Aging of the erythrocyte XVII. Binding of autologous immunoglobulin G

Increased amounts of autologous immunoglobulin (Ig) G were found on older bovine erythrocytes employing ¹²⁵I-labelled Protein A, ¹²⁵I-labelled anti(bovine IgG) antibodies and hemolysis in the presence of anti(bovine IgG) antibodies + complement. In vitro, desialylation, proteolysis and action of an O₂$\text{·}$ source but not glycosylation induced binding of extraneous IgG to the erythrocytes.     

Tryptophan-induced stimulation of hepatic ornithine decarboxylase activity in the rat

The effect of a single tube feeding of l-tryptophan on hepatic ornithine decarboxylase (ODC) activity in rats was investigated. The levels of ODC activity in the livers of control and experimental rats were assayed in vitro by measuring the release of ¹⁴CO₂ from DL-[1-¹⁴C]ornithine. Single tube feedings of varying levels of l-tryptophan ($\text{2.5–30 mg}\text{100 g}$ body wt) to overnight-fasted rats 1 hr before sacrifice exhibited increases in the hepatic ODC activities. l-Tryptophan ($\text{30 mg}\text{100 g}$ body wt) tube fed to overnight-fasted rats $\text{1}\text{6}$ to 12 hr before sacrifice induced hepatic ODC activities which were significantly elevated beginning at 1 hr and peaking at 2 hr (6.5-fold increase over controls). In vitro [¹⁴C]leucine incorporation into protein using hepatic microsomes of tryptophan-treated rats was significantly increased at 1 hr in comparison with that of controls. The tryptophan-induced stimulation of hepatic ODC activity was not affected by prior adrenalectomy but was abolished by pretreatment with cycloheximide. These studies demonstrate that a single feeding of l-tryptophan can significantly enhance in the rat the activity of ODC, a key enzyme in the biosynthesis of polyamines.     

Inhibition of neuraminidase activity by derivatives of 2-deoxy-2,3-dehydro-N-acetylneuraminic acid

Eighteen derivatives of 2-deoxy-2,3-dehydro-N-acetylneuraminic acid were assayed for inhibitory activity against neuraminidases from viral and bacterial sources. Twelve of these compounds were active against neuraminidases of Vibrio cholerae, influenza $\text{A}\text{Mel}\text{,}\text{B}\text{Lee}$, and Newcastle disease virus, causing 50% enzyme inhibition in concentrations ranging from 10^?3M to 10^?6M. The most active of them and the most potent neuraminidase inhibitor described so far is 2-deoxy-2,3-dehydro-N-trifluoroacetylneuraminic acid. This compound has an inhibitor constant (K_i) of 7,9 × 10^?7, M for influenza $\text{A}\text{Mel}$ virus neuraminidase whereas the K_m of the virus enzyme for the substrate is 1000 times weaker (K_m = 6, 9 × 10^?4M). The mechanism of inhibition is competitive, and enzyme inhibition is independent of enzyme concentration. 2-Deoxy-2,3-dehydro-N-trifluoroacetylneuraminic acid inhibits hemagglutination by NDV and SV5 but does not inhibit agglutination of red cells by Sendai virus or influenza A and B viruses.     

Formation of the guanylylated and methylated 5′-terminus of vaccinia virus mRNA

mRNA was synthesized in vitro by vaccinia virus particles in the presence of S-adenosyl-[methyl-³H]methionine and α- or β,γ-³²P-labeled ATP or GTP. The two 5′-terminal structures m⁷G(5′)ppp(5′)G^m and m⁷G(5′)ppp(5′)A^m were isolated after P₁ nuclease digestion of the RNA. The distribution of radioactivity between the methylated mononucleotides and inorganic phosphate released by nucleotide pyrophosphatase treatment of m⁷G(5′)ppp(5′)G^m and m⁷G(5′)ppp(5′)A^m was consistent with the formation of the latter structures by condensation of pG residues from GTP with ppG- and ppA- at the 5′-termini of vaccinia mRNAs. An alternative mechanism involving the transfer of ppG residues to pA- at the 5′-terminus of the RNA was ruled out by the ³²P-labeling data as well as by the formation of m⁷G(5′)ppp(5′)A^m- when β,γ-methylene-GTP was substituted for GTP. At limiting concentrations of S-adenosylmethionine, the predominant methylated 5′-terminal structure was m⁷G(5′)ppp(5′)N- in which the penultimate nucleoside was unmethylated; in the absence of S-adenosylmethionine, G(5′)ppp(5′)N- as well as unblocked ppN- termini were detected. On the basis of the structures formed under various conditions, the following sequence of reactions was considered:

${}^{\mathrm{\gamma \beta \alpha }}\text{ppG}\text{+}{}^{\mathrm{\beta \prime \alpha \prime }}\text{ppN}\text{?→}\text{G}\text{(5′)}\text{N}\text{?+}{}^{\mathrm{\gamma \beta }}\text{PPi}$

$\text{G}\text{(5′)}\text{ppp}\text{(5′)}\text{N}\text{?+}\text{AdoMet}\text{→}\text{m}{}^7\text{G}\text{(5′)}\text{ppp}\text{(5′)}\text{N}\text{?+}\text{AdoHcy}$

$\text{m}{}^7\text{G}\text{(5′)}\text{ppp}\text{(5′)}\text{N}\text{?+}\text{AdoMet}\text{→}\text{m}{}^7\text{G}\text{(5′)}\text{ppp}\text{(5′)}\text{N}{}^{\mathrm{m}}\text{+}\text{AdoHcy}$

     

The cellular site of sulfation of influenza viral glycoproteins.

The incorporation of ³⁵SO₄^2? into viral polypeptides in MDBK cells infected with influenza virus was analyzed by SDS-polyacrylamide gel electrophoresis. When infected cells were labeled in the absence of calf serum, three polypeptides, HA, NP, and M, were resolved in isolated plasma membranes, and HA was the only polypeptide in which ³⁵SO₄^2? incorporation was detected. Sulfation of HA was also demonstrated in both smooth and rough cytoplasmic membranes, whereas there was no detectable ³⁵SO₄^2? incorporation into unglycosylated proteins. These results indicate that at least partial sulfation of HA is already completed at rough membranes. However, when infected cells were doubly labeled with [³H]leucine and ³⁵SO₄^2?, the ${}^{\mathrm{<mtext>35</mtext><mtext>S</mtext>}}{}^3\text{H}$ ratio in the HA polypeptide was not uniform in virions and subcellular components. The ratio was highest in virions, and decreased in the order of virions, plasma membranes, smooth membranes, and rough membranes, suggesting that further sulfate incorporation may occur in smooth membranes and plasma membranes as well as in rough membranes. The 355/3H ratio of HA associated with plasma membranes varied with the length of labeling; higher ratios were observed with shorter labeling periods. This observation may be explained by sulfate incorporation into performed HA. Significant amounts of ³⁵SO₄^2? incorporation into HA were found in the presence of cycloheximide, at concentrations wich completely inhibited the synthesis of viral polypeptides. Further, pulse-labeling of infected cells with ³⁵SO₄^2? at various times after inhibition of protein sy thesis by cycloheximide showed that sulfation of HA polypeptides continues to occur as long as 30 min or more after synthesis, which also suggests that ³⁵SO₄^2? continues to be incorporated into HA polypeptides even after they migrate from rough membranes. The acceptors for sulfation appear to be oligosaccharide units of viral glycoproteins since almost all ³⁵S label was recovered in association with glycopeptides after exhaustive digestion of virions with Pronase followed by gel filtration. As was observed for HA, the incorporation of ³⁵SO₄^2? into cellular mucopolysaccharides was also observed in every subcellular fraction tested. Further, when either smooth or rough cytoplasmic membranes isolated from infected cells were incubated with 3′-phosphoadenosine-5′-phosphosulfate ([³⁵S]PAP) in vitro, sulfate incorporation into mucopolysaccharide was detected, which suggests that sulfation of mucopolysaccharide occurs in both smooth and rough membranes in vivo. Additionally, it was found that the rate of incorporation of ³⁵SO₄^2? into cellular mucopolysaccharide was markedly inhibited by influenza virus infection.     

Non-immune non-activated chicken macrophage destroy murine fibroblast

Non-immune non-activated chicken bone marrow-derived macrophages (BM MØ) killed murine embryonic fibroblasts $\text{in vitro}$. Following precultivation for 10–35 days chicken BM MØ had the capacity to destroy normal murine embryonic fibroblasts at effector: target ratios of 10:1 to 1:1. Optimal killing was observed following cocultivation of MØ and fibroblasts for 48–72 hrs. Addition of LPS neither initiated nor potentiated MØ-mediated killing. This study demonstrates that chicken MØ have the capacity to destroy, $\text{in vitro}$, cells of phylogenetically distant species, similarly to the ability of murine MØ to kill chicken fibroblasts. It is suggested that vertebrate MØ xenolytic potential is analagous to the capacity of invertebrate phagocytes to destroy xenografts.     

Physical properties of a minimal infectious RNA (viroid) associated with the exocortis disease

Purification of the pathogenic RNA (viroid) from exocortis disease has been accomplished. Composition analysis indicates the absence of any DNA or protein and a molar nucleotide composition of CMP, 29.4%; AMP, 21.5%; GMP, 28.8%; and UMP, 19.9%. Even though the $\text{AMP}\text{UMP}$ and $\text{GMP}\text{CMP}$ ratios approach unity the per cent hyperchromicity (22%) resembled a tRNA-like molecule and not a dsRNA, while the T_m (52°) in 0.1 SSC was higher than the pure tRNA species tested. Nuclease digestion similarly suggested the presence of a highly ordered structure for the pathogenic RNA. We also introduced low field NMR spectroscopy as a new technique in the study of pathogenic RNA's (viroids) of unknown sequence.     

Oxygen-ascorbic acid-Copper(II) initiating system. A kinetic study of the polymerisation of methyl acrylate in aqueous medium

Polymerisation of methyl acrylate (MA) initiated by O₂-ascorbic acid (AA)-Cu²⁺ system was studied in aqueous medium at 40°C. The rate of polymerisation R_p, was found to increase, remain constant, and then decrease with increasing [Cu²⁺]. R_p was found to depend on [Cu²⁺]^0,6, [AA]⁰, [O₂]^0,6, and [MA]^1,3 in the increasing region, on [Cu²⁺]⁰, [AA]⁰, [O₂]^0,6, and [MA]^1,6 in the constant region and on [Cu²⁺]^?0,9, [AA]⁰, [O₂]^0,7, and [MA]^2,0 in the decreasing region. Rate laws were derived assuming a plausible reaction mechanism for the different regions. R_p increased with ionic strength and decreased with [H₂SO₄,]. An initial increase, then steady state followed by a decrease in rate with temperature was noticed in the range 25 ? 55°C. k_p/k_t^1/2 values were calculated and compared with literature values. Chain lengths were determined viscometrically.     

Studies on lectins of amago (Oncorhyncusrhodurus) I. Amago ova lectin and its receptor on homologous macrophages

A lectin was found in the ova of amago, a Japanese trout ($\text{Oncorhyncus}$$\text{rhodurus}$), which agglutinates rabbit, rat and human B-type ery-throcytes. The hemagglutination was specifically inhibited by monosaccharides, L-rhamnose, D-galactose, and their C₂ and C₄ analogs, and p-nitrophenyl-α-D-galactoside and melibiose, indicating a binding specificity for α-L-ramnosyl or α-D-galactosyl type sugar moiety. To study its interaction with homologous cells, amago peritoneal macrophages were isolated from corn starch-stimulated peritoneal exudates. The lectin-rabbit erythrocyte complexes were found to adhere onto the macrophages harvested on the 4th day or later after the stimulation, but not to those obtained within 3 days; the latter macrophages acquired the complex-binding capacity when cultured for 3 to 4 days $\text{in}$$\text{vitro}$. These findings indicated that a lectin receptor is expressed on peritoneal macrophages after inflammatory stimulation. Similar lectin receptor-bearing macrophage-like-cells were also detected during $\text{in}$$\text{vitro}$ amago head kidney culture. This suggested that the inflammatory induced peritoneal macrophages may be differentiated from the head kidney macrophage precursor cells and during this process the ova lectin receptors also become expressed.     

Mitogenic responses of frog lymphocytes to crude and purified preparations of bacterial lipopolysaccharide (LPS)

We have compared $\text{in}$$\text{vitro}$ mitogenic responses of frog ($\text{Xenopus}$$\text{laevis}$ and $\text{Rana}$$\text{pipiens}$) lymphocytes to various preparations of lipopolysaccharide (LPS). Commercial LPS prepared from $\text{E}$. $\text{coli}$ (phenol extraction) and from $\text{S}$. $\text{abortus-equi}$ (phenol and TCA extraction procedures) was mitogenic for frog lymphocytes. After reextraction of these LPS preparations with phenol-water, the remaining LPS was either considerably less mitogenic or not mitogenic. Purified $\text{E}$. $\text{coli}$ 055:B5 LPS, prepared by phenol water extraction, enzyme treatment and column chromatography, was not mitogenic. Frog cells proliferated poorly or not at all with all concentrations of reextracted or purified LPS tested (0.5–400 ug/ml) and at all culture periods examined (days 1–7). All LPS preparations used were mitogenic for CAF₁ mouse lymphocytes, whereas reextracted and purified LPS preparations were not mitogenic for lymphocytes from C3H/HeJ cells. $\text{Xenopus}$ were also not susceptible to toxicity induced by parenterally administered LPS in concentrations which killed CAF₁ mice.     

Mechanism of inactivation of a Fasciola proteolytic enzyme by peptide aldehydes and alkylating agents

A proteolytic enzyme of the liver fluke Fasciola sp. was purified as described previously by ammonium sulfate fractionation, gel filtration on Sephadex G-200 column and l-phenylalanine-agarose chromatography. Leupeptin, a peptide aldehyde of microbial origin, competitively inhibited the enzyme activity with respect to the substrate $\text{α-N-}\text{benzoyl-}\text{l}\text{-argininamide}$; the apparent K_i value for leupeptin is 45 000-fold less than the apparent K_m for the substrate. Incubation of the enzyme with leupeptin resulted in time-dependent inactivation of the globinolytic activity, with an inactivation constant (K_inact) of 0.4 μM giving the half-maximum inactivation velocity, and with a minimum inactivation half-time (T) of 2.7 min at infinite concentration of this compound. The inactivated enzyme was not reactivated by extensive dialysis. These results imply that leupeptin yields an affinity labelling of an active site of the enzyme. The activity of the Fasciola proteolytic enzyme was also inactivated by other peptide aldehydes and alkylating agents and inactivation constants observed were 0.5 μM for chymostatin, 13 μM for antipain, 2 μM for $\text{p-}\text{toluenesulfonyl-}\text{l}\text{-lysine}$ chloromethyl ketone, 140 μM for $\text{p-}\text{toluenesulfonyl-}\text{l}\text{-phenylalanine}$ chloromethyl ketone and 40 μM for iodoacetate under the conditions used.     

Respiratory variability in preterm and term infants: Effect of sleep state, position and age

The influence of sleep state and position on respiratory variability (RV) was studied in 13 preterm infants (PTIs) and 19 term infants (TIs). Temporally matched epochs of nasal pressure and oxygen saturation (Sp_O2)$(\text{S}{\text{p}}_{{\text{O}}_2})$ data were extracted from nap polysomnography. Inspiratory onset times (I) were determined, and variability measures of the I–I   interval compared in quiet sleep and active sleep, prone and supine and with age. Sleep state influenced respiratory variability (RV) in PTI and TI but Sp_O2$\text{S}{\text{p}}_{{\text{O}}_2}$ only varied with sleep state in PTI (p = 0.03). Position had no effect on RV in TI but influenced the standard deviation of ventilatory frequency (SDf) in PTI (p = 0.04). Age did not influence RV in PTI but SDf and the coefficient of variation of ventilatory frequency (CVf) decreased in TI from birth to 3 months. These data confirm sleep state as the predominant influence on RV in healthy term and convalescent preterm infants, with horizontal prone positioning having little effect when sleep state is controlled for.     

Friend leukemia and endogenous viruses. I. Production of friend and endogenous C-type particles by mouse cells cultured in vitro.

Within 3 days after infection of $\text{BALB}\text{c}$ 3T3 mouse cells with Friend virus, formation of new virions is detectable by agglutination of [³H]uridine-labeled virus with Con A. Newly infected cells produce a heterogeneous population of particles which includes infectious leukemia virus that is selectively and quantitatively agglutinated by Con A. Particles that are nonagglutinable are released in gradually diminished amounts as cells are serially passaged in vitro. Continued cultivation of the cells results in “spontaneous” production of other particles of density 1.18 that are coproduced with FLV. C-type particles (also of density 1.18) are produced simultaneously by cells not infected with Friend virus. They are morphologically indistinguishable from FLV, bind ferritin Con A, and similarly contain 60–70 S RNA.