Three natural forms of vertebrate gonadotropin-releasing hormone (GnRH) provided the structural basis upon which to design new GnRH agonists: [His5,Trp7,Leu8]-GnRH, dogfish (df) GnRH; [His5,Asn8]-GnRH, catfish (cf) GnRH; and [His5,Trp7,Tyr8]-GnRH, chicken (c) GnRH-II. The synthetic peptides incorporated the position 6 dextro (D)-isomers D-arginine (D-Arg) or D-naphthylalanine (D-Nal) in combination with an ethylamide substitution of position 10. The in vitro potencies for LH and FSH release of these analogues were assessed using static cultures of rat anterior pituitary cells. Efficacious peptides were examined for their gonadotropin-II and growth hormone releasing abilities from perifused goldfish pituitary fragments. Rat LH and FSH release was measured using homologous radioimmunoassays, whereas goldfish growth hormone and gonadotropin-II release were determined using heterologous carp hormone radioimmunoassays. The receptor binding of the most potent analogues was determined in bovine pituitary membrane preparations. Substitution of D-Nal6 into [His5,Asn8]-GnRH increased the potency over 2200-fold compared with the native ligand (cfGnRH) in cultured rat pituitary cells. This was equivalent to a 55-fold greater potency than that of the native mammal (m) GnRH peptide. Substitution of D-Nal6 or D-Arg6 into dfGnRH or cGnRH-II resulted in potencies that were related to the overall hydrophobicity of the analogues. The [D-Nal6,Pro9NEt]-cfGnRH bound to the bovine membrane preparation with an affinity statistically similar to that of [D-Nal6,Pro9NEt]-mGnRH (kd = 0.40 +/- 0.04 and 0.55 +/- 0.10 nM, respectively) in cultured rat pituitary cells. All analogues tested released the same ratio of FSH to LH. In goldfish, the analogues did not possess superagonistic activity but instead desensitized the pituitary fragments at lower analogue doses than that of the sGnRH standard suggesting differences in receptor affinity or signal transduction.

Amino Acid Sequence↗

PubMed1995

Ig VH hypermutation is absent in the germinal centers of aged mice.

After injection with immunogenic conjugates of the hapten (4-hydroxy-3-nitrophenyl)acetyl (NP), two distinct B cell populations can be identified in the spleen during the primary response. One of these populations is specialized for Ab production; the other, the germinal centers (GCs), has been identified as the site of Ig somatic hypermutation. Ag-driven selection of GC B cells bearing mutated receptors with higher affinity leads to the affinity maturation of serum Ab and increased protective humoral immunity. Microdissection of GC B cell populations specific for NP and sequencing of the recovered Ig heavy chain variable region genes revealed that the somatic hypermutation process is absent in the GCs of aged C57BL/6 mice. However, selection for Ag appears to occur in the absence of hypermutation in the form of competition between unmutated clones of Ag-activated B lymphocytes. Thus, affinity maturation in these animals is limited to the affinities of Ab encoded by the germline.

Age Factors↗

PubMed1995

A new family of Conus peptides targeted to the nicotinic acetylcholine receptor.

In this work, a new family of Conus peptides, the alpha A-conotoxins, which target the nicotinic acetylcholine receptor, is defined. The first members of this family have been characterized from the eastern Pacific species, Conus purpurascens (the purple cone); three peptides that cause paralysis in fish were purified and characterized from milked venom. The sequence and disulfide bonding pattern of one of these, alpha A-conotoxin PIVA, is as follows: [formula: see text] where O represents trans-4-hydroxyproline. The two other peptides purified from C. purpurascens venom are the under-hydroxylated derivatives, [Pro13]alpha A-conotoxin PIVA and [Pro7,13]alpha A-conotoxin PIVA. The peptides have been chemically synthesized in a biologically active form. Both electrophysiological experiments and competition binding with alpha-bungarotoxin demonstrate that alpha A-PIVA acts as an antagonist of the nicotinic acetylcholine receptor at the postsynaptic membrane.

Amino Acid Sequence↗

PubMed1995

Immunochemical probing of the functional role of the 238-246 and 567-574 sequences of myosin heavy chain.

Polyclonal site-directed peptide antibodies were raised against the 567-574 and 238-246 sequences of the rabbit skeletal muscle myosin heavy chain. These sequences, which are located in the subfragment 1 (S1) segment of myosin, have been implicated by former studies in actin and nucleotide binding of the molecule and in the communication between the two binding sites. The antibodies obtained from rabbit sera were found to be conformation-sensitive since they specifically reacted with S1 in solid-phase binding assay but not in Western blot. The binding of both antibodies to S1 was strongly inhibited by actin. The antibody against the 567-574 sequence, Ab567-574, moderately decreased the binding of S1 to actin filaments in rigor but not in the weakly-attached state, while Ab238-246 did not influence the binding of S1 to actin under either conditions. Both antibodies inhibited the actin activation of the MgATPase of S1 but did not affect MgATPase without actin or the Ca- and K(EDTA)-activated ATPase activities of S1. The sliding velocity of actin filaments in the in vitro motility assays were also reduced in the presence of the antibodies. Ab567-574 had especially strong inhibitory effect on the movement of actin filaments. The results indicate that the binding of antibodies may induce conformational changes, which propagate in the S1 structure, perturb the coupling between the binding sites and impair the motor function of myosin.

Actins↗

PubMed1995

Association of kyphomelic dysplasia with severe combined immunodeficiency.

Kyphomelic dysplasia is a distinct, rare, skeletal dysplasia with short angulated femora, bowing of long bones, short ribs, narrow thorax, and metaphyseal abnormalities. While immune deficiency occurs in other short stature/short-limb skeletal dysplasias and cartilage-hair hypoplasia, it has not been described with kyphomelic dysplasia. We report on an infant with this disorder who had profound humoral and cellular immunologic abnormalities consistent with severe combined immune deficiency (SCID). The infant died at age 2 months of overwhelming cytomegalovirus pneumonia. Kyphomelic dysplasia, as with other short stature/short-limb skeletal dysplasias, can be associated with immune deficiency and immune function should be investigated when this disorder is identified.

Bone Diseases, Developmental↗

PubMed1995

FK506 hepatotoxicity in liver allograft recipients.

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Adult↗

PubMed1995

A novel 27/16 kDa form of subtilisin cleaved actin: structural and functional consequences of cleavage between Ser234 and Ser235.

A new 27/16 kDa form of cleaved actin was prepared by subtilisin cleavage between Ser234 and Ser235 of F(MgADP)-actin complexed with BeFx. The cleavage had little effect on actin-actin interactions as probed in polymerization measurements and by electron microscopy. In circular dichroism melting experiments the thermostability of F-actin was reduced by about 10 degrees C by this cleavage. The in vitro motility and Vmax, but not Km, of actomyosin ATPase were decreased by about 20% upon 27/16 kDa cleavage of F-actin. The binding of tropomyosin to actin was unchanged by this modification.

Actins↗

PubMed1995

An electron paramagnetic resonance method for quantification of copper isotopes in proteins: application to a mixed-isotope derivative of laccase.

We have developed a method of denaturing a copper-containing protein and sequestering the copper in the form of the bis(acetylacetonato)copper(II) complex. Because of the excellent resolution of the hyperfine structure in the resulting EPR spectrum, the method is useful for determining the 65Cu/63Cu isotope ratio in proteins such as tree laccase. Here we analyzed a mixed isotope sample prepared by reconstituting 63Cu-enriched type 2-depleted laccase with 65Cu(I) and found that it contained only 25% 65Cu. This proves that reconstitution of the type 2 copper is possible under conditions that permit little or no exchange between resident copper and the external pool. However, exchange does occur under reducing conditions when we expose the holoprotein to a large excess of exogenous copper.

Binding Sites↗

PubMed1995

Silver as a probe of pore-forming residues in a potassium channel.

In voltage-dependent potassium channels, the molecular determinants of ion selectivity are found in the P (pore) region, a stretch of 21 contiguous residues. Cysteine was introduced at each P region position in a Shaker potassium channel. Residues projecting side chains into the pore were identified by means of channel inhibition by a sulfhydryl-reactive potassium ion analog, silver ion. The pattern of silver ion reactivity contradicts a beta barrel architecture of potassium channel pores.

Animals↗

PubMed1995

Facultative role of germinal centers and T cells in the somatic diversification of IgVH genes.

The development of memory B cells takes place in germinal centers (GC) of lymphoid follicles where antigen-driven lymphocytes undergo somatic hypermutation and affinity selection, presumably under the influence of helper T cells. However, the mechanisms that drive this complex response are not well understood. We explored the relationship between GC formation and the onset of hypermutation in response to the hapten phosphorylcholine (PC) coupled to antigenic proteins in mice bearing different frequencies of CD4+ T cells. PC-reactive GC were identified by staining frozen splenic sections with peanut agglutinin (PNA) and with monoclonal Abs against AB1-2, a dominant idiotope of T15+ anti-PC antibody. The nucleotide sequences of rearranged T15 VH1 genes were determined from polymerase chain reaction amplifications of genomic DNA from microdissected GC B cells. T15+ GC became fully developed by day 6-7 after primary immunization of euthymic mice with either PC-keyhole limpet hemocyanin (KLH) or PC-chicken gamma globulin (CGG). Yet the VH1 gene segments recovered from the primary GC as late as day 10-14 had low numbers of mutations, in contrast to responses to the haptens nitrophenyl or oxazolone that sustain high levels of hypermutation after GC formation. PC-reactive B cells proliferate in histologically typical GC for considerable periods with no or little somatic hypermutation; the signals for GC formation are independent of those for the activation of hypermutation. We then examined GC 7 d after secondary immunization with PC-KLH in euthymic mice, in nu/nu mice reconstituted with limited numbers of normal CD4+ cells before priming (CD4(+)-nu/nu) and in nu/nu mice. All of these animals develop T15+ GC after antigen priming, however, the patterns of V gene mutations in the secondary GC reflected the levels of CD4+ cells present during the primary response. VDJ sequences from secondary GC of euthymic mice were heavily mutated, but most of these mutations were shared among all related (identical VDJ joints) sequences suggesting the proliferation of mutated, memory B cells, with little de novo somatic hypermutation. In contrast, the patterns of V gene diversity in secondary GC from CD4(+)-nu/nu mice suggested that there was ongoing mutation and clonal diversification during the first week after rechallenge. The secondary GC from T cell-deficient, nu/nu mice showed little evidence for mutational and/or recombinational diversity of T15+ B cells. We conclude that the participation of CD4+ helper cells is required for full activation of the mutator in GC and takes place in a dose-dependent fashion.

Amino Acid Sequence↗

PubMed1995

Evidence for a high density of secretoneurin-like immunoreactivity in the extended amygdala of the rat.

Secretoneurin is a novel 33-amino-acid neuropeptide produced by endoproteolytic processing from secretogranin II, which is a member of the chromogranin/secretogranin family. In this immunocytochemical study, we compared the distribution pattern of secretoneurin immunoreactivity with that of tyrosine hydroxylase, calbindin, substance P, and Leu-enkephalin in adjacent sections of rat forebrain. Secretoneurin appeared mainly in varicosities and fibers. Only a few cell bodies were stained. In the nucleus accumbens, a partial overlap of secretoneurin-immunoreactive patches with enkephalin-immunopositive areas was found. Secretoneurin displayed low to moderate levels of immunoreaction in calbindin-rich as well as in calbindin-immunonegative areas of the caudate-putamen. In the globus pallidus, entopeduncular nucleus, and substantia nigra, secretoneurin immunoreactivity was oriented ventromedially preferentially in woolly fibers. The dense immunostaining in the medial nucleus accumbens was directly continuous with dense secretoneurin immunoreactivity in the bed nucleus of the stria terminalis. Two strongly secretoneurin-immunopositive bands, one in the sublenticular portion and a smaller one along the posterior limb of the anterior commissure, interconnected the highly secretoneurin-immunopositive centromedial amygdala with the bed nucleus of the stria terminalis. Thus, the distribution pattern of secretoneurin immunoreactivity provides a marker of the extended amygdala that forms a continuum between the centromedial amygdala and the bed nucleus of the stria terminalis.

Amygdala↗

PubMed1995

Reversal of primary hyperoxaluria cardiomyopathy after combined liver and renal transplantation.

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Adult↗

PubMed1995

Genetic mechanisms involved in cellular recovery from oxidative stress.

Sophisticated biochemical networks allow organisms such as bacteria and insects to switch from very rapid growth and development in ideal environments to dormancy during severely unfavorable conditions. These switches may be accompanied by abrupt changes in oxidation/reduction involving reactive oxygen species (ROS). ROS have the potential of damaging nucleic acids, proteins, and membranes. In Escherichia coli, certain genetically regulated circuits (regulons) turn on synthesis of anti-oxidant enzymes to protect against distinct ROS excesses (superoxide, hydrogen peroxide, organic or lipid peroxides, etc.). As examples, the soxRS regulon controls synthesis of Mn-superoxide dismutase, oxyR controls catalase HPI, rpoS positively regulates HPII, and fur regulates several oxidative reactions that involve iron uptake. Our studies have focused on the regulatory role of rpoS, known to be a sigma factor (sigma 38) that combines with RNA polymerase and is a regulator of those gene products needed to protect cells during dormancy. Since insect cells, during both active growth and dormancy, endure severe environments, analogous protective gene products may be induced. Examples are presented of insect anti-oxidant metabolism, including those involved in the aging process. In addition, we searched several DNA and protein sequence data banks to compare resemblances between anti-oxidant gene products of bacteria and insects.

Animals↗