Presence and dynamics of leptin,GLP‐1, and PYY in human breast milk at early postpartum

Objective: The presence of appetite hormones, namely glucagon‐like peptide‐1 (GLP‐1), peptide YY (PYY), and leptin in breast milk may be important in infant feeding regulation and infant growth. This study evaluated whether concentrations of GLP‐1, PYY, and leptin change across a single feeding (from fore‐ to hindmilk), and are associated with maternal and infant anthropometrics. Design and Methods: Thirteen postpartum women (mean ± SD: 25.6 ± 4.5 years, 72.0 ± 11.9 kg) provided fore‐ and hindmilk samples 4‐5 weeks after delivery and underwent measurements of body weight and composition by Dual X‐ray Absorptiometry. GLP‐1, PYY, and leptin concentrations were measured using radioimmunoassay, and milk fat content was determined by creamatocrit. Results: Concentration of GLP‐1 and content of milk fat was higher in hindmilk than foremilk (P ≤ 0.05). PYY and leptin concentrations did not change between fore‐ and hindmilk. Both leptin concentration and milk fat content were correlated with indices of maternal adiposity, including body mass index (r = 0.65‐0.85, P < 0.02), and fat mass (r = 0.65‐0.84, P < 0.02). Hindmilk GLP‐1 was correlated with infant weight gain from birth to 6 months (r = ?0.67, P = 0.034). Conclusion: The presence of appetite hormones in breast milk may be important in infant appetite and growth regulation.     

Human mismatch repair and G*T mismatch binding by hMutSalpha in vitro is inhibited by adriamycin, actinomycin D, and nogalamycin

Loss of the human DNA mismatch repair pathway confers cross-resistance to structurally unrelated anticancer drugs. Examples include cisplatin, doxorubicin (adriamycin), and specific alkylating agents. We focused on defining the molecular events that link adriamycin to mismatch repair-dependent drug resistance because adriamycin, unlike drugs that covalently modify DNA, can interact reversibly with DNA. We found that adriamycin, nogalamycin, and actinomycin D comprise a class of drugs that reversibly inhibits human mismatch repair in vitro at low micromolar concentrations. The substrate DNA was not covalently modified by adriamycin treatment in a way that prevents repair, and the inhibition was independent of the number of intercalation sites separating the mismatch and the DNA nick used to direct repair, from 10 to 808 base pairs. Over the broad concentration range tested, there was no evidence for recognition of intercalated adriamycin by MutSalpha as if it were an insertion mismatch. Inhibition apparently results from the ability of the intercalated drug to prevent mismatch binding, shown using a defined mobility shift assay, which occurs at drug concentrations that inhibit repair. These data suggest that adriamycin interacts with the mismatch repair pathway through a mechanism distinct from the manner by which covalent DNA lesions are processed.     

Towards understanding the mechanisms of molecular recognition by computer simulations of ligand-protein interactions

The thermodynamic and kinetic aspects of molecular recognition for the methotrexate (MTX)-dihydrofolate reductase (DHFR) ligand-protein system are investigated by the binding energy landscape approach. The impact of 'hot' and 'cold' errors in ligand mutations on the thermodynamic stability of the native MTX-DHFR complex is analyzed, and relationships between the molecular recognition mechanism and the degree of ligand optimization are discussed. The nature and relative stability of intermediates and thermodynamic phases on the ligand-protein association pathway are studied, providing new insights into connections between protein folding and molecular recognition mechanisms, and cooperativity of ligand-protein binding. The results of kinetic docking simulations are rationalized based on the thermodynamic properties determined from equilibrium simulations and the shape of the underlying binding energy landscape. We show how evolutionary ligand selection for a receptor active site can produce well-optimized ligand-protein systems such as MTX-DHFR complex with the thermodynamically stable native structure and a direct transition mechanism of binding from unbound conformations to the unique native structure.     

Imaging the pharmacodynamics of HER2 degradation in response to Hsp90 inhibitors

The development of therapeutic inhibitors of key signaling pathways has been hampered by the inability to assess the effect of a drug on its target in the patient. 17-allylaminogeldanamycin (17-AAG) is the first Hsp90 inhibitor to be tested in a clinical trial. It causes the degradation of HER2 and other Hsp90 targets, and has antitumor activity in preclinical models. We have developed a method for imaging the inhibition of Hsp90 by 17-AAG. We labeled an F(ab')2 fragment of the anti-HER2 antibody Herceptin with 68Ga, a positron emitter, which allows the sequential positron-emission tomographic imaging of HER2 expression. We have used this method to quantify as a function of time the loss and recovery of HER2 induced by 17-AAG in animal tumors. This approach allows noninvasive imaging of the pharmacodynamics of a targeted drug and will facilitate the rational design of combination therapy based on target inhibition.     

Rapid glucocorticoid stimulation and GABAergic inhibition of hippocampal serotonergic response: in vivo dialysis in the lizard anolis carolinensis

Central serotonin (5-HT) is activated during stressful situations and aggressive interactions in a number of species. Glucocorticoids secreted peripherally during stressful events feed back on central systems and may affect 5-HT mediation of stress-induced behavioral events. To test the neuromodulatory effect of stress hormone secretion, serotonin overflow was measured from the hippocampus of the lizard Anolis carolinensis. Microdialysis was used to collect repeated samples from anesthetized lizards, with perfusate measured by HPLC with electrochemical analysis. Following initially high levels of 5-HT, concentrations stabilized to basal levels after approximately 2 h. Intracortical infusion of 200 ng/ml corticosterone evoked transient increases in 5-HT release of approximately 400%. The effect of corticosterone on 5-HT overflow appears to be dose dependent as 20 ng/ml stimulated an increase of 200%, whereas 2 ng/ml stimulated a 50% increase. Administration of 0.1 and 1 ng/ml GABA via the dialysis probe significantly inhibited 5-HT overflow by 20 and 40%, respectively. The duration of GABA inhibition is greater than the stimulatory response for glucocorticoids. Short-lived glucocorticoid stimulation of 5-HT release suggests a possible mechanism for endocrine mediation of continuously changing social behavioral events.     

Mode of reproduction and amplified fragment length polymorphism variation in purple needlegrass (Nassella pulchra): utilization of natural germplasm sources

A dominant plant of the California grasslands, purple needlegrass [Nassella pulchra (Hitchc.) Barkworth] is an important revegetation species in its native range. The amplified fragment length polymorphism (AFLP) method was used to elucidate mode of reproduction and nucleotide variation among 11 natural populations and three selected natural germplasm releases of N. pulchra. A total of 12 co-dominant AFLPs, informative within eight populations, failed to reveal any heterozygous individuals, indicating very high selfing rates (S(H)=1). Estimates of nucleotide diversity within populations ranged from 0 to 0.00069 (0.00035 average), whereas the total nucleotide divergence among populations ranged from 0.00107 to 0.00382 (0.00247 average). Measures of population differentiation (GS) in terms of Shannon-Weaver diversity values and estimated nucleotide substitutions were 0.90 and 0.86, respectively. Although some of the sample populations contained a mixture of true breeding genotypes, most populations could be distinguished unambiguously. Moreover, geographical distance between the natural source populations was significantly correlated with genetic distance (r = 0.60) among the corresponding sample populations. Results indicate that inbreeding, combined with founder effects and/or selection, has contributed to the differentiation of N. pulchra populations. Foundation seed populations of the selected natural germplasm releases were genetically well defined and most similar to natural seed collected near the corresponding source populations. Thus, these commercial germplasm sources will be made practically available and useful for conservation plantings within the intended areas of utilization.     

The crystallographic structure of brome mosaic virus

The structure of brome mosaic virus (BMV), the type member of the bromoviridae family, has been determined from a single rhombohedral crystal by X-ray diffraction, and refined to an R value of 0.237 for data in the range 3.4-40.0 A. The structure, which represents the native, compact form at pH 5.2 in the presence of 0.1 M Mg(2+), was solved by molecular replacement using the model of cowpea chlorotic mottle virus (CCMV), which BMV closely resembles. The BMV model contains amino acid residues 41-189 for the pentameric capsid A subunits, and residues 25-189 and 1-189 for the B and C subunits, respectively, which compose the hexameric capsomeres. In the model there are two Mg ions and one molecule of polyethylene glycol (PEG). The first 25 amino acid residues of the C subunit are modeled as polyalanine. The coat protein has the canonical "jellyroll" beta-barrel topology with extended amino-terminal polypeptides as seen in other icosahedral plant viruses. Mass spectrometry shows that in native BMV virions, a significant fraction of the amino-terminal peptides are apparently cleaved. No recognizable nucleic acid residue is visible in the electron density maps except at low resolution where it appears to exhibit a layered arrangement in the virion interior. It is juxtaposed closely with the interior surface of the capsid but does not interpenetrate. The protein subunits forming hexameric capsomeres, and particularly dimers, appear to interact extensively, but the subunits otherwise contact one another sparsely about the 5-fold and quasi 3-fold axes. Thus, the virion appears to be an assembly of loosely associated hexameric capsomeres, which may be the basis for the swelling and dissociation that occurs at neutral pH and elevated salt concentration. A Mg ion is observed to lie exactly on the quasi-3-fold axis and is closely coordinated by side-chains of three quasi-symmetry-related residues glutamates 84, with possible participation of side-chains from threonines 145, and asparagines 148. A presumptive Mg(2+) is also present on the 5-fold axis where there is a concentration of negatively charged side-chains, but the precise coordination is unclear. In both cases these cations appear to be essential for maintenance of virion stability. Density that is contiguous with the viral interior is present on the 3-fold axis at the center of the hexameric capsomere, where there is a pore of about 6 A diameter. The density cannot be attributed to cations and it was modeled as a PEG molecule.     

Control of germination and lipid mobilization by COMATOSE,the Arabidopsis homologue of human ALDP

Embryo dormancy in flowering plants is an important dispersal mechanism that promotes survival of the seed through time. The subsequent transition to germination is a critical control point regulating initiation of vegetative growth. Here we show that the Arabidopsis COMATOSE (CTS) locus is required for this transition, and acts, at least in part, by profoundly affecting the metabolism of stored lipids. CTS encodes a peroxisomal protein of the ATP binding cassette (ABC) transporter class with significant identity to the human X-linked adrenoleukodystrophy protein (ALDP). Like X-ALD patients, cts mutant embryos and seedlings exhibit pleiotropic phenotypes associated with perturbation in fatty acid metabolism. CTS expression transiently increases shortly after imbibition during germination, but not in imbibed dormant seeds, and genetic analyses show that CTS is negatively regulated by loci that promote embryo dormancy through multiple independent pathways. Our results demonstrate that CTS regulates transport of acyl CoAs into the peroxisome, and indicate that regulation of CTS function is a major control point for the switch between the opposing developmental programmes of dormancy and germination.     

The structure of tomato aspermy virus by X-ray crystallography

The three-dimensional structure of tomato aspermy virus (TAV) has been solved by X-ray crystallography and refined to an R factor of 0.218 for 3.4-40 A data (effective resolution of 4A). Molecular replacement, using cucumber mosaic virus (Smith et al., 2000), provided phases for the initial maps used for model building. The coat protein of the 280 A diameter virion has the canonical "Swiss roll" beta-barrel topology with a distinctive amino-terminal alpha-helix directed into the interior of the virus where it interacts with encapsidated RNA. The N-terminal helices are joined to the beta-barrels of protein subunits by extended polypeptides of six amino acids, which serve as flexible hinges allowing movement of the helices in response to local RNA distribution. Segments of three nucleotides of partially disordered RNA interact with the capsid, primarily through arginine residues, at interfaces between A and B subunits. Side chains of cys64 and cys106 form the first disulfide observed in a cucumovirus, including a unique cysteine, 106, in a region otherwise conserved. A positive ion, putatively modeled as a Mg(+)ion, lies on the quasi-threefold axis surrounded by three quasi-symmetric glutamate 175 side chains.