlier events in B-cell activation share this requirement, and which are the result of direct interaction of mitogen with the B-cell. Having previously shown that the acceleration of lecithin synthesis in human B-cells at 16-20 hr requires both T-cells and monocytes, we now examine whether B-cells require similar interactions to increase their protein synthetic rate, another important activation event. At 21-24 hr of PWM stimulation, the stimulation index (SI) for incorporation of [35S]methionine into protein was 2.1 +/- 0.4 for unfractionated cells, 1.7 +/- 0.1 for B-cells, 2.5 +/- 0.1 for T-cells, and 3.4 +/- 0.5 for monocytes. Thus monocytes contributed substantially to early mitogen-induced protein synthesis by human peripheral blood mononuclear cells. When the monocyte/B-cell fraction (MB) and T-cell fraction (T) were mixed at various ratios in PWM-stimulated cultures, synergy was apparent at MB:T ratios of 1:1 and 1:2, indicating cell interactions augmented early mitogen-driven protein synthesis in at least one of these cell types. However, much or all of this synergy could be attributed to T-cells, whose protein synthetic response was augmented by B-cells and monocytes. In contrast, the early increase in B-cell protein synthesis appeared to be independent of cell interactions, since their SI of 1.7 was not influenced by varying the proportion of M- or T-cells over a 50-fold range. These contrasting results between two contemporary events fits the hypothesis that one (accelerated phospholipid synthesis) requires a first signal plus one or more cell interaction signals, whereas the other (accelerated protein synthesis) requires only the first signal.     

Cytotoxic effects of erythromycin estolate and chlorpromazine on hepatocytes isolated from rats of varying ages: A brief note

Hepatocytes isolated from young (1 month) rats were as sensitive to the cytotoxic effects of erythromycin estolate and chlorpromazine as were liver cells obtained from older (3, 10 and 24 months) rats. The hepatocytes from the 24-month-old rats released aspartate transaminase more slowly than did parenchymal cells isolated from the younger rats.     

Plasticity of the ipsilateral retinotectal projection in early enucleated opossums: Changes in retinotopy and magnification factors

The aberrant ipsilateral retinocollicular projection in early enucleated opossums was studied with both electrophysiological and radioautographic techniques. It was shown that the expanded projection originates from the same restricted region of temporal retina that gives origin to the normal ipsilateral projection. Furthermore, it was observed that the aberrant projection conveys a well-ordered retinotopy, with changes of the magnification factors compatible with the interpretation that ganglion cells at the borders of the ispilaterally-projecting region display the most intense plastic response after enucleation.     

Decrease of inhibitory driving force in crayfish stretch reception: a mechanism of the convulsant action of penicillin

The effect of penicillin on the evoked IPSP was investigated in the isolated crayfish stretch receptor. The IPSP driving force (IPSP reversal potential minus membrane potential) was reduced in a dose-dependent fashion but, when necessary correction was made for the decrease in resting membrane conductance, the synaptic conductance was only slightly reduced. The possibility that a penicillin-induced intracellular acidification was responsible for the decrease in IPSP driving force is considered.     

Age-linked changes in the activity of enzymes of the tricarboxylate cycle and lipid oxidation, and of carnitine content, in muscles of the rat

The activities of citrate synthase, NAD-isocitrate dehydrogenase and 2-oxoglutarate dehydrogenase were measured in homogenates of soleus, diaphragm and heart muscles of the rat, in an attempt to define potential tricarboxylate cycle activity and its response to aging. Activities were significantly decreased in 24-month animals versus 6-month controls in every case (except 2-oxoglutarate dehydrogenase in heart muscle). Age-linked decrements were greatest in the soleus and least in heart. Cytochrome oxidase was measured as an index of total respiratory chain activity and decreased significantly in each case, with the smallest decrease in the heart. Acyl-CoA dehydrogenase and 3-hydroxyacyl-Co-A dehydrogenase were measured as an index of beta-oxidative activity; the former decreased in soleus and diaphragm, the latter in soleus and heart, with the decrease in the soleus being the greater. Carnitine acetyl- and palmitoyltransferases were measured, together with the muscle content of carnitine and acylcarnitine, as determining the potential rate of entry of acyl groups into the mitochondria for oxidation. Carnitine acetyltransferase activity was decreased with age in each of the muscles, but to the greatest extent in the heart. Carnitine palmitoyltransferase was decreased in both soleus and diaphragm. Carnitine content was decreased most in the soleus and the heart and to a lesser extent in the diaphragm. It is concluded that there is a generalized decline in oxidative activity in all of these muscles with age, on the basis of wet weight; this occurs to the greatest extent in the soleus and to the least extent in the heart. There is, in addition, a specific deficiency in the ability to oxidize fatty acids, relative to other substrates, in heart muscle.     

Retention of visual discrimination learning reinforced by brain stimulation in mice

Visual discrimination learning of stripes vs black were compared in two groups of C57Bl/6J mice, one group (n = 11) bar-pressed for 10 electrical self-stimulations of the brain (ESB) and the other (n = 10) earned a 20 mg pellet of food reward in a T maze. The ESB animals had a stereotaxically placed electrode in the medial forebrain bundle lateral hypothalamic area and the food reward mice had been reduced to 80% body weight. Comparison of ESB to food reinforcement showed fewer trials to criterion in the ESB group for acquisition, reacquisition after one day and retention after 7 days. The results demonstrate sufficiency of ESB to support a pattern of learning and excellent retention in mice.     

Effect of denervation on the endogenous phosphorylating activity in the cytosol of rat skeletal muscle

An endogenous phosphorylating activity is demonstrated in the cytosol from soleus muscle of the rat which is markedly stimulated after severing the motor nerve fibers to this muscle. The [γ-³²P]ATP phosphotransferase reaction is heat-labile, dependent upon Mg²⁺ but not Ca²⁺ or cyclic GMP, inhibited by a cyclic AMP dependent protein kinase inhibitor, and directly related to the amount of cytosolic protein which provides the endogenous source of both the protein kinase enzyme, ATP, cyclic AMP and phosphorylatable protein substrate. The time-course of the delayed transitory stimulation of the cytosolic phosphorylating activity of the denervated soleus may involve neurotropic factors.     

Ultrastructural analysis of platelets in nonhuman primates. III. Stereo microscopy of microtubules during platelet adhesion and the release reaction

The role of microtubules in adhesion and in the adhesion-stimulated release reaction of platelets from African green monkeys has been studied using conventional (100 kV) and high voltage (1000 kV) stereo electron microscopy. Upon exposure in vitro to either glass or a carbon-stabilized formvar surface, platelets were rapidly activated and extended numerous filopodia. The adhesion process evolved over a period of 10–20 min with extension of a delicate hyalomere across the surface between adjacent filopodia. Microtubules, which form a circumferential ring in the resting cell, were dissociated upon cell activation and then reassembled in patterns which conformed to the general cell shape. In dendritic cells with numerous filopodia the microtubules were oriented radially. With subsequent hyalomere development, the microtubules paralleled major cell axes. This morphological transition of platelet adhesion was accompanied by the release of β-thromboglobulin (B-TG) which was maximal at 20 min, a time that corresponded to maximal hyalomere extension. Treatment of platelets with colchicine or vinblastine sulfate prior to adhesion dissociated the microtubules, but affected neither the morphological transition nor β-TG release. Our observations separate microtubules from adhesion-related events and suggest that during adhesion the microtubules, by conforming to cell shape, may simply provide cytoskeletal support.     

Effect of estrogen and corticosterone on the lymphoid system in neonatal mice

Neonatal female NMRI mice were injected with varying doses of estradiol-17β (E₂), diethylstilbestrol (DES), or corticosterone (CC). All three substances reduced the body weight and the weight of thymus and spleen. On a dose level, DES was more potent than E₂ or CC. DES treatment resulted in pronounced degeneration in thymus cortex, reduced incorporation of [³H]thymidine in thymus and spleen, reduced mitotic rate in thymus, and reduced number of leukocytes in peripheral blood with a decreased percentage of mononuclear cells. Nine-week-old animals, injected with DES neonatally, had persistent changes in their peripheral leukocyte population. Cytosol fraction of thymus and spleen from 4- to 5-day-old females contained a DES and E₂-binding macromolecule with a sedimentation coefficient of approximately 4.5S and binding characteristics similar to α-fetoprotein. Autoradiograms of thymus after an [³H]DES injection did not show any labeling of thymocytes. The concentration of radioactivity in thymus and spleen after a single [³H]DES injection was very low and constant from 10 min to 4 hr after the injection, while a pronounced radioactivity occurred in uterus and skeletal muscle. A model is discussed for the estrogen effect on the lymphoid tissue in neonatal mice. The results underline the importance of studying the immune system in offspring of women treated with DES during pregnancy.     

Cardiac morphologic alterations in acute minoxidil cardiotoxicity in miniature swine

Minoxidil, a vasodilating antihypertensive drug, was given orally at 10 mg/kg daily for 2 days to twelve 25- to 35-kg miniature pigs. Twelve control pigs were also studied. Minoxidil-treated pigs had tachycardia and hypotension and were killed 24 hr after the second dose. Gross examination showed diffuse hemorrhage in left atrial epicardium in all pigs, and also in ventricular epicardium (2 of 12 pigs) and endocardium (3 of 12 pigs). Pale areas of necrosis were observed on incision of the left ventricular papillary muscles in 3 pigs. Light and electron microscopic studies showed acute vascular damage with hemorrhage in the left atrial epicardium. Affected arterioles had endothelial cell swelling and transmural and perivascular accumulations of leukocytes, edema fluid, fibrin clumps, and erythrocytes. The swollen endothelial cells had large, irregularly shaped nuclei with abundant euchromatin; mitotic figures were frequent. The cytoplasm contained numerous polysomes and cisterns of rough endoplasmic reticulum. Fibroblasts adjacent to damaged vessels had edematous cytoplasm and increased amounts of rough endoplasmic reticulum. In the affected left ventricular papillary muscles, necrotic myocytes showed contraction bands, mitochondrial matrical densities, lipid accumulation, initial lysis of I bands, and pyknotic nuclei. The lesions were judged to result from two mechanisms: (1) hemorrhagic lesions from drug-induced vascular injury centered on epicardial and subepicardial arterioles and (2) papillary muscle necrosis from ischemic injury from hypoperfusion during minoxidil-induced tachycardia and hypotension.     

Recording of dorsal raphe unit activity in vitro

The spontaneous electrical activity of single neurons of the midbrain dorsal raphe nucleus in the rat was recorded in vitro in 400 μm thick slices. The majority of raphe neurons exhibited the slow and regular discharge pattern that characterizes the activity of raphe neurons when recorded in vivo. The viability of these neurons in vitro thus provides a preparation for analyzing endogenous and exogenous factors that regulate the activity of these serotonin-containing neurons.     

Retention of passive avoidance behavior in rats following alpha- and gamma-endorphin administration: effects of post-learning treatments

Neuropeptides related to beta-endorphin (beta-LPH61-91) profoundly affect avoidance behavior. That gamma- and alpha-endorphin (beta-LPH61-77 and beta-LPH61-76, respectively) influence memory processes, has been investigated on rats. Using a one-trial learning, step-through passive avoidance paradigm, it was found that gamma-endorphin attenuates later retention of passive avoidance behavior when administered immediately or 3 h after learning; alpha-endorphin facilitates this response when given immediately after learning. Neither gamma- nor alpha-endorphin influence retention when the treatment is postponed for 6 h after learning. These data are interpreted as suggesting an opposite effect of gamma- and alpha-endorphin on consolidation processes.