Morphometrical and molecular evidence suggests cryptic diversity among hookworms (Nematoda: Uncinaria) that parasitize pinnipeds from the south-eastern Pacific coasts.

Hookworms of the genus Uncinaria parasitize pinniped pups in various locations worldwide. Four species have been described, two of which parasitize pinniped pups in the southern hemisphere: Uncinaria hamiltoni parasitizes Otaria flavescens and Arctocephalus australis from the South American coast, and Uncinaria sanguinis parasitizes Neophoca cinerea from the Australian coast. However, their geographical ranges and host specificity are unknown. Uncinaria spp. are morphologically similar, but molecular analyses have allowed the recognition of new species in the genus Uncinaria. We used nuclear genetic markers (internal transcribed spacer (ITS) and large subunit (LSU) rDNA) and a mitochondrial genetic marker (cytochrome c oxidase subunit I (COI)) to evaluate the phylogenetic relationships of Uncinaria spp. parasitizing A. australis and O. flavescens from South American coasts (Atlantic and Pacific coasts). We compared our sequences with published Uncinaria sequences. A Generalized Mixed Yule Coalescent (GMYC) analysis was also used to delimit species, and principal component analysis was used to compare morphometry among Uncinaria specimens. Parasites were sampled from A. australis from Peru (12°S), southern Chile (42°S), and the Uruguayan coast, and from O. flavescens from northern Chile (24°S) and the Uruguayan coast. Morphometric differences were observed between Uncinaria specimens from both South American coasts and between Uncinaria specimens from A. australis in Peru and southern Chile. Phylogenetic and GMYC analyses suggest that south-eastern Pacific otariid species harbour U. hamiltoni and an undescribed putative species of Uncinaria. However, more samples from A. australis and O. flavescens are necessary to understand the phylogenetic patterns of Uncinaria spp. across the South Pacific.

GHRH receptor of little mice contains a missense mutation in the extracellular domain that disrupts receptor function.

The growth hormone-releasing hormone receptor (GHRHR) is a member of the family of G protein-coupled receptors that is expressed on pituitary somatotrope cells and mediates the actions of GHRH in stimulating growth hormone (GH) synthesis and secretion. We report that the Ghrhr gene is located in the middle of mouse chromosome 6 in the same region as the little mutation. Mice homozygous for this mutation have reduced GH secretion and a dwarf phenotype. A missense mutation was identified in the extracellular domain of the little GHRHR that disrupts receptor function, suggesting that the growth deficit in these mice results from a defect in the GHRHR. Similar alterations in GHRHR might explain some isolated GH deficiencies in humans.

Suggestive synergy between genetic variants in TF and HFE as risk factors for Alzheimer's disease.

Alzheimer's disease (AD) is a complex disease that is likely influenced by many genetic and environmental factors. Citing evidence that iron may play a role in AD pathology, Robson et al. [Robson et al. (2004); J Med Genet 41:261-265] reported that epistatic interaction between rs1049296 (P589S) in the transferrin gene (TF) and rs1800562 (C282Y) in the hemochromatosis gene (HFE) results in significant association with risk for AD. In this study we attempted to replicate their findings in a total of 1,166 cases and 1,404 controls from three European and European American populations. Allele and genotype frequencies were consistent across the three populations. Using synergy factor analysis (SFA) and Logistic Regression analysis we tested each population and the combined sample for interactions between these two SNPs and risk for AD. We observed significant association between bi-carriers of the minor alleles of rs1049296 and rs1800562 in the combined sample using SFA (P = 0.0016, synergy factor = 2.71) and adjusted SFA adjusting for age and presence of the APOE epsilon 4 allele (P = 0.002, OR = 2.4). These results validate those of the previous report and support the hypothesis that iron transport and regulation play a role in AD pathology.

Dynamic changes in inhibin messenger RNAs in rat ovarian follicles during the reproductive cycle.

The alterations in morphology and function of the ovarian follicle as it matures, ovulates, and becomes a corpus luteum are dramatic. A variety of steroid and polypeptide hormones influence these processes, and the ovary in turn produces specific hormonal signals for endocrine regulation. One such signal is inhibin, a heterodimeric protein that suppresses the secretion of follicle-stimulating hormone from pituitary gonadotrophs. Rat inhibin complementary DNA probes have been used to examine the levels and distribution of inhibin alpha-and beta A-subunit messenger RNAs in the ovaries of cycling animals. Striking, dynamic changes have been found in inhibin messenger RNA accumulation during the developmental maturation of the ovarian follicle.

Regulation of jun-B messenger RNA and AP-1 activity by light and a circadian clock.

The suprachiasmatic nuclei (SCN) of the hypothalamus comprise the primary pacemaker responsible for generation of circadian rhythms in mammals. Light stimuli that synchronize this circadian clock induce expression of the c-fos gene in rodent SCN, which suggests a possible role for Fos in circadian entrainment. Appropriate light stimuli also induce the expression of jun-B messenger RNA in the SCN of golden hamsters but only slightly elevate c-jun messenger RNA levels. In addition, light increases the amount of a protein complex in the SCN that binds specifically to sites on DNA known to mediate regulation by the AP-1 transcription factor. The photic regulation of both jun-B messenger RNA expression and AP-1 binding activity is dependent on circadian phase: only light stimuli that shift behavioral rhythms induce jun-B and AP-1 expression. Thus, light and the circadian pacemaker interact to regulate a specific set of immediate-early genes in the SCN that may participate in entrainment of the circadian clock.

Regulation of CREB phosphorylation in the suprachiasmatic nucleus by light and a circadian clock.

Mammalian circadian rhythms are regulated by a pacemaker within the suprachiasmatic nuclei (SCN) of the hypothalamus. The molecular mechanisms controlling the synchronization of the circadian pacemaker are unknown; however, immediate early gene (IEG) expression in the SCN is tightly correlated with entrainment of SCN-regulated rhythms. Antibodies were isolated that recognize the activated, phosphorylated form of the transcription factor cyclic adenosine monophosphate response element binding protein (CREB). Within minutes after exposure of hamsters to light, CREB in the SCN became phosphorylated on the transcriptional regulatory site, Ser133. CREB phosphorylation was dependent on circadian time: CREB became phosphorylated only at times during the circadian cycle when light induced IEG expression and caused phase shifts of circadian rhythms. These results implicate CREB in neuronal signaling in the hypothalamus and suggest that circadian clock gating of light-regulated molecular responses in the SCN occurs upstream of phosphorylation of CREB.

Photic and circadian regulation of c-fos gene expression in the hamster suprachiasmatic nucleus.

Photic information entrains a circadian pacemaker located in the suprachiasmatic nucleus (SCN) of the mammalian hypothalamus to environmental light/dark cycles. To determine whether light regulates c-fos gene expression in the SCN, we have measured c-fos mRNA levels in the SCN of the golden hamster. We report that, during the subjective night, light causes a rapid increase in levels of c-fos mRNA in the SCN. Light pulses of 5 min duration are sufficient to induce c-fos mRNA, and the highest mRNA levels occur 30 min following the onset of light. The minimum level of illumination required to induce an increase in c-fos mRNA is indistinguishable from the minimum irradiance that produces a phase shift in the hamster's circadian rhythm of activity. In addition, the induction of c-fos mRNA in the SCN by light is itself under circadian regulation. Light induction of c-fos mRNA occurs only during the subjective night, at circadian times when photic phase shifting of activity occurs. Taken together, these data suggest that c-fos may be a molecular component of the photic pathway for entrainment of mammalian circadian rhythms.

Carp hemoglobin. V. Effects of pH and P6-inositol on autooxidation.

Autooxidation of carp hemoglobin has been measured from 4--25 degrees C with and without P6-inositol. The rate was accelerated by the increase of proton concentration and/or the addition of P6-inositol. As the rate increases the kinetics become more complex. In acidic media with P6-inositol, the autooxidation is initially rapid which becomes slower subsequently but eventually proceeds very fast. The activation energy for autooxidation is about 19 kcal. mol-1. The complicated kinetics are partly attributable to the fact that carp hemoglobin is incompletely oxygenated under these conditions and that various liganded molecules including partially oxidized species have different susceptibilities toward autooxidation.

Photo-CIDNP nuclear magnetic resonance as a probe for conformational changes in epidermal growth factor.

Photo-chemically induced dynamic nuclear polarization (CIDNP)-NMR spectroscopy at 360 MHz has been used to investigate pH-induced conformational transitions in mouse epidermal growth factor. At about pH 9, all five tyrosine residues and both tryptophan residues are, to various extents, solvent-exposed, while the His-22 residue is buried in the protein matrix. Tyr-13 is the least exposed of the tyrosine residues and also the most immobilized. As the pH is decreased to 5.9, the tryptophan residues gradually become less exposed, while the Tyr-13 residue becomes internalized in the protein. These data suggest that the C-terminus and part of the N-terminal structural domain are affected by a conformational transition in mouse epidermal growth factor occurring between pH 6 and 8 via breakage of the His-22 inter-residue linkage. Above pH 9, a decreased photo-CIDNP effect is evident for both tryptophans and for Tyr-10 and Try-13; this information suggests that a second conformational change takes place at basic pH, which may simply be incipient denaturation.

B/PI-derived synthetic peptides: synergistic effects in tethered bactericidal and endotoxin neutralizing peptides.

Human neutrophil bactericidal protein (B/PI) is known for its ability to kill bacteria and to neutralize the action of endotoxin. Short linear peptides derived from residues 80-109 have been synthesized and their bactericidal and endotoxin neutralizing activities have been assayed. A series of 'walk-through' decapeptides, overlapping 3 to 4 residues, indicates that endotoxin neutralizing and partial bactericidal activities can be localized within the N- and C-terminal portions, respectively, of the 80-109 sequence. Bactericidal activity toward Pseudomonas aeruginosa was localized in central peptides of the walk-through series and greatest in peptide 90-99. By using longer peptides, residues 86-104 and 82-108, both bactericidal and endotoxin neutralizing activities are significantly enhanced. Bactericidal activity of peptide 82-108 is now only 6-fold less than that of parent B/PI and 9-fold more potent than peptide 86-104. The 82-108 peptide was 7-fold more active at endotoxin neutralization than 86-104 but showed less enhanced activity, being approx. 470-times less active than B/PI. Cyclized 82-108 peptide retained bactericidal activity but did not improve in capacity to neutralize endotoxin.

Designed beta-sheet-forming peptide 33mers with potent human bactericidal/permeability increasing protein-like bactericidal and endotoxin neutralizing activities.

Novel peptide 33mers have been designed by incorporating beta-conformation stabilizing residues from the beta-sheet domains of alpha-chemokines and functionally important residues from the beta-sheet domain of human neutrophil bactericidal protein (B/PI). B/PI is known for its ability to kill bacteria and to neutralize the action of bacterial endotoxin (lipopolysaccharide, LPS) which can induce septic shock leading to eventual death. Here, the goal was to make short linear peptides which demonstrate good beta-sheet folding and maintain bioactivity as in native B/PI. A library of 24 peptide 33mers (betapep-1 to betapep-24) were synthesized with various amino acid substitutions. CD and NMR data acquired in aqueous solution indicate that betapep peptides form beta-sheet structure to varying degrees and self-associate as dimers and tetramers like the alpha-chemokines. Bactericidal activity toward Gram-negative Pseudomonas aeruginosa was tested, and betapep-19 was found to be only about 5-fold less potent (62% kill at 1.2 x 10(-7) M) than native B/PI (80% kill at 2.9 x 10(-8) M). At LPS neutralization, betapep-2 and -23 were found to be most active (66-78% effective at 1.2 x 10(-6) M), being only about 50-100-fold less active than B/PI (50% at 1.5 x 10(-8) M). In terms of structure-activity relations, beta-sheet structural stability correlates with the capacity to neutralize LPS, but not with bactericidal activity. Although a net positive charge is necessary for activity, it is not sufficient for optimal activity. Hydrophobic residues tend to influence activities indirectly by affecting structural stability. Furthermore, results show that sequentially and spatially related residues from the beta-sheet domain of native B/PI can be designed into short linear peptides which show good beta-sheet folding and retain much of the native activity. This research contributes to the development of solutions to the problem of multiple drug-resistant, opportunistic microorganisms like P. aeruginosa and of agents effective at neutralizing bacterial endotoxin.

NOE-derived conformation of GRGDSP cell adhesion recognition site in the presence of SDS micelles and integrin receptor GPIIB/IIIA.

The tripeptide RGD is well known for its role in integrin receptor-mediated cell-cell surface adhesion. Here, NMR and transferred NOE studies have been done with the fibrinogen/fibronectin-derived hexapeptide GRGDSP in the presence of sodium dodecyl sulfate (SDS) and purified platelet glycoprotein integrin receptor GPIIb/IIIa. In the presence of SDS and absence of receptor, GRGDSP gives NOE-based distance geometry-generated structures characteristic of two "nested' beta-turns centered at RG and GD. In the presence of integrin GPIIb/IIIa, GRGDSP resonances are chemically shifted and broadened consistent with a dynamic equilibrium between free and receptor "bound' peptide. NOEs characteristic of the nested beta-turns are either absent or weaker indicating a significant conformational change in GRGDSP in the receptor bound state. GRGDSP appears to bind the receptor in a more extended backbone conformation which positions aspartic acid and arginine residues spatially close for potential electrostatic interactions.

Recommendations for cancer prevention trials using potentially ototoxic test agents.

PURPOSE: Preventive oncology applies pharmacologic agents to reverse, retard, or halt progression of neoplastic cells to invasive malignancy, a process that may require administration of agents over long periods of time. Although ototoxicity may be a tolerable side effect of anticancer or antimicrobial therapy, even modest ototoxicity may not be acceptable in agents developed for preventive oncology that are routinely administered to subjects who neither are, nor necessarily will become, clinically ill. MATERIALS AND METHODS: Age-related shifts in hearing may occur over the course of longterm or open-ended therapy; consequently, age-adjusted norms enable researchers to better distinguish hearing loss caused by drugs from that caused by aging. Norms for hearing sensitivity are derived from the Baltimore Longitudinal Study of Aging and are the basis for the proposed audiologic monitoring recommendations. RESULTS: Audiologic monitoring recommendations are presented that standardize patient selection, adverse event reporting, posttreatment follow-up, and audiologic testing for potentially ototoxic investigational agents. CONCLUSION: These recommendations are applicable to trials of investigational agents as well as various classes of drugs used in routine clinical care.

Three-dimensional organization of retroviral capsid proteins on a lipid monolayer.

We have used a method for the two-dimensional crystallization of retroviral structural proteins to obtain a three-dimensional structure of negatively stained, membrane-bound, histidine-tagged Moloney murine leukemia virus (M-MuLV) capsid protein (his-MoCA) arrays. Tilted and untilted micrographs from crystals formed by purified his-MoCA proteins incubated beneath lipid monolayers containing nickel-chelating lipids were used in 3D reconstructions. The 2D crystals had unit cell dimensions of a=72.6 A, b=72.5 A and gamma=119.5 degrees, but appeared to have no intrinsic symmetry (p1) in 3D, in contrast to the trigonal or hexagonal appearance of their 2D projections. Membrane-bound his-MoCA proteins showed a strand-like organization, apparently with dimer building blocks. Membrane-proximal regions, or putative N-terminal domains (NTDs), dimerized with different partners than the membrane-distal putative C-terminal domains (CTDs). Evidence also suggests that CTDs can adopt alternate orientations relative to their NTDs, forming interstrand connections. Our results are consistent with helical-spiral models for retrovirus particle assembly, but are not easily reconcilable with icosahedral models.

Further evidence for lack of gene expression in the Tetrahymena micronucleus.

Certain galA mutations in the ciliated protozoan Tetrahymena thermophila confer an almost total loss of galactokinase activity in homozygotes. Heterokaryons have been constructed that are homogeneous for the galA1 mutation in the (45n) macronucleus, but which contain a galA+ (2n) micronucleus. Soluble cell extracts prepared from these heterokaryons have been assayed for galactokinase activity, using a radiometric assay for the conversion of galactose to galactose-1-phosphate (gal-1-P). No galactokinase activity attributable to the micronuclear genes is observed in such heterokaryons. These results, obtained with the galA1 marker, provide the first direct, quantitative evidence for the lack of micronuclear (germ line) gene expression in Tetrahymena during vegetative growth, and substantiate the predictions of previous phenotypic observations on heterokaryons and autoradiographic studies of micronuclear RNA synthesis. The generality of this conclusion will be established in the future when other enzymically assayable mutations become available for similar studies.

Inhibin and activin Molecular aspects of regulation and function.

Inhibins and activins exhibit a broad spectrum of biologic activities, many of which affect the reproductive axis. Within the ovary and testis, the synthesis of the inhibin and activin alpha and beta subunits is regulated by circulating hormones, such as the pituitary gonadotropins, and by diverse paracrine factors. Considerable progress has been made in establishing patterns of inhibin and activin subunit gene expression in the gonads and in cultured gonadal cells. Analysis of the inhibin and activin subunit genes is now providing insight into the signaling pathways and molecular mechanisms by which inhibin and activin subunit gene expression is modulated in the ovary and testis. Genetic manipulation of the inhibin and activin subunit genes promises additional revelations on the biologic functions of these intriguing hormones.

Molecular cloning and expression of a pituitary-specific receptor for growth hormone-releasing hormone.

A novel cDNA was isolated from rat pituitary mRNA using the polymerase chain reaction to amplify sequences encoding G protein-coupled receptors. The human homolog of this cDNA was isolated and expressed in human kidney 293 cells, and membrane fractions from these cells were found to bind human GH-releasing hormone (GHRH) with high affinity and specificity. GHRH also stimulates intracellular cAMP production in these transfected cells. The encoded receptor protein contains seven potential membrane-spanning domains, a hallmark of G protein-coupled receptors, and is homologous to previously identified receptors for secretin and vasoactive intestinal peptide, ligands that are related to GHRH. The rat GHRH receptor mRNA is expressed predominantly, if not exclusively, in the anterior pituitary gland, the major target for GHRH action. These results define a mechanism for cellular signaling by GHRH and provide the opportunity to examine the role of the GHRH receptor in growth abnormalities that involve the GH axis.

Transient expression of progesterone receptor messenger RNA in ovarian granulosa cells after the preovulatory luteinizing hormone surge.

The ovarian steroid progesterone affects reproductive physiology by regulating the expression of specific genes in target tissues. In an attempt to address the question of whether the ovary itself is a target tissue for progesterone action, we have examined the localization and regulation of progesterone receptor (PR) mRNA in the rat ovary. We used the polymerase chain reaction to clone the steroid-binding domain of the rat PR from uterine cDNA and used this as a probe to isolate a larger cDNA from a rat placental cDNA library. We used RNA filter hybridization, a quantitative reverse transcription-polymerase chain reaction amplification assay, and in situ hybridization to detect PR mRNA in the rat ovary. Expression of the PR gene was initially studied in an immature animal model; 23-day-old rats were treated with either PMSG or PMSG followed by hCG. We found little or no PR mRNA in the ovaries of control or PMSG-treated animals; however, the mRNA was highly expressed in the granulosa cells of large follicles in the ovaries of animals treated with PMSG followed by hCG. Other cell types, including thecal and interstitial cells, did not express detectable levels of PR mRNA. The PR mRNA was induced more than 20-fold in the immature ovary 5 h after hCG administration and was down-regulated to near-basal levels by 12 h after hCG administration. In a subsequent series of experiments, we examined PR gene expression in adult rats during the estrous cycle. The expression of PR mRNA was transient and was tightly coupled to the preovulatory LH surge on proestrous evening. PR mRNA was localized to the granulosa cells of mature ovarian follicles during the estrous cycle. In cycling animals treated with pentobarbital to block the preovulatory LH surge, no induction of PR mRNA on proestrous evening was observed. This transient, hormonally regulated, and cell-specific expression of the PR gene in the rat ovary strongly suggests an important intraovarian function for progesterone during the rat reproductive cycle.

Identification of binding domains of the growth hormone-releasing hormone receptor by analysis of mutant and chimeric receptor proteins.

The hypothalamic peptide GH-releasing hormone (GHRH) stimulates the release of GH from the pituitary through binding and activation of the GHRH receptor, which belongs to the family of G protein-coupled receptors. The objective of this study was to identify regions of the receptor critical for interaction with the ligand by expressing and analyzing truncated and chimeric epitope-tagged GHRH receptors. Two truncated receptors, GHRHdeltaN, in which part of the N-terminal domain between the putative signal sequence and the first transmembrane domain was deleted, and GHRHdeltaC, which was truncated downstream of the first intracellular loop, were generated. Both the receptors were deficient in ligand binding, indicating that neither the N-terminal extracellular domain (N terminus) nor the membrane-spanning domains with the associated extracellular loops (C terminus) are alone sufficient for interaction with GHRH. In subsequent studies, chimeric proteins between the receptors for GHRH and vasoactive intestinal peptide (VIP) or secretin were generated, using the predicted start of the first transmembrane domain as the junction for the exchange of the N terminus between receptors. The chimeras having the N terminus of the GHRH receptor and the C terminus of either the VIP or secretin receptor (GNVC and GNSC) did not bind GHRH or activate adenylate cyclase after GHRH treatment. The reciprocal chimeras having the N terminus of either the VIP or secretin receptors and the C terminus of the GHRH receptor (VNGC and SNGC) bound GHRH and stimulated cAMP accumulation after GHRH treatment. These results suggest that although the N-terminal extracellular domain is essential for ligand binding, the transmembrane domains and associated extracellular loop regions of the GHRH receptor provide critical information necessary for specific interaction with GHRH.