A new determination of G using two methods.

We present the results of a measurement of G made with a torsion-strip balance used in two substantially independent ways. The two results agree to within their respective uncertainties; the correlation coefficient of the two methods is -0.18. The result is G = 6.675 59(27)x10(-11) m(3) kg(-1) s(-2) with a standard uncertainty of 4.1 parts in 10(5). Our result is 2 parts in 10(4) higher than the recent result of Gundlach and Merkowitz.

Chronic effects of a nonpeptide corticotropin-releasing hormone type I receptor antagonist on pituitary-adrenal function, body weight, and metabolic regulation.

CRH, the principal regulator of the hypothalamic-pituitary-adrenal axis and modulator of autonomic nervous system activity, also participates in the regulation of appetite and energy expenditure. Antalarmin, a pyrrolopyrimidine compound, antagonizes CRH type 1 receptor-mediated effects of CRH, including pituitary ACTH release, stress behaviors, and acute inflammation. We administered antalarmin chronically to evaluate its effects on hypothalamic-pituitary-adrenal axis function and metabolic status. Adult male rats were treated twice daily with 20 mg/kg of i.p. antalarmin or placebo over 11 days. The animals were weighed; plasma ACTH, corticosterone, leptin, and blood glucose levels were determined; and morphometric analyses were performed to determine adrenal size and structure, including sizing, histochemistry, immunohistochemistry, and electron microscopy. Leptin messenger RNA expression in peripheral fat was analyzed by Northern blot. Antalarmin decreased plasma ACTH (mean +/- SD, 2.62 +/- 0.063 pg/ml) and corticosterone concentrations (10.21 +/- 1.80 microg/dl) compared with those in vehicle-treated rats [respectively, 5.3 +/- 2.0 (P < 0.05) and 57.02 +/- 8.86 (P < 0.01)]. Antalarmin had no significant effect on body weight, plasma leptin, or blood glucose concentrations or fat cell leptin messenger RNA levels. The width of the adrenal cortex of animals treated with antalarmin was reduced by 31% compared with that in controls without atrophy of the gland. On the ultrastructural level, adrenocortical cells were in a hypofunctional state characterized by reduced vascularization, increased content of lipid droplets, and tubulovesicular mitochondria with fewer inner membranes. The apoptotic rate was increased in the outer zona fasciculata of animals treated with the antagonist (26.6 +/- 3.58%) compared with that in placebo-treated controls (6.8 +/- 0.91%). We conclude that chronic administration of antalarmin does not affect body weight, carbohydrate metabolism, or leptin expression, whereas it reduces adrenocortical function mildly, without anatomical, clinical, or biochemical evidence of causing adrenal atrophy. These results are promising for future uses of such an antagonist in the clinic.

Immunogenicity and efficacy trials in Aotus nancymai monkeys with model compounds representing parts of a 75-kD merozoite surface antigen of Plasmodium falciparum.

We tested the ability of a recombinant DNA-encoded fragment (C7Ag) of a Plasmodium falciparum merozoite protein (p75) and of two carrier-free peptide models (28-mer and 76-mer) to stimulate boostable antibody responses in Aotus nancymai monkeys. In addition, we evaluated protection against challenge with the Uganda Palo Alto (FUP) strain of this parasite. The data indicate that C7Ag elicited a strong and boostable IgG antibody response in all the monkeys immunized. However, studies with the peptide models demonstrated that various animals produce antibodies to different portions of this structure. When the post-boost sera from monkeys immunized with C7Ag were analyzed for reactivity against two major portions of C7Ag, most of the antibody response was observed against the disulfide-bonded 76-residue region that forms a conformational immunogenic epitope. In the same sera, antibody levels against the charged helical region modeled with a 28-mer were generally low. Immunization with synthetic peptides revealed that the 76-mer stimulated an antibody response almost as strong as C7Ag, with substantial cross-reactivity against the parasite antigen. The 28-mer evoked a response that was not efficient or uniform, and showed little reactivity with the authentic parasite antigen. Aotus nancymai was shown to be susceptible to infection with the Uganda Palo Alto strain of P. falciparum; however, maximum parasitemia varied markedly in both immunized and control monkeys. Statistical analysis failed to recognize differences in maximum parasitemia between the vaccine and control groups. The variation in maximum parasitemia suggests that the FUP strain in this species of Aotus is a poor model for the detection of differences in efficacy based on maximum parasitemia. This initial study with structures based on parts of the 75-kD merozoite surface antigen of P. falciparum indicated that both the recombinant-produced protein C7 and the 76-mer synthetic peptide, when combined with a Syntex adjuvant formulation, were safe and immunogenic in A. nancymai monkeys. However, the data emphasize the problems of using animal models to evaluate the potential effects of immunogens in humans.

Antibody responses stimulated in rabbits, guinea-pigs and mice by recombinant and synthetic portions of a 75 kDa malarial merozoite protein.

The 75 kDa heat-shock-related protein (p75) of Plasmodium falciparum is an abundant, highly conserved, merozoite surface protein. A bacterial clone, C7, produces a polypeptide (C7Ag) of approximately 30 kDa representing the C-terminal 40% of p75. In several species of animals, the C7Ag stimulated high titre IgG antibodies which cross-react with p75. Two major portions of the C7Ag, theoretically predicted to have strong secondary structural preferences, were modelled with four synthetic peptides. An alpha-helical, hydrophilic region, modelled with a 28-mer, proved a poor immunogen in guinea-pigs and several strains of inbred mice, even though it had been a strong immunogen in rabbits. A disulphide-bonded region of the C7Ag was modelled with three peptides of increasing length, namely 49-, 64- and 76-amino acid residues. In general, the order of immunogenicity was 49 less than 64 less than 76-mer. Antibodies to the 76-mer and the 64-mer reacted strongly with the native parasite protein. The data also suggested that the 76-mer was a good model for the region of the molecule it was made to represent.

Peptide mapping of conformational epitopes in a human malarial parasite heat shock protein.

A protein of 75 kDa is found in large quantities throughout the blood stages of the human malarial parasite, Plasmodium falciparum. Based on a partial amino acid sequence for p75, previously deduced from a cDNA clone encoding approximately 40% of the molecule, secondary structural predictions were made. The potential role of long range effects on the tertiary structure of the protein stabilized by disulfide bridges was determined by reduction and alkylation of the fusion protein. Five regions were then chosen for peptide modeling. Peptides of 16, 28, 49, 64, and 76 residues were synthesized and used to immunize rabbits. All but the 16-residue peptides were capable of stimulating boostable IgG antibody responses in rabbits, but the antibody produced against the 49 mer did not react with the native parasite protein. Thus, the 28, 64, and 76 residue peptides represent good immunologic models for portions of the P. falciparum 75-kDa protein capable of stimulating both T and B cells in rabbits. The peptides were also used to probe whether any of the selected regions contain epitopes which react with antibodies from owl monkeys immune to P. falciparum. Of these peptides, two were found to be consistently recognized in ELISA by four owl monkey antisera raised in response to malarial infection. Because these two peptides model a cysteine-containing region of the protein, owl monkey sera were also used as probes of the importance of disulfide bonding in maintaining the native structure. The results obtained were consistent with a folding pattern for p75 that incorporates a disulfide bond between cysteines 161 and 194. These results also suggest that most of the epitopes recognized in this part of p75 by the immune system of the monkey are created by folding of the molecule.

Immunologic modeling of a 75-kDa malarial protein with carrier-free synthetic peptides.

A protein of 75 kDa is produced in large quantities by the human malarial parasite Plasmodium falciparum and is present on the surface of the merozoite, whose function is to infect erythrocytes. Based on nucleotide sequence coding for 40% of this protein, two nonoverlapping model peptides 13 and 19 residues long were synthesized, coupled to a keyhole limpet hemocyanin carrier, and used to immunize rabbits. Although both antisera had high titers of anti-peptide antibodies, only that raised against the 13-residue peptide showed good reactivity against the original protein. Although the 19-mer adopted the helical secondary structure predicted for the corresponding protein region, antisera against this peptide reacted with the native protein weakly or not at all. Concluding that the poor anti-protein reactivity was due to modification of lysine-containing epitopes by glutaraldehyde conjugation, we used a carrier-free 28-residue peptide presented as a 56-residue disulfide-bonded dimer to model the same region. This peptide, in contrast to the conjugated 19-mer, stimulated the production of IgG antibodies that reacted at high dilution with the authentic protein in immunoblots, ELISA, and radioimmunoprecipitation assays. These data indicate that large carrier-free peptides may be successfully used as immunogens. In addition, our results show that this strategy may greatly improve the ability of conjugation-sensitive peptides to stimulate antibodies reactive with the original protein and therefore has substantial practical application.

A 75 kd merozoite surface protein of Plasmodium falciparum which is related to the 70 kd heat-shock proteins.

Proteins on the merozoite surface of the human malarial parasite Plasmodium falciparum are targets of the host's immune response. The merozoite surface location of p75, a 75 kd P. falciparum protein, was established by immunoelectron microscopy using antisera raised to the expressed product of a cDNA clone. Immunoprecipitation from protein extracts biosynthetically labeled during different periods of the asexual cycle showed that p75 is made continuously, although ring-stage parasites appear to synthesize larger quantities. p75 is conserved and invariant in size in eight isolates of P. falciparum. The 880 bp cDNA sequence encoding part of p75 reveals one open reading frame containing a repetitive sequence unit of four amino acids. The predicted reading frame is correct since antisera to a synthetic peptide corresponding to the repetitive region recognize p75 in immunoblots. The sequence of p75 is homologous with the sequences of proteins from the ubiquitous, highly conserved family of 70 kd heat-shock proteins, suggesting an important physiological function for p75. The cDNA fragment encoding part of p75 hybridizes with multiple genomic fragments, whose sizes are identical in DNA from nine P. falciparum strains, suggesting that the gene for p75 is well conserved and may be part of a gene family.

Synthesis and biological activity of linear and cyclic enkephalins modified at the Gly3-Phe4 amide bond.

As part of our continuing effort to define structure-activity relationships for enkephalin and design enzymatically resistant analogs, we report the synthesis and biological activities of linear and cyclic enkephalin analogs modified at the Gly3-Phe4 amide bond. The partial retro-inverso enkephalin analog Tyr-D-Ala-gGly-(R,S)-mPhe-Leu-NH2 and its cyclic counterpart, Tyr-cyclo[D-A2 bu-gGly-(R,S)-mPhe-Leu-], were synthesized as diastereomeric mixtures using solution methodology. The racemic benzylmalonate allowed the linear analog to be synthesized by fragment coupling at the reversed bond. Cyclization of the second analog was carried out at high concentration, eliminating formation of polymer by the use of an insoluble base. All gem-diaminoalkyl residues were prepared by conversion of peptidyl amides with benzene iodonium bis(trifluoroacetate). Diastereomers of both compounds were separable by reverse phase HPLC but those of the linear compound racemized rapidly under conditions of testing and were therefore tested together. All analogs tested had activities ranging from 6 to 14% of the activity of Leu enkephalin, indicating that the Gly3-Phe4 amide bond is important, though not crucial, for receptor binding.

Orality, impulsivity and cigarette smoking in men: further findings in support of a theory.

PIP: The hypothesis of this investigation is that heavy cigarette smoking in men reflects part of a pattern of underlying oral craving defended against by active, impulsive behavior. The element of selective cultural reinforcement is also considered. 2 personality and parental attitudes questionnaires were used with 134 males, average age 26. 76 were college students and 58 were industrial workers. Classification of these subjects into smoking categories revealed no differences in socioeconomic status (in the case of students) but resulted in significant differences in the age variable, since 58% of the college students were nonsmokers. 59% of this group were between age 18 and 20. Oldest subjects tended to be former smokers, and youngest tended to be nonsmokers. 2 homogeneous groups could be compared -- 1 consisting of 51 cigarette smokers and the other of 80 nonsmokers. On all measures of self-reports of personality traits, the differences are significant in the expected direction. For the concept of maternal over-control, coldness and harshness, the mean differences are nonsignificant. Smokers, regardless of intensity of habit, report that they are more defiant, impetuous, thrill-and-danger seeking, emotionally labile and preoccupied with oral concerns that are non and former smokers. Taking into account certain confounding influences, such as age differences and sex of interviewer assoicated with the method of data collection, the study replicates the findings of 2 previous investigations that a constellation of factors, termed "oral craving" coupled with an active, impulsive defensive structure is associated with heavy cigarette smoking in males. EVen when these confounding effects are ignored, 5 of the 6 variables hypothesized to be relevant were found to be associated with smoking behavior.^ieng