Adjuvant activity of some nonpyrogenic hydrophobic analogues of muramyl dipeptide in enhancing the primary humoral and cell mediated immune responses in guinea pig model

Five muramyl dipeptide analogues synthesized by derivatization of gamma-carboxyl of D-isoglutamine residue of MDP into alkyl amides or incorporation of lysine residue at the site via epsilon-NH2 function were evaluated for immuno-adjuvant activity. Derivatization of gamma-carboxyl of D-isoglutamine into butyl, octyl and dibutyl residues stimulated delayed type of hypersensitivity (DTH) response, the maximum stimulation being observed with octyl amide. Introduction of lauryl amide residue abolished DTH response. The antibody response was impaired with all the alkyl amide analogues except for the lysyl amide derivative with which the response was higher than MDP. Correlation was observed between DTH response and macrophage migration.     

Effects of cancer chemotherapeutic agents on testicular DNA synthesis in the rat. Evaluation of a short-term test for studies of the genetic toxicity of chemicals and drugs in vivo.

Changes in the rate of testicular DNA synthesis in the rat were studied at various times after single doses of 12 cancer chemotherapeutic agents. The animals were given intravenous injections of [14C]thymidine and [3H]thymidine 24 and 3 h, resp., before they were killed. By combining measurements of the free serum radioactivity and the testicular incorporation of the differentially labelled precursor, different response patterns were obtained for agents with different modes of action. The DNA-damaging agents cyclophosphamide, chlorambucil, thio-TEPA, busulphan, CCNU and procarbacine, after some delay, caused a decrease of testicular thymidine incorporation and a corresponding increase of free serum radioactivity. The non-DNA-damaging agents 5-fluorouracil, methotrexate, hydroxyurea and cytosine arabinoside had a rapid effect on testicular thymidine incorporation and produced diverse response patterns different from that of the DNA-damaging agents. Actinomycin D and also vinblastine caused changes in testicular thymidine incorporation and showed response patterns different from those of the other agents. These results show that simple measurements of testicular DNA synthesis may provide useful information for the evaluation of genotoxic effects of chemical compounds and may help one to distinguish between DNA-damaging agents and metabolic inhibitors of DNA synthesis.     

Hormonal control of testicular protein synthesis in developing Tenebrio molitor

During differentiation, the testes of Tenebrio molitor were found to exhibit increases in biosynthetic capacity reflected by changes in the testicular protein content. A gradual increase in testicular protein content was observed during the prepupal stage. The observed increase was more dramatic in the pupal stage and reached its maximum level between days 4 and 7 of pupal development. During the adult stage, the biosynthetic processes for producing protein were apparently reduced following the first few days after adult emergence.The incorporation of radioactive leucine into TCA-precipitable testicular protein was not affected by the administration of exogenous juvenile hormone alone (JH I, 1 μg/animal) during the pupal stage. However, the administration of exogenous ecdysterone (0.5 μg/animal) to pupal T. molitor resulted in an increase in radioactive leucine incorporated into TCA-precipitable testicular proteins, particularly during the first five days after pupal ecdysis.Simultaneous administration of both JH and ecdysterone to mealworm pupae at specific ages indicated that no apparent interaction, synergistic or antagonistic, occurred between these two hormones with respect to [³H]-leucine incorporation. Furthermore, the amount of [³H]-leucine incorporated approximated to that obtained following injection of ecdysterone alone in all the pupal ages studied.     

Synthesis and phosphorylation of histone H1 and high mobility group proteins in the regenerating rat liver after X irradiation

A rat was irradiated to the upper abdomen including the liver and then partially hepatectomized. The subsequent synthesis and phosphorylation of histone H1 and nonhistone chromosomal high mobility group (HMG) proteins were investigated. Incorporation of [3H]lysine into histone H1 was increased and reached its peak at 27 h after hepatectomy, and 14 Gy of X rays inhibited the increase. Increase in the incorporation of [3H]lysine into HMG (1 + 2), 14, and 17 which occurred around 27 h after hepatectomy was not inhibited by 14 Gy irradiation. Phosphorylation of histone H1, measured with 32Pi incorporation in vivo, was maximal between 21 and 24 h, and it was inhibited by 4.8 Gy of X rays and delayed with 1.9 Gy. Phosphorylation of HMG 14, which was the only HMG protein phosphorylated under present conditions, was not affected by X irradiation. The [3H]thymidine incorporation into nuclear DNA started increasing at 21 h and reached its maximum at 27 h after hepatectomy. X irradiation with 4.8 Gy inhibited the incorporation, and 1.9 Gy lowered it.     

Effects of imprinting on lysine uptake and incorporation into protein in chick brain

One-day old chicks were exposed for either 30, 60, or 120 min to an imprinting stimulus or kept in darkness in similar conditions. At the end of this time they were injected peripherally with ¹⁴C-lysine and killed 20 min later. The radioactivity of free lysine and that incorporated into protein was measured; incorporation was found to differ between exposed and dark birds only in the anterior part of the forebrain roof after 60-min treatment (E/D = 1.25). However, more free radioactive lysine was found in all brain regions of exposed birds at this time. When the specific radioactivity of the free lysine (dpm/nmol lysine) was measured there were no differences between the two types of birds, indicating that the incorporation difference was not due to a change in precursor radioactivity. The use of ¹⁴C-2-aminoisobutyrate confirmed that even with a nonincorporated amino acid pool size changes still occurred. The greater lysine incorporation in anterior forebrain roof was largely restricted to the cytoplasmic soluble fraction.     

Incorporation of radioactive precursors into filarial larvae of Brugia developing in susceptible and refractory mosquitoes.

The incorporation of tritiated precursors injected into mosquito hosts parasitized by developing filarial larvae of Brugia patei has been studied by autoradiography in 2 species of mosquito, Aedes togoi in which filarial development was normal and Anopheles labranchiae atroparvus in which filarial development was abnormal. In both mosquito hosts there was significant incorporation into 4--5-day-old developing larvae of uridine and amino acids (isoleucine, leucine, valine, arginine, lysine, cystine, methionine, phenylalanine, tyrosine, tryptophan, histidine, and proline), although lower incorporation of methionine, tyrosine, and tryptophan was found during abnormal development. No incorporation of thymidine, hydroxytryptophan, dopa, or carbohydrate was found at this stage of larval development. Some incorporation of glucose and dopa was found in or around earlier stages of development in An. l. atroparvus. Mosquito flight muscle showed lower incorporation of glucose, but not of amino acids, around the site of filarial parasite development. The flight muscle of An. l. atroparvus showed a higher level of incorporation of lysine compared to that in A. togoi and higher levels of lysine and valine were found in the abnormally developing filarial larvae in the refractory mosquito.     

CYTOPLASMIC GRANULE FORMATION IN MYELOCYTES : An Electron Microscope Radioautographic Study on the Mechanism of Formation of Cytoplasmic Granules in Rabbit Heterophilic Myelocytes

The intracellular flow of tritiated lysine as revealed by electron microscope radioautography was studied in heterophilic myelocytes of rabbit marrow. Label over the Golgi complex rose to a maximum of 37% of total cytoplasmic grains 30 min after initial exposure to the tracer and fell to 11% after 3 to 4 hr of incubation. Coincident with decrease in label over the Golgi complex, grain counts over granules rose to 32% after 3 to 4 hr. The time sequence of incorporation and flow of tritiated lysine and the per cent distribution of label was similar in bone marrow myelocytes under in vivo and in vitro conditions. The results demonstrate a function of the Golgi complex in incorporating or packaging certain basic amino acids or proteins into cytoplasmic granules of heterophilic myelocytes.     

Amino acid uptake and protein synthesis in germinating spores of Saccharomyces cerevisiae.

Spores transferred to germination medium incorporated exogenous lysine into protein within 20 min but required 2-3 to begin incorporation of exogenous proline or alanine. During this time considerable uptake of amino acids into the intracellular pool occurred. Cycloheximide added to the germination medium inhibited incorporation of lysine into protein but did not lessen in accumulation in the pool. Spore germination was inhibited by cycloheximide.     

Effects of electroconvulsive shock and cycloheximide on the incorporation of amino acids into proteins of mouse brain subcellular fractions.

—The incorporation of [4,5-³H]lysine and [1-¹⁴C]leucine into the proteins of subcellular fractions of mouse brain was examined following a single electroconvulsive shock (ECS) or following cycloheximide injections. When the [³H]lysine was injected intraperitoneally immediately after the ECS the incorporation into total brain proteins was decreased by more than 50% as compared to sham controls. The proportion of lysine incorporated into the microsomal fraction was increased, but no changes were observed in the other subcellular fractions including the synaptosomal fraction. With extended pulses administered at various times after the ECS there was no change in total incorporation nor were selective effects seen in any subcellular fractions. With intracranial injections of both [³H]lysine and [¹⁴C]leucine the decreased incorporation caused by ECS was not observed, neither were there selective changes in any subcellular fraction. This lack of inhibition occurred because the intracranial injection itself severely inhibited [³H]lysine incorporation. Cycloheximide (30 mg/kg) which depressed [³H]lysine incorporation into brain proteins by 84% caused a selective depression of the incorporation into the cell-sap fraction and selective elevations into the microsomal and synaptosomal fractions. Similar changes were seen with a higher (amnestic) dose of cycloheximide (150 mg/kg) which inhibited incorporation by 94%. These data are interpreted in terms of the diverse mechanisms by which ECS and cycloheximide inhibit protein synthesis.     

Aspartokinase III repression and lysine analogs utilization for protein synthesis

The extents of thialysine and selenalysine incorporation into cell proteins were compared in E. coli KL16 and in a mutant able to grow equally well in the presence or in the absence of both lysine analogs. The mutant differs from the parental strain in the repression of aspartokinase III (AKIII), the first enzyme of the lysine biosynthetic pathway. No analog incorporation into proteins was observed in mutant cells grown in the presence of either analog, whereas a marked analog incorporation was observed in the parental strain, where up to 17% and 12% of protein lysine can be substituted by thialysine and selenalysine respectively. In the parental strain grown in media containing either analog at different concentration the extent of analog incorporation into proteins is related to the extent of AKIII repression.     

Biochemical consequences of follicle-stimulating hormone binding to testicular macrophages in culture

The present studies demonstrate that testicular macrophages respond to follicle-stimulating hormone (FSH) by: 1) stimulating the rate of incorporation of amino acids into secreted proteins; 2) increasing the rate of incorporation of uridine into RNA; and 3) enhancing the accumulation of intracellular cyclic adenosine monophosphate (cAMP; which was potentiated by the addition of 1 mM 3-isobutyl-1-methylxanthine; MIX). In addition, dibutyryl cAMP (dbcAMP) enhanced the incorporation of amino acids into secreted proteins; however, this cAMP analog had no effect on the incorporation of uridine into RNA. Finally, it was demonstrated that testicular macrophages possess specific receptors with a high affinity for FSH.     

Luteinizing hormone response to an oestradiol challenge in 5 intersex pigs possessing ovotestes

After challenge with oestradiol benzoate, the mean maximum LH concentration in 5 XX intersex pigs possessing ovarian and testicular tissue, or only testicular tissue, was 2.10 (+/- 0.41) ng/ml compared with 8.9 ng/ml in mature domestic gilts. These results indicate that exposure of the pig brain to testosterone before Day 30 of gestation is important, or that early testicular secretions other than testosterone are involved in the determination of brain gender. The observation that some intersex pigs show normal oestrous cycles implies that the response to these prenatal factors is primarily quantitative rather than qualitative.     

SUB-CELLULAR LOCALISATION OF ENHANCED LYSINE INCORPORATION INTO CEREBRAL CORTEX PROTEINS IN DARK-REARED AND LIGHT-EXPOSED RATS

Incorporation of lysine into acid-insoluble material from subcellular fractions of rat cerebral cortex has been studied using double and single-labelling techniques, in littermates reared for 50 days in the dark and then dark-maintained or light-exposed for 1 h. When light-exposed animals were compared to dark controls the only subcellular fraction from the whole cortex in which lysine incorporation shows a significant elevation (168%, P < 0.05) was located in the ribosomal pellet of the cerebral cortex. A similar comparison of subcellular fractions from visual and motor cortices showed that the elevation was again in the ribosomes and confined to visual cortex only. Motor cortex of light-exposed animals showed a small depression of incorporation in ribosomes as compared to dark controls. Sub-fractionation of nuclei from whole cortex preparations showed varying, but non-significant elevations in light-exposed animals in all but the histone fraction in which there was negligible incorporation of precursor. It is concluded that enhancement of incorporation of precursor into proteins of the cerebral cortex, which accompanies first exposure to light, is a complex response. At exposure for 1 h it involves a number of particular protein species located in the visual cortex, a major proportion of which are ribosomally bound.     

Histone synthesis in isolated neuronal perikaryon relative to the postnatal appearance of a short DNA repeat length

Neuronal perikaryon were purified from rabbit cerebral hemispheres at early postnatal stages of brain development. When incubated in vitro these neuronal cells demonstrated the ability to incorporate labeled amino acids into total protein at a linear rate, however, incorporation of labeled thymidine was not apparent. Isolated neuronal perikaryon showed a transient ability to incorporate labeled lysine and arginine into both core (H3, H2B, H2A, H4) and linker (H1) histones between 30 and 90 hr following birth, with a maximum incorporation at 42 hr. This period of histone synthesis may correlate with the conversion of neuronal chromatin to a short DNA repeat length which occurs between 60 and 84 hr following birth.     

Biosynthesis of proteins and ribonucleic acids in red and white rat skeletal muscles]

Protein biosynthesis is studied in red and white rat shank muscles in vitro. It is found that the incorporation rate of 14C-lysine in red muscle was 2-fold higher than that in white muscle. The difference in the lysine incorporation rate into muscle proteins studied increased with the increase of lysine molar concentration in the incubation medium, which was probably due to a selective protein synthesis activation in the red muscle. A higher level of 14C-lysine incorporation in red muscle proteins was found under similar uptake of the labelled amino acid in both red and white muscles. RNA synthesis rate was the same in both muscles and its inhibition with actinomycin D did not affect the ratio of protein synthesis rates in red and white muscles.     

Lysine biosynthesis in Penicillium chrysogenum is regulated by feedback inhibition of α-aminoadipate reductase

A partially purified preparation of alpha-aminoadipate reductase (EC 1.2.1.31) from Penicillium chrysogenum is competitively inhibited by lysine (Ki of 0.26 mM). Exogenous addition of 10 mM L-lysine to resting mycelia of P. chrysogenum increased the intracellular lysine pool concentration 2-fold, but decreased the incorporation of (6-14C)-alpha-aminoadipate into protein-bound lysine to a fifth. The distribution of radioactivity in the pathway metabolites alpha-aminoadipate, saccharopine and lysine was consistent with the assumption of a lysine sensitive enzyme step in vivo between alpha-aminoadipate and saccharopine. Hence lysine inhibition of alpha-aminoadipate reductase may be of physiologic importance.     

Inhibition of protein synthesis in Krebs 2 ascites cells and cell-free systems by phenylalanine and its effect on leucine and lysine in the amino acid pool

1. The inhibition of incorporation of ¹⁴C-labelled amino acids into protein of whole cells by phenylalanine has been reproduced in a cell-free system. In both cases only the l-isomer was inhibitory. 2. The effect of phenylalanine on incorporation of [¹⁴C]leucine and [¹⁴C]lysine into protein was different in both whole cells and cell-free systems. 3. In whole cells inhibition of incorporation of leucine at 2·5μg./ml. was very rapid, but when the concentration was increased to 100μg./ml. the inhibition was not apparent for about 1hr. The kinetics of inhibition of lysine was the same at both these concentrations and was similar to that found with leucine at 100μg./ml. 4. Neither a lower specific radioactivity of the two amino acids in the pool nor a decrease in their pool size could be consistently related with inhibition of protein synthesis. 5. In the cell-free system l-phenylalanine inhibited the incorporation of leucine but not of lysine. 6. Charging of transfer RNA by leucine was markedly decreased in the presence of phenylalanine, whereas charging of transfer RNA by lysine was not.     

Increased Incorporation of [3H]Lysine into Synaptic Membranes of the Visual Cortex Following First Exposure of Dark-Reared Rats to Light Is Confined to Particular Polypeptides

Abstract: The incorporation of [³H]lysine into separated polypeptides of synaptic-membrane fractions prepared from the visual cortices of dark-reared rats and littermates exposed to light for 1 h was examined. Increased incorporation of [³H]lysine was found in synaptic membranes from light-exposed compared to dark-reared rats in polypeptides of four molecular weights: 100,000, 71,000, 44,000, and 38,000. The 44,000-molecular-weight peak has been suggested to be actin on the basis of its comigration with pure brain actin. These results indicate that increased incorporation of [³H]lysine into synaptic membranes of the visual cortex, following first exposure of dark-reared rats to light, is confined to particular polypeptides.     

Unique Functional Properties of Conserved Arginine Residues in the Lentivirus Lytic Peptide Domains of the C-terminal Tail of HIV-1 gp41

A previous study from our laboratory reported a preferential conservation of arginine relative to lysine in the C-terminal tail (CTT) of HIV-1 envelope (Env). Despite substantial overall sequence variation in the CTT, specific arginines are highly conserved in the lentivirus lytic peptide (LLP) motifs and are scarcely substituted by lysines, in contrast to gp120 and the ectodomain of gp41. However, to date, no explanation has been provided to explain the selective incorporation and conservation of arginines over lysines in these motifs. Herein, we address the functions in virus replication of the most conserved arginines by performing conservative mutations of arginine to lysine in the LLP1 and LLP2 motifs. The presence of lysine in place of arginine in the LLP1 motif resulted in significant impairment of Env expression and consequently virus replication kinetics, Env fusogenicity, and incorporation. By contrast, lysine exchanges in LLP2 only affected the level of Env incorporation and fusogenicity. Our findings demonstrate that the conservative lysine substitutions significantly affect Env functional properties indicating a unique functional role for the highly conserved arginines in the LLP motifs. These results provide for the first time a functional explanation to the preferred incorporation of arginine, relative to lysine, in the CTT of HIV-1 Env. We propose that these arginines may provide unique functions for Env interaction with viral or cellular cofactors that then influence overall Env functional properties.