Recruitment of lysozyme as a major enzyme in the mouse gut: Duplication,divergence, and regulatory evolution

Summary Two major types of lysozymec (M and P) occur in the mouse genus,Mus, and have been purified from an inbred laboratory strain (C58/J) ofM. domesticus. They differ in physical, catalytic, and antigenic properties as well as by amino acid replacements at 6 of 49 positions in the amino-terminal sequence. Comparisons with four other mammalian lysozymesc of known sequence suggest that M and P are related by a gene duplication that took place before the divergence of the rat and mouse lineages. M lysozyme is present in most tissues; achieves its highest concentration in the kidney, lung, and spleen; and corresponds to the lysozyme partially sequenced before from another strain ofM. domesticus. InM. domesticus and several related species, P lysozyme was detected chiefly in the small intestine, where it is probably produced mainly by Paneth cells. A survey of M and P levels in 22 species of muroid rodents (fromMus and six other genera) of known phylogenetic relationships suggests that a mutation that derepressed the P enzyme arose about 4 million years ago in the ancestor of the housemouse group of species. Additional regulatory shifts affecting M and P levels have taken place along lineages leading to other muroid species. Our survey of 187 individuals of wild house mice and their closest allies reveals a correlation between latitude of origin and level of intestinal lysozyme.     

Effect of thiols on micronucleus frequency in gamma-irradiated mammalian cells

The effects of the thiols cysteamine, WR-1065, and WR-255591 on radiation-induced micronucleus (MN) frequency and cell killing were compared in cultured Chinese hamster ovary cells. MN were measured using the cytochalasin B assay of Fenech and Morley (1985), which minimizes the effect of cytokinetic perturbations on MN expression. The dose-response curves for MN induction were curvilinear both for control cells at doses between 1.5 and 4.5 Gy and for thiol-treated cells at doses between 3 and 9 Gy. Protection against MN induction by each thiol was independent of radiation dose. Furthermore, there was a close correlation between the degree of modification of MN induction and cell survival by each thiol, i.e., the MN frequency closely predicted the survival level regardless of the presence of absence of the thiols. A similar predictive relationship has also been reported by us for cell survival and DNA double-strand break (DSB) induction in this cell line following treatment with these same thiols. Collectively, these data support the hypothesis that, for DNA-repair-proficient mammalian cells treated with radiomodifying agents that do not alter DNA-repair processes, MN and DSB induction are predictive of the level of radiation lethality and of each other.     

Amino acid sequences of stomach and nonstomach lysozymes of ruminants

Summary Complete amino acid sequences are presented for lysozymesc from camel and goat stomachs and compared to sequences of other lysozymesc. Tree analysis suggests that the rate of amino acid replacement went up as soon as lysozyme was recruited for the stomach function in early ruminants. The two lysozymes from goat stomach are the products of a gene duplication that probably took place before the divergence of cow, goat, and deer about 25 million years ago. Partial sequences of three lysozymes from goat tears indicated that (a) the goat tear family of lysozymes may have diverged from the stomach lysozyme family by an ancient duplication and (b) later duplications are probably responsible for the multiple forms of tear and milk lysozymes in ruminants.     

Phylogenetic diversification of immunoglobulin genes and the antibody repertoire

Immunoglobulins are encoded by a large multigene system that undergoes somatic rearrangement and additional genetic change during the development of immunoglobulin-producing cells. Inducible antibody and antibody-like responses are found in all vertebrates. However, immunoglobulin possessing disulfide-bonded heavy and light chains and domain-type organization has been described only in representatives of the jawed vertebrates. High degrees of nucleotide and predicted amino acid sequence identity are evident when the segmental elements that constitute the immunoglobulin gene loci in phylogenetically divergent vertebrates are compared. However, the organization of gene loci and the manner in which the independent elements recombine (and diversify) vary markedly among different taxa. One striking pattern of gene organization is the "cluster type" that appears to be restricted to the chondrichthyes (cartilaginous fishes) and limits segmental rearrangement to closely linked elements. This type of gene organization is associated with both heavy- and light-chain gene loci. In some cases, the clusters are "joined" or "partially joined" in the germ line, in effect predetermining or partially predetermining, respectively, the encoded specificities (the assumption being that these are expressed) of the individual loci. By relating the sequences of transcribed gene products to their respective germ-line genes, it is evident that, in some cases, joined-type genes are expressed. This raises a question about the existence and/or nature of allelic exclusion in these species. The extensive variation in gene organization found throughout the vertebrate species may relate directly to the role of intersegmental (V<==>D<==>J) distances in the commitment of the individual antibody-producing cell to a particular genetic specificity. Thus, the evolution of this locus, perhaps more so than that of others, may reflect the interrelationships between genetic organization and function.      

Studies with a human plasma-derived immunosuppressive,anti-lymphoma factor

Summary A low-molecular-weight (1,400) factor isolated from a human plasma -globulin concentrate by acid-salt dissociation and ultrafiltration inhibits proliferation of mitogen-stimulated T cells and L1210 leukemia cells. The factor (UM05R) inhibits DNA, RNA, and protein synthesis in sensitive cells, acts in G1 of the cell cycle, and appears to suppress mitogen-responsive T cells without an accessory cell requirement. UM05R activity is enhanced by known cAMP-elevating agents and by sulfhydryl compounds. The results of the present study are consistent with the hypothesis that the plasma-derived agent inhibits lympho-proliferation as a result of elevation of intracellular cAMP.     

Numerical simulation of circulation in a Caribbean-type backreef lagoon

A numerical model based on the vertically intergrated equations of motion and continuity was used to simulate circulation in a shallow, well-mixed, Caribbean-type backreef lagoon. As the focus of previous research, Great Pond Bay, St. Croix, provided a suitable study site. Tides, wind, and the effect of waves impinging on the reef were incorporated within the model. Direction of simulated flow under various wind and wave regimes agrees well with patterns found during current meter and drogue tracking experiments. When momentum transfer over the reef due to wave breaking and set-up is added, the magnitude of flow within the lagoon, ranging 5–30 cm/s, also compares favorably to in situ measurements. Model results indicate a relatively rapid and realistic flushing period of less than one day. Results of this study indicate that numerical modeling techniques potentially offer an accurate and cost-effective means to predict the pattern of water movement within coral-reef lagoons.     

Human plasma-derived immunosuppressive factors having anti-lymphoma activity

Summary Extraction of Cohn IV-1, an -globulin enriched fraction of human plasma, with a high-salt, low-pH solution, followed by sequential ultrafiltration steps yielded an immunosuppressive preparation (UM05R) of mol.wt. 500–10,000. UM05R inhibited antibody formation in the mouse in vivo and transformation in vitro of lymphocytes treated with either T-or B-cell stimulants. Suppression of lymphocyte transformation, indicated by inhibition of ³H-thymidine incorporation into DNA, was confirmed by inhibition of blast cell formation. From dose-response curves the UM05R concentration to produce 50% suppression of lymphocyte blast transformation was 15–50 g protein/ml. Selectivity for lymphoid cells was suggested by growth inhibition in vitro of L1210 and P1798 leukemias but not murine neuroblastoma or human fetal fibroblasts. This observation also rules out the presence of an agent which is broadly cytotoxic. Fractionation of UM05R on Sephadex G-25 in 10% acetic acid yielded an early-emerging fraction, mol. wt. 5,000–10,000, containing B-cell inhibitor, and a late fraction, mol. wt. 1,400, inhibitory for both T- and B-cell transformation and growth of L1210. The inhibitory activity for B cells was removed from the other two activities by 5% trichloroacetic acid (TCA). The possibility is raised that the inhibitory activity for T cells and L1210 may reside in the same molecule. Sensitivity of the early-emerging B-cell inhibitor to carboxypeptidase B suggests that it is a polypeptide, but resistance of the T-cell inhibitor to various treatments leaves its nature uncertain. The properties of these factors suggest consideration of them as lymphocyte chalones occurring in plasma complexed to high-molecular-weight components.     

Calcium gradient-dependent and calcium gradient-independent phosphorylation of sarcoplasmic reticulum by orthophosphate. The role of magnesium.

Phosphorylation of the calcium-transport ATPase of skeletal muscle sarcoplasmic reticulum by inorganic phosphate was investigated in the presence or absence of a calcium gradient. The maximum phosphoprotein formation in the presence of a calcium gradient at 20 degrees C and pH 7.0 is approximately 4 nmol/mg sarcoplasmic reticulum protein, but only between 2.4 and 2.8 nmol/mg protein in the absence of a calcium gradient, using Ionophore X-537 A or phospholipase-A-treated sarcoplasmic reticulum vesicles. Maximum phosphoprotein formation independent of calcium gradient at 20 degrees C and pH 6.2 is in the range of 3.6--4 nmol/mg protein. Half-maximum phosphoprotein formation dependent on calcium gradient was achieved with 0.1--0.2 mM free orthophosphate at 10 mM free magnesium or at 0.1--0.2 mM free magnesium at 10 mM free orthophosphate. Phosphoprotein formation independent of calcium gradient is in accordance with a model which assumes, firstly, the formation of a ternary complex of the ATPase protein with orthophosphate and magnesium (E . Pi . Mg) in equilibrium with the phosphoprotein (E-Pi . Mg) and, secondly, an interdependence of both ions in the formation of the ternary complex. The apparent equilibrium constant was 0.6 and the apparent dissociation constants KMg, KMg', KPi and KPi' were 8.8, 1.9, 7.2 and 1.5 mM respectively, assuming a total concentration of the phosphorylation site per enzyme of 7 nmol/mg protein.     

Glycogen metabolism in the placenta of streptozotocin diabetic rats.

Administration of STZ prior to mating, induced significant impairment in glycogen metabolism in term rat placentae. Glycogen retention was observed in the treated placentae, while glycogen synthetase activity was reduced. The glycogenolytic pathway seemed to proceed mainly through amyloglucosidase enzyme whose activity increased threefold. Glucose-6-phosphatase showed a moderate activation, mainly in the labyrinth zone, while phosphorylase was slightly inhibited. These changes were accompanied by a decrease in placental insulin levels. The possible defensive role played by the placenta in cases of maternal hyperglycemia was postulated.     

Congruency of phylogenies derived from different proteins

Summary This communication examines the question of phylogenetic congruency- i.e., whether or not the branching order of evolutionary trees is independent of the protein studied. It was found that trees constructed for birds on the basis of immunological comparison of their transferrins, albumins, and ovalbumins agree approximately with a published tree based on the amino acid sequences of their lysozymesc. This congruency is especially noteworthy with respect to the phylogenetic position of the chachalaca, a Mexican bird classified on morphological grounds in the family Cracidae of the order Galliformes. At the protein level, this species differs as much from non-cracid galliform birds as does the duck, which belongs to another order. Despite the organismal similarity between cracid and non-cracid galliform birds, the molecular relationship is remote. If this contrast between organismal and molecular results had been based on comparative studies with only lysozyme, one could have ascribed the contrast to the possibility that chachalaca lysozyme was paralogous, rather than orthologous, to the other bird lysozymesc. Examination of several proteins is thus desirable in cases of possible paralogy.This work was supported in part by grants GB-42028X from NSF and GM-21509 from NIH