The augmentation of tumor-specific immunity using haptenic muramyl dipeptide (MDP) derivatives

Summary A new haptenic compound, a muramyl dipeptide (MDP) derivative (designated as L4-MDP-ONB) cross-reactive with Bacillus Calmette Guerin (BCG) was synthesized. The cross-reactivity of L4-MDP hapten to BCG was demonstrated from the following evidence; (a) lymph node cells from BCG-primed C3H/HeN mice exhibited appreciable L4-MDP-specific proliferative responses to the in vitro stimulation of L4-MDP-modified syngeneic cells (L4-MDP-self); (b) inoculation of L4-MDP-self into footpads of BCG-primed C3H/HeN mice elicited ample delayed type-hypersensitivity (DTH) responses in vivo as measured by footpad swelling; and (c) BCG-primed mice contained L4-MDP-reactive helper T cell activity which functions to augment the generation of effector T cell responses to cell surface antigens. This crossreactivity between L4-MDP hapten and BCG as measured by the helper T cell activity was applied to enhanced induction of tumor-specific immunity. When BCG-primed C3H/HeN mice were immunized with L4-MDP-modified syngeneic X5563 tumor cells, these mice could generate augmented tumor-specific in vivo protective (tumor neutralizing) immunity as well as in vitro cytotoxic T cell responses. These results indicate the effectiveness of L4-MDP hapten in augmenting tumor-specific immunity. The present approach is discussed in the context of potential advantages of this new hapten for its future application to clinical tumor systems.     

Allelic variants of Ly-5 in inbred and natural populations of mice

Allelic variants of Ly-5 in inbred commensal and other natural populations of mice were analyzed by patterns of restriction fragment length polymorphisms (RFLP) and Southern hybridization using an Ly-5 cDNA probe and by cell-surface staining with a panel of antibodies directed against polymorphic and nonpolymorphic Ly-5 determinants. New Ly-5 alleles were defined by RFLPs generated by both Eco RI and Bam HI restriction enzyme digests. The Mus musculus subspecies and other species within the genus Mus showed a strong correlation between allelic variants defined by restriction enzymes and serologic specificities. The data also suggest the conservation of the Ly-5 gene throughout the genus Mus.     

In vivo biotinylation of fusion proteins expressed in Escherichia coli with a sequence of Propionibacterium freudenreichii transcarboxylase 1.3S biotin subunit.

Biotinylation of fusion proteins in E. coli was studied using a sequence of Propionibacterium freudenreichii transcarboxylase 1.3S biotin subunit. As the biotinylation sequence, we examined two sequences: one was of amino acid residues [84-123] of 1.3S, a partial sequence containing a region from a conserved tetrapeptide (Ala-Met-Bct-Met) around the biotinyl lysine (Bct) to the carboxyl terminal; the other was of an almost entire sequence [18-123]. We constructed recombinant plasmids for fusion proteins of beta-galactosidase, of chloramphenicol acetyltransferase, and of alkaline phosphatase. We found the biotinylation in the [18-123] sequence fused to alkaline phosphatase.     

Changes in whole body concentrations of thyroid hormones and cortisol in metamorphosing conger eel

Summary To clarify the hormonal regulation of metamorphosis of the conger eel (Conger myriaster), changes in whole body concentrations of thyroid hormones, thyroxine (T₄) and triiodothyronine (T₃), and cortisol during metamorphosis were examined, as well as the changes in the histological activity of the thyroid gland. In larvae before metamorphosis, T₄ and T₃ levels were less than 5 and 0.15 ng·g^-1 respectively. Levels of T₄ increased to about 30 ng·g^-1 during early metamorphosis, and decreased subsequently. Levels of T₃ increased gradually in early metamorphosis, and then increased abruptly to about 2.0 ng·g^-1 in late metamorphosis. Before metamorphosis, cortisol levels of the leptocephali less than 11 cm in total length were greater than 200 ng·g^-1. Cortisol levels decreased rapidly in larger premetamorphic leptocephali, and low levels were maintained throughout the metamorphic period. Histological observation revealed an activation of the thyroid gland in early metamorphosis; thyroid follicle epithelial cells became columnar and their nuclei larger. Active uptake of colloid by these cells and intensive vascularization of the gland were also observed. By the end of metamorphosis, follicle epithelial cells became squamous, indicating a low level of glandular activity. These results suggest that thyroid hormone plays an important role in regulation of conger eel metamorphosis.Abbreviations AL anal length - TL total length - T ₃ triiodothyronine - T ₄ thyroxine     

Neural coding of speech sound in the telencephalic auditory area of the mynah bird

Summary The responses of neurons in field L in the auditory neostriatum of the mynah bird, Gracula religiosa, were recorded during presentation of intact or manipulated mimic voices. A typical mimic voice konnichiwa elicited responses in most of the neurons. Neurons in the input layer (L2) of field L showed many peaks on peristimulus time histograms while those in other layers (L1 and L3) exhibited only one or two peaks. Several neurons in L1 and L3 responded only to the affricative consonant /t/ in the intact mimic voices. They did not respond to the affricative consonant in the isolated segment or to the one in the playbacked voice in reverse. Forty-five percent of the neurons (33/ 73) decreased in firing rates at the affricative consonant in the isolated segment compared with in the intact voice. Some of these neurons, in which neither the affricative consonant in the isolated segment nor bursts of noise alone elicited responses, exhibited clear phasic responses to /t/ in the case when bursts of noise with particular central frequencies preceded the affricative consonant. The responsiveness of these neurons appears to receive temporal facilitation. These results suggest that these neurons code the temporal relationship of speech sound.Abbreviations HVc hyperstriatum ventrale, pars caudale - TFN temporally facilitated neuron - TSN temporally suppressed neuron     

Recessive Nonsense Suppressors in SACCHAROMYCES CEREVISIAE : Action Spectra, Complementation Groups and Map Positions

Three genes SUP111, SUP112 and SUP113 of Saccharomyces cerevisiae have been identified that can mutate to give recessive omnipotent nonsense suppressors. Alleles of these loci can also act as allosuppressors; that is, different phenotypes, due apparently to different efficiencies of suppression, can result from different alleles at a given locus. The SUP111, SUP112 and SUP113 loci map to the right arms of chromosomes VIII, VII and XIII, respectively.     

Molecular cloning and sequence analysis of full-length cDNA for rabbit liver NADPH-cytochrome P-450 reductase mRNA

The nucleotide sequence of the mRNA for NADPH-cytochrome P-450 reductase from rabbit liver was determined from a full-length cDNA clone (pFP105). The clone contains 2,269 nucleotides complementary to rabbit liver reductase mRNA. The single open reading frame of 2,037 nucleotides codes for a 679-amino acid polypeptide with a calculated molecular weight of 76,583 daltons. The cloned cDNA contains the complete 3'-noncoding region of 193 nucleotides, including 68 nucleotides of poly(A), and 39 nucleotides of the 5'-noncoding region. The nucleotide sequence in the coding region of cDNA of rabbit reductase (pFP105) showed 85% homology to that of rat reductase (Porter, T.D. & Kasper, C.B. (1985) Proc. Natl. Acad. Sci. U.S. 82, 973-977, and Murakami, H. et al. (1986) DNA 5, 1-10). Rabbit reductase has one more amino acid residue than the rat enzyme, and the amino acid compositions of the two enzymes are similar. The amino acid sequence of the rabbit enzyme showed 91% identity with that of the rat enzyme. The segment related to binding of FMN and FAD was well conserved among rabbit, rat, and pig reductases. The sequence related to AMP moiety-binding was also conserved among these species, and was found in the amino acid sequence of NADH-cytochrome b5 reductase, another flavoenzyme in the microsomal electron transport system.     

Effect of calmodulin on aldosterone synthesis by a cytochrome P-45011 beta-reconstituted system from bovine adrenocortical mitochondria

Bovine adrenocortical calmodulin was purified and its general properties were examined. The latter were similar to those of bovine brain calmodulin. When added to a cytochrome P-450(11)beta-reconstituted system in the presence of dilauroylphosphatidylcholine, calmodulin decreased the rate of aldosterone production from corticosterone from 0.8 to 0.1 nmol/(min X nmol P-450), while it increased the rate of 18-hydroxycorticosterone production from 1.8 to 4.6 nmol/(min X nmol P-450). This effect of calmodulin on steroid production was maximum at a concentration of 1 microM, when 1 microM cytochrome P-450(11)beta was used. The effect was dependent on the presence of Ca2+, and maximal response was observed at less than 1 microM Ca2+. There was essentially no difference in the effect when bovine brain calmodulin was used. Calmodulin induced a change in the activity of cytochrome P-450(11)beta in the presence of a wide concentration range of corticosterone as a substrate. As for 18-hydroxycorticosterone production, calmodulin increased both the maximal activity and the apparent Km for corticosterone, but it decreased the apparent Km for adrenodoxin. Adrenodoxin at a concentration of less than 20 microM did not fully abolish the effect of calmodulin. A small type I difference spectrum appeared when calmodulin was added to cytochrome P-450(11)beta. The difference spectrum increased significantly in the presence of both Ca2+ and adrenodoxin. These results suggest that calmodulin interacts with cytochrome P-450(11)beta in the presence of adrenodoxin and then modulates the activity of aldosterone synthesis catalyzed by cytochrome P-450(11) beta.     

A spin-label study on human high density lipoprotein

Human plasma high density lipoproteins (HDL) have been labeled with N-(1-oxyl-2,2,6,6-tetramethyl-4-piperidinyl)maleimide (NEM-TEMPO). The spin-labeled HDL exhibited an ESR spectrum containing signals of both strongly immobilized and weakly immobilized components by the reaction with a high concentration of NEM-TEMPO, while an ESR spectrum containing only signals of a strongly immobilized component range between 4 degrees C and 37 degrees C, the signal height of the strongly immobilized component exhibited reversible temperature-dependent changes, whereas that of the weakly immobilized component changed irreversibly at temperatures above 25 degrees C. The activation energy of the irreversible change was estimated to be 26 kcal per mol. The strongly immobilized component was derived from NEM-TEMPO which modified apolipoprotein A-I covalently, while the weakly immobilized component was derived from NEM-TEMPO noncovalently bound to HDL. The rate of binding of NEM-TEMPO to either the strongly binding or weakly binding sites and the number of the strongly binding sites in apolipoprotein A-I were estimated to be 125 M-1.day-1 and 1.78, respectively. The binding of NEM-TEMPO to the strongly binding sites was suppressed greatly by pretreatment of HDL with 2,4,6-trinitrobenzene sulfonic acid (TNBS). The slow reaction and suppression with TNBS suggest that NEM-TEMPO binds to some amino acid residue, probably a lysine residue, in apoprotein A-I. The strongly immobilized and weakly immobilized components were reduced almost completely by ascorbate at the same rate, 0.048 min-1 at pH 7.4 and at 4 degrees C.     

Interpretation of the stokes radius of macromolecules determined by gel filtration chromatography

From a comparison of the gel chromatographic properties of large randomly-coiled polypeptides in 6 M guanidine hydrochloride and of large globular proteins, we found that the distribution coefficient was more closely correlated with the intrinsic viscosity-based Stokes radius than with the translational frictional coefficient-based Stokes radius. This means that the effect of the hydrodynamic flow of dissolved molecules during gel chromatography should be considered. The ratio of transport of solute by bulk flow as compared with that by net diffusion (i.e., Brownian motion) is large under some conditions. On the other hand, we consider that the distribution coefficient obtained in static equilibrium experiments should be determined by the translational frictional coefficient-based Stokes radius, since the solvent does not flow. On this basis, we discuss the meaning of the Stokes radius and the separation mechanism of macromolecules by gel filtration.