The metabolic fate of 14C-labeled immunoadjuvant peptidoglycan monomer. II. In vitro studies

Peptidoglycan monomer (GlcNAc-MurNAc-L-Ala-D-isoglutamine-meso-diaminopimelic acid-D-Ala-D-Ala), labeled with 14C both in the disaccharide and pentapeptide portions, was incubated with slices of mouse liver, kidney or spleen as well as with mouse and human blood, blood cells plasma and serum. Peptidoglycan monomer was isolated unchanged after incubations with mouse organs and blood cells. However, upon incubation with mouse or human blood, 10-50% of the peptidoglycan monomer underwent hydrolysis to the corresponding disaccharide and pentapeptide. After incubations with plasma and serum more than 90% of the [14C]peptidoglycan monomer was metabolized: about 50% of the administered radioactive dose was recovered in the disaccharide unit and about 35% in the pentapeptide part. These results suggest that in blood, plasma and serum of mouse and man, an N-acetylmuramoyl-L-alanine amidase (mucopeptide amidohydrolase, EC 3.5.1.28) exists which splits the amide bond between the lactyl carboxyl group of the muramyl residue and the amino group of the peptide moiety in the peptidoglycan molecule.     

Effect of glycl-tRNA concentration on in vitro serine incorporation into the peptidoglycan of S. epidermidis

Seven species of seryl-tRNA have been isolated from $\text{S. epidermidis}$ strain Texas 26 by the use of Plaskon CTFE powder as the support for Adogen 464 in reversed-phase chromatography. All but one of these seven seryl-tRNA incorporated ¹⁴C-serine into $\text{in vitro}$ biosynthesized peptidoglycan. This suggested that at least one of the functional roles of six of the seven seryl-tRNA peaks isolated may be involved in $\text{in vivo}$ cell wall synthesis. It has also been shown that the ratio of glycine to serine in the pentapeptide bridge depended upon the concentration of glycyl-tRNA in the assay mixture. By increasing the concentration of glycyl-tRNA the amount of serine inserted into the bridge is increased. Thus the amount of serine incorporated into the pentapeptide bridge appears to be dependent upon the concentration of glycyl-tRNA.     

Encapsulation of immunoadjuvant [24C]peptidoglycan monomer into liposomes: Effect on metabolism and immune response in mice

¹⁴C-labeled peptidoglycan monomer was encapsulated into negatively charged, multilamellar liposomes composed of egg phosphatidylcholine, cholesterol and dicetylphosphate. Excretion and tissue distribution of the label in mice were studied after intravenous injections. Encapsulation of peptidoglycan monomer into liposomes as compared to free peptidoglycan monomer, resulted in increased retention of the label, particulary in the liver and to a lesser extent in spleen. The excretion was drastically reduced and delayed even after 4 days when cholesterol-rich (phosphatidylcholine/cholesterol, 7:5 molar ratio) liposomes were used for encapsulation of peptidoglycan monomer. Peptidoglycan monomer and liposomes, when tested separately, stimulate the immune response to sheep erythrocytes in mice. However, there was no significant additive or synergistic effect when peptidoglycan monomer was encapsulated into liposomes.     

Controlled reduction of cytochrome b in succinate-cytochrome c reductase complex by succinate in the presence of ascorbate and antimycin.

$\text{Bacillus subtilis}$ membranes can transfer either N-acetylmuramyl-pentapeptide phosphate or N-acetylglucosaminyl phosphate from UMP directly onto undecaprenyl phosphate. Tunicamycin blocks only the latter transfer and inhibits peptidoglycan synthesis by toluenized cells of $\text{Bacillus megaterium}$ utilizing added nucleotide sugar precursors or cell wall synthesis by intact cells of $\text{B. subtilis}$. Tunicamycin prevents formation of the cell wall disaccharide lipid intermediate by blocking transfer of N-acetylglucosamine onto undecaprenyl muramyl pentapeptidyl pyrophosphate.     

Displacement of palmitate from albumin by chlorophenoxyisobutyrate.

The effect of chlorophenoxyisobutyrate, a hypolipidemic drug that decreases plasma free fatty acids, triglycerides, and cholesterol, on the partitioning of [¹⁴C]-palmitate between hexane and bovine serum albumin was studied at 37°. In this system, hexane served as a hydrophobic trap for free fatty acids displaced from BSA by chlorophenoxyisobutyrate, allowing less than 0.3% to remain in the aqueous phase. As the concentration of chlorophenoxy isobutyrate was raised from 0.4 to 3.2 mM, there was a progressive displacement of palmitate from the [¹⁴C]-palmitate-BSA complex into hexane, the magnitude being dependent on the initial $\text{V}$ value (moles palmitate bound/mole BSA). Beginning with [¹⁴C]-palmitate in hexane, chlorophenoxyisobutyrate (2 mM) decreased the moles palmitate bound/mole of BSA by 16% at $\text{V}$ = 0.2, and 34% at $\text{V}$ = 3.0.     

The enzyme stability of dehydropeptides

A pentapeptide, Z-Gly-Gly-Phe-Phe-Ala · OH $\text{(1b)}$ and the corresponding unsaturated pentapeptide, Z-Gly-Gly-Phe-Δ^ZPhe-Ala · OH $\text{(1a)}$, have been synthesized. The saturated compound $\text{(1b)}$ was rapidly hydrolyzed by both chymotrypsin and thermolysin to the expected products, but the dehydropeptide was completely unhydrolyzed by either enzyme even after thirty hours. A new method of peptide stabilization to enzymolysis is made available.     

11-dehydrocorticosterone (compd. A) formation by the Microtus adrenal

No 11β-hydroxysteroids were detected after 30 minutes incubations of progesterone-4-¹⁴C and pregnenolone-7α-³H with adrenals of $\text{Microtus pennsylvanicus}$. 11-Dehydrocorticosterone (Compd. A) was isolated as the major product and its identity confirmed by crystallization to constant specific activity. A tetrahydro derivative, 3α, 21-dihydroxy-5β-pregnane-11, 20-dione and an 18-hydroxy derivative, 18, 21-dihydroxy-4-pregnene-3,11, 20-trione were tentatively identified based-on Chromatographic behavior. The same products were observed with male adrenal and NADPH and with female adrenal using a NADPH generating system. Since the plasma manifested the typical fluorescence characteristics of corticosterone, the in vitro production of 11-keto steroids is considered to be the result of unusually high activity of the 11β-hydroxysteroid dehydrogenase in the $\text{Microtus}$ adrenal.     

Endorphins in brains of decapitated and microwave-killed mice

The size distribution of opioid peptides (endorphins) in mouse brain was determined by gel filtration, using the inhibition of stereospecific ³H-etorphine binding to guinea pig brain membranes as a measure of endorphin activity. The largest endorphin was about the same size as β-LPH-(61–91), the next largest was about half this size and the smallest was the size of the pentapeptide enkephalins. There was no important difference in the size distribution between brains of mice killed by rapid microwave irradiation and those killed by decapitation. Thus, opioid peptides of all three size classes appear to be present in the brain $\text{in}$$\text{vivo}$.     

Preparation of novel conjugates involving immunomodulating peptidoglycan monomer.

Peptidoglycan monomer, the disaccharide pentapeptide beta-D-Glcp-N-Ac-(1-->4)-D-Murp-N-Ac-L-Ala-D-mesoA2pm- (epsilonN H2)-D-Ala-D-Ala (PGM) is an immunomodulator. PGM and/or its derivative N-tert-butyloxycarbonyl-L-tyrosyl peptidoglycan monomer (Boc-Tyr-PGM) were coupled to two polysaccharides: the glucuronoxylomannan (GXM) from Cryptococcus neoformans, type B, solubilized by ultrasonic irradiation (MW 12-400 kDa) and to the dextran FP 70 (MW 70 kDa). Both polysaccharides were activated by CNBr. Initially, unprotected PGM was coupled via its amino group to GXM. The reactions yielded 42%-52% of the conjugate, containing only 0.18%-0.31% of PGM. In another approach Boc-Tyr-PGM (having its amino group blocked) was reacted via its free carboxyl group. Both CNBr-activated polysaccharides were first coupled to adipic acid dihydrazide (ADH) and then subsequently coupled to Boc-Tyr-PGM. The dextran conjugate (approximately 80% yield ) contained 6.3% of Boc-Tyr-PGM. The isolation of GXM conjugate required several modifications and it was obtained in lower yield (approximately 30%) but contained 13.7% of Boc-Tyr-PGM. Both conjugates were water soluble and apyrogenic and suitable for further testing of biological activity.     

Actions of morphine on prostate gland fructose metabolism

Varying doses of morphine sulfate (10, 20 or 40 mg/kg daily × 10) were observed to suppress metabolic activities in the mouse prostate gland. Prostate gland fructose, an index of androgenic activity, was significantly reduced by these dose regimes of morphine (P < 0.01). Injections of morphine sulfate (20 mg/kg daily × 10) led to an inhibitition in the $\text{in vitro}$ synthesis of both fructose^?14C and sorbitol^?14C from glucose^?14C by the prostate gland, part of which may have been due to decreased uptake of glucose by the gland. The $\text{in vitro}$ assimilation of 2-deoxyglucose^?14C by the prostate was also reduced by morphine treatment. The $\text{in vitro}$ actions of morphine (2 × 10^?3M) on the metabolism of radioactive glucose by the mouse prostate gland likewise revealed a significant reduction in the formation of sorbitol^?14C, but no decrease in fructose^?14C formation. These results indicate that both the $\text{in vitro}$ and $\text{in vivo}$ actions of morphine can inhibit fructose metabolism in the prostate gland.     

Metabolism of 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine by cell cultures

The metabolism of 1-alkyl-2-acetyl-$\text{sn}$-glycero-3-phosphocholine (platelet-activating factor) was studied using various cultured cell lines. All incubations were done in the presence of bovine serum albumin and serum-free media, since albumin eliminates the adsorption of 1-alkyl-2-acetyl-$\text{sn}$-glycero-3-phosphocholine to cultureware and serum enzymes interfere. Human leukemia (HL-60) cells, MDCK canine kidney cells, and transformed and nontransformed clones of mouse C3H1OT1/2 cells display varying rates of uptake, degradation, and capacities for reacylation of 1-alkyl-2-acetyl-$\text{sn}$-glycero-3-phosphocholine. HL-60 cells displayed the highest uptake rate (24.6 pmol/mg cell protein/15 min). Whereas C3H10T1/2 cells in culture showed uptake rates comparable to other cells tested, they displayed a relative metabolic inertness towards 1-alkyl-2-acetyl-$\text{sn}$-glycero-3-phosphocholine.     

Amphomycin inhibits phospho-N-acetylmuramyl-pentapeptide translocase in peptidoglycan synthesis of Bacillus.

Amphomycin, a selective inhibitor of peptidoglycan synthesis of bacteria, inhibited the lipid intermediates accumulation and the peptidoglycan synthesis from UDP-N-acetylmuramyl-L-Ala-D-Glu-[³H]-meso-Dpm-D-Ala-D-Ala (UDP-MurNAc-pentapeptide) and UDP-N-acetylglucosamine (UDP-GlcNAc) with a particulate fraction from $\text{Bacillus}\text{megaterium}$ KM, and also inhibited the formation of MurNAc (-pentapeptide)-P-P-lipid in the absence of UDP-GlcNAc. But it did not inhibit the formation of peptidoglycan from MurNAc(-pentapeptide)-P-P-lipid and UDP-GlcNAc with the same system of the organism.Thus, it is concluded that the site of action of amphomycin is phospho-MurNAc-pentapeptide translocase in peptidoglycan synthesis.     

Uteroplacental dysfunction and prostaglandin metabolism in zinc deficient pregnant rats

Relationships between perinatal mortality, disrupted uteroplacental function and prostaglandin metabolism have been studied in Zn-deficient rats. Uterine contractility $\text{in vitro}$, placental blood flow $\text{in viro}$, and uterine and placental prostaglandin synthesis from [1?¹⁴C] arachidonic acid $\text{in vitro}$ were investigated at day 22 of pregnancy. High amplitude uterine contractions were almost completely eliminated and utero-placental blood flow was decreased by 85% by Zn deficiency. Synthesis of [1?¹⁴C]-prostaglandin E₂, F_2α and 6-keto-F_1α from [1?¹⁴C] arachidonic acid decreased significantly in uterine tissue but increased in placentae. These possibly inter-related effects may contribute to the high perinatal mortality observed in Zn deficiency.     

Conversion of exogenous insulin into high molecular weight forms in vivo

[¹²⁵I]-insulin, injected in rats, was converted into high molecular weight forms as judged by gel filtration of blood serum samples collected at various intervals. These forms represented 26% (10 min. after injection) to 81% (240 min. after injection) of the total immunoprecipitable radioactivity. Their molecular weights were not affected by rechromatography in 0.1 M borate buffer (pH 8) or in 8 M urea-1 M acetic acid (pH 2.4). On incubation of [¹²⁵I]-insulin with blood serum $\text{in}\text{vitro}$, no high molecular weight forms could be observed.     

Assay of liver microsomal cholesterol 7 alpha-hydroxylase using deuterated carrier and gas chromatography-mass spectrometry

The activity of cholesterol 7α-hydroxylase in rat liver microsomes was assayed by measuring the mass of 5-cholestene-3β, 7α-diol formed from endogenous cholesterol under standardized incubation conditions. After termination of incubations, a known amount of 5-[24,25,7β-²H₃]cholestene-3β,7α-diol was added. A chloroform extract of the incubation mixture was subjected to thin layer chromatography and the fraction containing 5-cholestene-3β,7α-diol was converted into trimethylsilyl ether. The trimethylsilyl ether was subjected to combined gas chromatography-mass spectrometry and the amount of unlabeled 5-cholestene-3β,7α-diol in the mixture was calculated from the ratio between the relative intensitics of the peaks at $\text{m}\text{e}$ 456 (M-90) and $\text{m}\text{e}$ 459 [M-(90 + 3)]. The precision of the method was ±2.2% (SD). The results with this method of assay of cholesterol 7α-hydroxylase were compared with those obtained with a method based on conversion of a trace amount of added [4-¹⁴C]cholesterol into 5-cholestene-3β,7α-diol.     

A radioimmunoassay for leukotriene B4

A radioimmunoassay for leukotreine B₄ has been developed. The assay is sensitive; 5 pg LTB₄ caused significant inibition of binding of [³H]-LTB₄ and 50% displacement occurred with 30 pg. The specificity of the assay has been critically examined; prostaglandins, thromboxane B₂ and arachidonic acid do not exhibit detectable cross-reactions (< 0.03%). Flowever, some non-cyclic dihydroxy- and monohydroxy-eicosatetraeonic acids do cross-react slightly (e.g. diastereomers of 5,12-dihydroxy-6,8,10-$\text{trans}$-14-$\text{cis}$-elcosatetraenoic and 12-hydroxy-5,8,10,14-elcosatetraenoic acids cross-react 3.3% and 2.0% respectively). The assay has been used to monitor the release of LTB₄ from human neutrophils in response to the divalent cation ionophore, A23187. The immunoreactive material released during these incubations was confirmed as LTB₄ by reverse-phase high liquid chromatography follwing solvent extraction and silinic acid chromatography.     

Assembly of the vesicular stomatitis virus envelope: incorporation of viral polypeptides into the host plasma membrane

Incorporation of viral polypeptides into the host plasma membrane is an essential step in the formation of the lipoprotein envelope of vesicular stomatitis virus. A quantitative study of this process was carried out using a double-isotope labeling procedure. Infected cells were incubated for two hours with ¹⁴C-labeled amino acids, pulse-labeled with [³H]leucine and incubated for various times with an excess of non-radioactive leucine. The ${}^3\text{H}{}^{14}\text{C}$ ratio was determined for each viral polypeptide in isolated plasma membranes and in the whole cell by polyacrylamide gel electrophoresis. It was found that [³H]leucine-labeled viral polypeptides could be detected in the plasma membranes immediately following a 30-second pulse but that the ${}^3\text{H}{}^{14}\text{C}$ ratios of polypeptides in the plasma membrane did not reach the ${}^3\text{H}{}^{14}\text{C}$ ratios in the whole cells until the end of a two-minute chase period. The addition of puromycin to the cultures at the end of the pulse period did not affect subsequent incorporation of [³H]leucine-labeled polypeptides into the plasma membrane. The incorporation of various amino acid analogs into the viral polypeptides did not affect the efficiency with which they were incorporated into the plasma membranes. It is proposed that viral polypeptides are selected for incorporation into the plasma membrane from a small interior pool of completed molecules.     

Transfer of N, N'-diacetylchitobiose from dolichyl diphosphate into a gray matter membrane glycoprotein

Gray matter and white matter membranes catalyze the transfer of label from UDP-N-acetyl-[${}^{14}\text{C}$] glucosamine into N-acetyl[${}^{14}\text{C}$]glucosaminyl-pyrophosphoryl-dolichol, N,N′-diacetyl [${}^{14}\text{C}$]chitobiosyl-pyrophosphoryl-dolichol, and N-acetyl[${}^{14}\text{C}$]glucosamine-labeled glycoprotein. Gel filtration of the Pronase digests of gray matter N-acetyl[${}^{14}\text{C}$]glucosamine-labeled glycoprotein reveals two N-acetyl[${}^{14}\text{C}$]glucosamine-labeled glycopeptide fractions. One fraction (A) contains approximately eight glycose units. All of the radioactivity is at nonreducing termini and can be released by treatment with an exo-β-N-acetylglucosaminidase. A smaller N-acetyl[${}^{14}\text{C}$]glucosamine-labeled glycopeptide (B) is recovered in the elution volume expected for an asparaginyl disaccharide. Structural studies show that the labeled saccharide unit in glycopeptide B is N,N′-diacetyl[${}^{14}\text{C}$]chitobiose. The linkage between the ${}^{14}\text{C}$-labeled disaccharide and the polypeptide has the properties of an N-glycosidic attachment to asparagine. Only the larger N-acetyl[${}^{14}\text{C}$]glucosamine-labeled glycopeptide (A) is found in Pronase digests of white matter membrane N-acetyl[${}^{14}\text{C}$]glucosamine-labeled glycoprotein after incubation with UDP-N-acetyl[${}^{14}\text{C}$]glucosamine. When gray matter membranes are incubated with UDP-N-acetyl[${}^{14}\text{C}$]glucosamine in the presence of tunicamycin or UMP, the labeling of glycolipid and the asparaginyl disaccharide is inhibited. UMP and tunicamycin have no effect on the transfer of N-acetyl[${}^{14}\text{C}$]glucosamine to external acceptor sites of the larger glycopeptide (A). The transfer of N,N′-diacetyl[${}^{14}\text{C}$]-chitobiose from carrier lipid to protein is observed when extensively washed membranes containing endogenous, prelabeled ${}^{14}\text{C}$-labeled glycolipids are incubated in the presence or absence of unlabeled GDP-mannose. UMP treatment of the prelabeled membranes selectively discharged over 80% of the label from N-acetyl[${}^{14}\text{C}$]glucosaminyl-pyrophosphoryl-dolichol, but had no effect on the transfer of the ${}^{14}\text{C}$-labeled disaccharide to protein. All of these results are concordant with transfer of N,N′-diacetylchitobiose from dolichyl diphosphate to gray matter glycoprotein. The major membrane glycoprotein labeled by the lipid-mediated [${}^{14}\text{C}$]disaccharide transfer reaction has an apparent molecular weight of 24,000. Tunicamycin prevents the enzymatic labeling of the gray matter glycoprotein having an apparent molecular weight of 24,000.     

Cell surface antigens on erythroid cells: A comparison of normal and anemic (WW) mice

Cell surface antigens of normal and anemic ($\text{W}\text{W}$) mouse erythroid cells have been examined in cytotoxicity assays with two rat antisera. When tested on fetal liver cells, a rat anti-erythroblast serum recognized antigen(s) present on erythroid cells early in development, while rat anti-adult red blood cell serum recognized antigen(s) present on mature erythroid cells. Each of these sera had different activity on normal (+/+ or $\text{W}\text{+}$) as compared to anemic ($\text{W}\text{W}$) erythroid cells.     

Identification of albumin-synthesizing polysomes from mouse liver and a mouse hepatoma cell line.

Albumin-synthesizing polysomes from mouse liver and mouse hepatoma cells in in tissue culture have been localized on sucrose gradients with ¹²⁵I-labeled antimouse serum albumin used as a marker. Competition studies show that the ¹²⁵I-labeled antibody binds specifically to albumin-synthesizing polysomes from both tissues. The ¹²⁵I-labeled polysomes from liver and hepatoma cells have identical sedimentation properties on sucrose gradients, which indicates that the polysomes range in size from 9–14 ribosomes. This is comparable in size to polysomes from rat liver and Morris hepatoma. One significant difference between these albumin-synthesizing polysomes is that those extracted from hepatoma cells bind 70% less antibody than equivalent amounts of polysomes from liver cells. Since the level of albumin synthesis in the hepatoma cells is comparable to the level of albumin synthesis $\text{in vivo}$, this difference in antibody-binding capacity is not likely to be due to differences in polysomal content, but appears to be a characteristic difference between hepatoma and normal mouse liver cells.