A new role for PGRP-S (Tag7) in immune defense: lymphocyte migration is induced by a chemoattractant complex of Tag7 with Mts1

PGRP-S (Tag7) is an innate immunity protein involved in the antimicrobial defense systems, both in insects and in mammals. We have previously shown that Tag7 specifically interacts with several proteins, including Hsp70 and the calcium binding protein S100A4 (Mts1), providing a number of novel cellular functions. Here we show that Tag7–Mts1 complex causes chemotactic migration of lymphocytes, with NK cells being a preferred target. Cells of either innate immunity (neutrophils and monocytes) or acquired immunity (CD4⁺ and CD8⁺ lymphocytes) can produce this complex, which confirms the close connection between components of the 2 branches of immune response.     

19F nuclear magnetic resonance as a probe of structural transitions and cooperative interactions in heavy meromyosin

An 19F NMR probe has been attached to the reactive sulfhydryl SH1 of the globular heads of rabbit skeletal heavy meromyosin. It serves as a sensitive monitor of the conformational state of the heads of heavy meromyosin in a manner similar to that seen for subfragment-1 (Shriver, J.W., and Sykes, B.D. (1982) Biochemistry 21, 3022-3028; Tollemar, U., Cunningham, K., and Shriver, J.W. (1986) Biochim. Biophys. Acta 873, 243-251). The NMR spectra indicate that there are at least two states for the heads in the SH1 region. The energetics of the interconversion of the two states of heavy meromyosin (HMM) differs significantly from that of S-1. In HMM in the absence of divalent cations, there are two reversible paths between the low temperature and high temperature states with a hysteresis-like behavior. One path is consistent with the head groups behaving independently and similar to S-1 alone. The second path indicates a coupling of the globular head region observed in S-1 with a second region forming a distinctly different cooperative unit. Upon addition of Ca(II) the hysteresis effect is lost and only the second cooperative unit is observed. Two explanations are offered for these results: the globular heads in HMM may couple with the S-2 segment, or the two globular heads of HMM may couple to form a larger cooperative unit. The ability to stabilize the larger cooperative unit with a divalent metal ion implicates a role for the LC2 light chain in coupling regions of the myosin molecule.     

Mitochondrial acetyl-CoA carboxylase. Time course of mobilization/activation in liver of refed rats.

Fasted (48 h) rats were killed at 0, 2, 4, 6, 8, 12, 16, 20 and 24 h after they were refed on a high-carbohydrate diet. An increase in the maximal activity and quantity of cystolic acetyl-CoA carboxylase was found in liver of refed rats after a lag time of about 8 h. The increased quantity of cytosolic enzyme was attributable primarily to mobilization of mitochondrial storage forms and not to substantial increase in the rate of synthesis of acetyl-CoA carboxylase.     

Mutational analysis reveals only one catalytic histidine in neutral endopeptidase ("enkephalinase").

Aside from serving as zinc ligands, kinetic data has implicated one or more additional histidines as catalytic residues in neutral endopeptidase ("enkephalinase") action. One of these histidines has previously been identified as histidine 704 (Bateman et al., J. Biol. Chem., 265:8365-8368, 1990). In order to determine whether a second histidine is involved in catalysis each of these residues not previously changed have been converted to glutamine by site directed mutagenesis. The resultant recombinant enzymes possess full catalytic activity indicating that histidine 704 is the only catalytic histidine in the enzyme.     

Segregation distortion of the CTG repeats at the myotonic dystrophy locus.

Myotonic dystrophy (DM), an autosomal dominant neuromuscular disease, is caused by a CTG-repeat expansion, with affected individuals having > or = 50 repeats of this trinucleotide, at the DMPK locus of human chromosome 19q13.3. Severely affected individuals die early in life; the milder form of this disease reduces reproductive ability. Alleles in the normal range of CTG repeats are not as unstable as the (CTG)(> or = 50) alleles. In the DM families, anticipation and parental bias of allelic expansions have been noted. However, data on mechanism of maintenance of DM in populations are conflicting. We present a maximum-likelihood model for examining segregation distortion of CTG-repeat alleles in normal families. Analyzing 726 meiotic events in 95 nuclear families from the CEPH panel pedigrees, we find evidence of preferential transmission of larger alleles (of size < or = 29 repeats) from females (the probability of transmission of larger alleles is .565 +/- 0.03, different from .5 at P approximately equal .028). There is no evidence of segregation distortion during male meiosis. We propose a hypothesis that preferential transmission of larger CTG-repeat alleles during female meiosis can compensate for mutational contraction of repeats within the normal allelic size range, and reduced viability and fertility of affected individuals. Thus, the pool of premutant alleles at the DM locus can be maintained in populations, which can subsequently mutate to the full mutation status to give rise to DM.     

Vntr Allele Frequency Distributions under the Stepwise Mutation Model: A Computer Simulation Approach

Variable numbers of tandem repeats (VNTRs) are a class of highly informative and widely dispersed genetic markers. Despite their wide application in biological science, little is known about their mutational mechanisms or population dynamics. The objective of this work was to investigate four summary measures of VNTR allele frequency distributions: number of alleles, number of modes, range in allele size and heterozygosity, using computer simulations of the one-step stepwise mutation model (SMM). We estimated these measures and their probability distributions for a wide range of mutation rates and compared the simulation results with predictions from analytical formulations of the one-step SMM. The average heterozygosity from the simulations agreed with the analytical expectation under the SMM. The average number of alleles, however, was larger in the simulations than the analytical expectation of the SMM. We then compared our simulation expectations with actual data reported in the literature. We used the sample size and observed heterozygosity to determine the expected value, 5th and 95th percentiles for the other three summary measures, allelic size range, number of modes and number of alleles. The loci analyzed were classified into three groups based on the size of the repeat unit: microsatellites (1-2 base pair (bp) repeat unit), short tandem repeats [(STR) 3-5 bp repeat unit], and minisatellites (15-70 bp repeat unit). In general, STR loci were most similar to the simulation results under the SMM for the three summary measures (number of alleles, number of modes and range in allele size), followed by the microsatellite loci and then by the minisatellite loci, which showed deviations in the direction of the infinite allele model (IAM). Based on these differences, we hypothesize that these three classes of loci are subject to different mutational forces.     

The effect of polyamines on the poly(adenylic acid)-induced inhibition of ribonuclease activity.

Segments of poly(A) at the 3'-termini of 5 S rRNA inhibit the activities of ribonucleases from Citrobacter, Enterobacter, bovine pancreas, human spleen and human plasma. Certain polyamines, or compounds containing polyamine substructures, mediate reversal of this inhibition. Effective compounds contain three amino groups, at least two of which are charged and are separated from the others by no less than three carbon atoms. Spermidine and 9-aminoacridines, which contain substituted propyl- or butylamino moieties at the 9-amino position and which bear two positive charges per molecule, are efficacious at low concentrations (5 microM). A decrease in effectiveness is associated with the removal of one aromatic ring from the 9-aminoacridines. However, the resulting 4-aminoquinolines, unlike the acridines, do not inhibit enzyme activity when present in concentrations above 30 microM. Relocating the diamino side chain from the 4- to the 8-position of the quinoline nucleus causes a decrease in charge density to +1, with the result that such compounds are ineffective. The orders of polyamine efficacy of reversal of inhibition were similar for enzymes from Citrobacter, bovine pancreas, and human plasma, and paralleled the order of binding of polyamines to either poly(A) or 5 S rRNA. This was not the case with Enterobacter and human spleen RNAases, indicating that the identity of the most effective polyamines depends on the RNAase studied. The combination of variable 3'-terminal poly(A) segment length and polyamine identity and concentration constitutes a system by which RNAase activities, and, therefore, substrate-degradation rates, may be easily varied.     

In situ enzymatic removal of orthophosphate by the nucleoside phosphorylase catalyzed phosphorolysis of nicotinamide riboside

An enzymatic orthophosphate removal system is described which can be effectively used to continuously remove orthophosphate from biochemical samples. The phosphorolysis of nicotinamide riboside is catalyzed by calf spleen nucleoside phosphorylase to give ribose-1-PO4 and nicotinamide along with a proton. At pH 8 the production of ribose-1-PO4 from orthophosphate is essentially quantitative. This reaction can be monitored optically or by 31P nuclear magnetic resonance (NMR). Equations are given for determining the time required to remove a given amount of phosphate from a typical NMR sample with a known amount of nucleoside phosphorylase. The effects of a competing orthophosphate-producing reaction are considered.     

Analysis of the importance of arginine 102 in neutral endopeptidase (enkephalinase) catalysis.

Neutral endopeptidase 24.11 contains an active site arginine believed to function in substrate binding. This arginine is thought to form an ionic interaction with the COOH-terminal carboxylate of NEP substrates. The functionality of arginine 102 has been investigated by using site-directed mutagenesis to produce mutants in which this residue was converted to a lysine, glycine, glutamine, or glutamate. All of the mutants exhibited essentially full activity as determined with a synthetic peptide amide, glutaryl-Ala-Ala-Phe-4-methoxy-2-naphthylamide. In contrast, activity was detected only with the wild-type enzyme and the lysine mutant using a synthetic substrate containing a free COOH-terminal carboxylate, dansyl-Gly-Trp-Gly. Inhibition studies with the physiologically active peptide substrates substance P, endothelin, and angiotensin I, as well as substance P free acid, [D-Ala2,Leu5]enkephalin, and [D-Ala2,Leu5]enkephalinamide indicated a lack of importance of arginine 102 in substrate binding. With [D-Ala2,Met5]enkephalin and the chemotactic peptide, N-formyl-Met-Leu-Phe, a significant decrease in affinity is observed with the arginine 102 mutants. These results suggest that the contribution of arginine 102 to substrate binding is dependent upon the strength of other subsite interactions. Examination of dipeptides as inhibitors indicates that the nature and orientation of the P'2 residue is important in determining the strength of the interaction of arginine 102 with its substrates.