The location of muscarinic receptors in the rat pituitary gland was examined with an autoradiographic technique. Slides containing 10 or 20 μm horizontal sections of tissue were incubated in a solution of 1 nM [3H]quinuclidinyl benzilate (QNB) in phosphate-buffered saline to label muscarinic receptors. Autoradiograms were produced by placing the slides into X-ray cassettes with tritium-sensitive film and processing the film 8-10 weeks later. Minimal binding occurred when sections were incubated in 1 n M [3H]QNB plus 1 μM atropine. Highest specific binding of QNB was found in the anterior and intermediate lobes along their border with the pituitary cleft. Patches of high binding were also seen penetrating the intermediate lobe and at the border between the intermediate and neural lobes. The remainder of the intermediate lobe had background density of autoradiographic grains. Moderate density of specific binding was found throughout the anterior lobe. Low specific binding occurred in the neural lobe.

Isotretinoin (13‐cis‐RA) is known to be teratogenic in humans and laboratory animals. The relatively low potency of 13‐cis‐RA in NRMI mice in comparison to the all‐trans isomer has been proposed to be due to minimal transfer across the placenta (Creech‐Kraft et al., '87). To further delineate the teratogenic potential of 13‐cis‐RA, a dose‐response, temporal study was conducted in vivo and in vitro using submerged limb culture and image analysis evaluation of development. Dose‐dependent embryotoxicity was produced by treatment on GD 7, while later treatments produced inconsistent effects on resorption rate and fetal weight. Treatment on either GD 7 or GD 8 produced a number of malformations in dose‐dependent manner. Most common were tail and cleft palate defects, which were produced by 13‐cis‐RA on each of the days tested (GD 7–GD 11), with peak malformations occurring on GD 9 and GD 10 for tail and cleft palate, respectively. Most limb defects were produced after GD 10 and GD 11 exposure. The observed frequency of defects confirmed that in ICR mice 13‐cis‐RA is about 10‐fold less potent than all‐trans‐RA as a limb teratogen (Kwasigroch and Kochhar, '80; Kochhar and Penner, '87). Effects observed via image analysis following maintenance of limbs in serum‐free culture medium were dose dependent. Low dose treatment produced occasional polydactyly. The intermediate dose caused somewhat variable region‐dependent increases in cartilaginous bone anlagen area. The high dose of 13‐cis‐RA produced irregular limb outlines, a reduction in bone anlagen area, and an inhibition of alcian blue staining of cartilage without affecting morphogenesis of bone anlagen. These results confirm that, when the effects of the administered doses are evaluated, 13‐cis‐RA is a much less potent teratogen in comparison to the all‐trans isomer. More importantly, the results show that retinoids can enhance (at low and intermediate doses), depress (at high doses), or eliminate (high dose) chondro‐genenic expression during limb morphogenesis in vitro. This indicates that retinoids such as 13‐cis‐RA can manipulate events in development in a variety of ways (i. e., produce malformations, interfere with chondrogenic expression without affecting morphogenesis, and stimulate growth) in a dose‐ and time‐dependent manner. Although the ability of RA to act as a true morphogen has recently been questioned (Wanek et al., '91; Noji et al., '91), the results presented here support the position that RA can modulate the development of the limb (and probably other organ systems) in several vertebrate species.

The autosomal mutation brachypod (bpH/bpH) in the mouse affects the development of precartilage mesenchymal condensation in the limb‐bud. We have previously shown that this defect is localized to the expression of terminal N‐acetylglucosamine (GlcNAc) glycoproteins in the plasma membrane (Elmer and Wright, '83). The present study is focused on cell surface galactosyltransferase (GalTase), an ectoenzyme that transfers galactose to its GlcNAc substrate. Purified plasma membrane preparations derived from wild‐type ( +/+ ), heterozygote (+ /bpH) and brachypod (bpH/bpH) embryonic mouse limb cells were assayed for GalTase activity during in vitro and in utero chondrogenesis using High‐Performance Liquid Chromatography (HPLC). On embryonic day E12, prior to overt expression of the mutant gene, no significant difference in GalTase activity was observed. By the third day in culture, all major chondrogenic elements of the autopod were present in +/+ and +/bpH embryos, whereas the mutant autopods were markedly deficient in staining and appeared consistently shorter. The accumulation of alcianophilic cartilage matrix in the wild‐type was accompanied by a 29% increase in GalTase activity, which reflected the net change (29%) observed during development from days E12 to E13 in utero. The GalTase activity for the in utero E13 mutant (13%) was significantly different from control. In culture, day E12 mutant autopods actually decreased in their GalTase level by 3 days so that the activity was reduced to only 57% of the wild‐type. Though GalTase activity in the heterozygote showed an intermediate expression, optical image analysis did not reveal consistent differences in cartilage development when compared to +/+, arguing against a gene‐dosage effect at the gross anatomical level. These data indicate that an increase in plasma membrane GalTase activity is a natural developmental event that occurs during limb‐bud chondrogenesis and a decrease in GalTase activity contributes to the dysmorphogenesis in brachypod limb‐buds.

Caprine arthritis encephalitis virus (CAEV) is a lentivirus which is closely related by nucleotide sequence and biological properties to visna virus and is more distantly related to the human AIDS virus, HIV-1. Previous studies indicated that the CAEV Rev protein (Rev-C) functions as a trans-activator of mRNA cytoplasmic transport and expression. The function of Rev-C is mediated through an RNA element (RRE-C) present between nucleotides (nt) 7906 and 8110 in the CAEV env gene. In this study, RNA/protein immunoprecipitation experiments were used to demonstrate that Rev-C binds directly to the 204-nt RRE-C in vitro. Competition assays illustrate that this interaction is specific for the positive sense RRE-C RNA. Glutaraldehyde crosslinking studies demonstrate that the wildtype Rev-C protein can also form multimeric complexes in vitro. Deletions or amino acid alterations within the basic domain of Rev-C reduce affinity for the RRE and disrupt assembly of Rev-C multimers in vitro, indicating that this domain is involved in RRE binding and Rev multimer formation. Mutations within the leucine-rich domain of Rev-C do not greatly effect RRE-C binding or self-assembly. However, previous results demonstrate that some leucine-rich domain mutants are unable to trans-activate. These data are consistent with the hypothesis that the leucine domain is the effector domain of Rev-C.

The kinetics of recovery after inhibition of growth by erythromycin and clarithromycin were examined in Staphylococcus aureus cells. After inhibition for one mass doubling by 0.5 μg of the antibiotics/ml, a postantibiotic effect (PAE) of 3 and 4 h duration was observed for the two drugs before growth resumed. Cell viability was reduced by 25% with erythromycin and 45% with clarithromycin compared with control cells. Erythromycin and clarithromycin treatment reduced the number of 50S ribosomal subunits to 24 and 13% of the number found in untreated cells. 30S subunit formation was not affected. Ninety minutes was required for resynthesis to give the control level of 50S particles. Protein synthesis rates were diminished for up to 4 h after the removal of the macrolides. This continuing inhibition of translation was the result of prolonged binding of the antibiotics to the 50S subunit as measured by 14C-erythromycin binding to ribosomes in treated cells. The limiting factors in recovery from macrolide inhibition in these cells, reflected as a PAE, are the time required for the synthesis of new 50S subunits and the slow loss of the antibiotics from ribosomes in inhibited cells.

Macrolide antibiotics like erythromycin can prevent the formation of the 50S ribosomal subunit in growing bacterial cells, in addition to their inhibitory effect on translation. The significance of this novel finding has been further investigated. The 50% inhibitory doses of erythromycin for the inhibition of translation of 50S subunit assembly in Staphylococcus aureus cells were measured and were found to be identical. Together they account quantitatively for the observed effects of erythromycin on cell growth rates. There is also a direct relationship between the loss of rRNA from the 50S subunit and its accumulation as oligoribonucleotides in cells. The importance of this second site for erythromycin inhibition of bacterial cell growth is discussed.

Macrolide antibiotics are clinically important antibiotics which are effective inhibitors of protein biosynthesis in bacterial cells. We have recently shown that some of these compounds also inhibit 50S ribosomal subunit formation in Escherichia coli. Now we show that certain macrolides have the same effect in two gram-positive organisms, Bacillus subtilis and Staphylococcus aureus. Assembly in B. subtilis was prevented by erythromycin, clarithromycin, and azithromycin but not by oleandomycin, 50S subunit formation in S. aureus was prevented by each of seven structurally related 14-membered macrolides but not by lincomycin or two streptogramin antibiotics. Erythromycin treatment did not stimulate the breakdown of preformed 50S subunits in either organism. The formation of the 30S ribosomal subunit was also unaffected by these compounds. Assembly was also inhibited in a B. subtilis strain carrying a plasmid with the ermC gene that confers macrolide resistance by rRNA methylation. These results suggest that ribosomes contain an additional site for the inhibitory functions of macrolide antibiotics.

This chapter examines whether or not the transplant circuitry, including both the intrinsic connections among transplant neurons and the afferent and efferent connections of the principal neurons of the transplant with the host brain, might be necessary for the recovery of function associated with the transplantation of fetal tissue into cavities created by the bilateral aspiration of the hippocampus in adult rats. The procedures used to produce the lesions in the adult rats and obtain the fetal brain tissue have been described in detail in the chapter. Pregnant female hooded rats serve as donors for the fetal material. Rats to be given lesions are anesthetized with sodium pentobarbital. After the scalp is incised and small holes drilled in the skull, a lesion that encompasses most of the septa1 end of the hippocampus and as much of the ventrocaudal hippocampus as can be removed without damage to lateral thalamic structures is produced by aspiration. A photomicrograph of 2 levels through such a lesion is presented in the chapter. Histological analysis indicates that bilateral transplants are present in all of the grafted rats. The mean number of responses, number of reinforcements, and percent correct for each 5 consecutive days is calculated for each rat to provide data for the statistical analysis of the differential reinforcement of low rates of responding (DRL) performance. The integrity of the transplant circuitry is not necessary for continuation of the ability to perform DRL once recovery has been established.

A variety of chemical agents and surgical procedures have dramatic influences on the ontogenetic development of noradrenergic neurons. Because of space limitations, in-depth descriptions of each of the neurotoxins could not be given, and the reader is referred to review articles which present a different perspective on uses of the agents (Breese, 1975; Jonsson, 1980; Kostrzewa, 1988; Kostrzewa and Jacobowitz, 1974; Thoenen and Tranzer, 1973).

Male hooded rats were trained on a schedule of differential reinforcement of low response rates (DRL) until a stable baseline level of responding was obtained. The rats then received a bilateral intraseptal injection of an ascorbic acid solution. They were returned to the DRL schedule for 10 days, after which they received a bilateral intraseptal injection of either the ascorbic acid solution or the vehicle containing 6-hydroxydopamine (6-OHDA). After 10 additional days on the DRL schedule, a bilateral hippocampal ablation was performed on all rats. Performance on the DRL schedule was assessed, and rats that had received 6-OHDA prior to the hippocampal ablation emitted significantly fewer responses under the DRL schedule subsequent to the lesion than those that had received just the vehicle. Fluorescence microscopy reveals a substantial loss of the catecholamines in animals receiving 6-OHDA injections as compared to normals or vehicle-treated rats. The results are discussed within the context of 6-OHDA-induced denervation supersensitivity and the possible disruption of ablation-induced collateral axonal sprouting in the septum.