Metabolism of daidzein by intestinal bacteria from rhesus monkeys (Macaca mulatta)

PURPOSE: To identify the metabolites produced from an isoflavonoid, daidzein, by colonic bacteria of rhesus monkeys. METHODS: The metabolism of daidzein by the fecal bacteria of nine monkeys was investigated. Daidzein was incubated anaerobically with fecal bacteria, and the metabolites were analyzed by use of liquid chromatography and mass spectrometry. RESULTS: The fecal bacteria of all of the monkeys metabolized daidzein to various extents. Dihydrodaidzein was found in cultures of fecal bacteria from two monkeys; dihydrodaidzein and equol were found in cultures from four monkeys; dihydrodaidzein, equol, and an unknown metabolite (MW = 244) were found in cultures from one monkey; and dihydrodaidzein and the unknown metabolite were found in cultures from two monkeys. CONCLUSIONS: Similar to that in humans, variation was evident in the metabolism of isoflavonoids by fecal bacteria from rhesus monkeys. Some metabolites produced by fecal bacteria from monkeys were the same as those produced by fecal bacteria from humans.     

Quantitative analysis of tryptic protein mixtures using electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry

For the first time, quantitative analysis of tryptic protein mixtures, labeled with Quantification-Using-Enhanced-Signal-Tags (QUEST)-markers, were performed with electrospray ionization and a 9.4 T Fourier Transform Ion Cyclotron Resonance (FTICR) mass spectrometer. Coupling a High-Pressure Liquid Chromatography (HPLC) separation step prior to mass analysis resulted in an increased amount of identified labeled tryptic peptides. The range for the determined intensity ratios of two peptides in a labeled pair was large, but the obtained median intensity ratio correlated very well with the corresponding concentration ratio. This method can be used for observing protein dynamics in a specific cell type, tissue, or in body fluids.     

Pollinator attractiveness increases with distance from flowering orchids

Orchids are extraordinary among plants because many species are pollinated through sexual duplicity by producing flowers that mimic female insects to lure unsuspecting males. Previous work showed that sexual deception by the orchid Chiloglottis trapeziformis can have a negative impact on its wasp pollinator Neozeleboria cryptoides. We report that female wasps may be capable of mitigating the cost of the orchids' deception. Although male wasps quickly habituated to areas planted with unrewarding flower decoys, we found that the effectiveness of the chemical cue used by the wingless females to attract males increases with increasing distance from an orchid patch. The apparent specificity of the males' site-based avoidance strategy means that females emerging in areas occupied by flowering orchids could, potentially, leave the orchid colony by walking to increase their attractiveness.     

NO mediates downregulation of RBF after a prolonged reduction of renal perfusion pressure in SHR

The aim of the study was to investigate mechanisms underlying the downregulation of renal blood flow (RBF) after a prolonged reduction in renal perfusion pressure (RPP) in adult spontaneously hypertensive rats (SHR). We tested the effect on the RBF response of clamping plasma ANG II in sevoflurane-anesthetized SHR. We also tested the effect of general cyclooxygenase (COX) inhibition and inhibition of the inducible COX-2. Furthermore, we assessed the effect of clamping the nitric oxide (NO) system. A prolonged period (15 min) of reduced RPP induced a downregulation of RBF. This was unchanged after clamping of plasma ANG II concentrations, general COX inhibition, and specific inhibition of COX-2. In contrast, clamping the NO system diminished the ability of SHR to downregulate RBF to a lower level. The downregulation of RBF was not associated with a resetting of the lower limit of autoregulation in the control group, in the ANG II-clamped group, or the NO clamped group. However, general COX inhibition and specific COX-2 inhibition enabled downward resetting of the lower limit of autoregulation. In conclusion, in SHR the renin-angiotensin system does not appear to play a major role in the downregulation of RBF after prolonged reduction of RPP. This response appears to be mediated partly by the NO system. We hypothesize that, in SHR, lack of downward resetting of the lower limit of autoregulation in response to a prolonged lowering of RPP could be the result of increased COX-2-mediated production of vasoconstrictory prostaglandins.     

A model system for increased meiotic nondisjunction in older oocytes

For at least 5% of all clinically recognized human pregnancies, meiotic segregation errors give rise to zygotes with the wrong number of chromosomes. Although most aneuploid fetuses perish in utero, trisomy in liveborns is the leading cause of mental retardation. A large percentage of human trisomies originate from segregation errors during female meiosis I; such errors increase in frequency with maternal age. Despite the clinical importance of age-dependent nondisjunction in humans, the underlying mechanisms remain largely unexplained. Efforts to recapitulate age-dependent nondisjunction in a mammalian experimental system have so far been unsuccessful. Here we provide evidence that Drosophila is an excellent model organism for investigating how oocyte aging contributes to meiotic nondisjunction. As in human oocytes, nonexchange homologs and bivalents with a single distal crossover in Drosophila oocytes are most susceptible to spontaneous nondisjunction during meiosis I. We show that in a sensitized genetic background in which sister chromatid cohesion is compromised, nonrecombinant X chromosomes become vulnerable to meiotic nondisjunction as Drosophila oocytes age. Our data indicate that the backup pathway that normally ensures proper segregation of achiasmate chromosomes deteriorates as Drosophila oocytes age and provide an intriguing paradigm for certain classes of age-dependent meiotic nondisjunction in humans.     

The refined structure of a protein catenane: the HK97 bacteriophage capsid at 3.44 A resolution

The HK97 bacteriophage capsid is a unique example of macromolecular catenanes: interlocked rings of covalently attached protein subunits. The chain mail organization of the subunits stabilizes a particle in which the maximum thickness of the protein shell is 18A and the maximum diameter is 550A. The electron density has the appearance of a balloon illustrating the extraordinary strength conferred by the unique subunit organization. The refined structure shows novel qualities of the HK97 shell protein, gp5 that, together with the protease gp4, guides the assembly and maturation of the virion. Although gp5 forms hexamers and pentamers and the subunits exist in different structural environments, the tertiary structures of the seven protein molecules in the viral asymmetric unit are closely similar. The interactions of the subunits in the shell are exceptionally complex with each subunit interacting with nine other subunits. The interactions of the N-terminus released after gp5 cleavage appear important for organization of the loops that become crosslinked to the core of a neighboring subunit at the maturation. A comparison with a model of the Prohead II structure revealed that the surfaces of non-covalent contact between the monomers that build up hexamers/pentamers are completely redefined during maturation.     

TCR comodulation of nonengaged TCR takes place by a protein kinase C and CD3 gamma di-leucine-based motif-dependent mechanism

One of the earliest events following TCR triggering is TCR down-regulation. However, the mechanisms behind TCR down-regulation are still not fully known. Some studies have suggested that only directly triggered TCR are internalized, whereas others studies have indicated that, in addition to triggered receptors, nonengaged TCR are also internalized (comodulated). In this study, we used transfected T cells expressing two different TCR to analyze whether comodulation took place. We show that TCR triggering by anti-TCR mAb and peptide-MHC complexes clearly induced internalization of nonengaged TCR. By using a panel of mAb against the Ti beta chain, we demonstrate that the comodulation kinetics depended on the affinity of the ligand. Thus, high-affinity mAb (K(D) = 2.3 nM) induced a rapid but reversible comodulation, whereas low-affinity mAb (K(D) = 6200 nM) induced a slower but more permanent type of comodulation. Like internalization of engaged TCR, comodulation was dependent on protein tyrosine kinase activity. Finally, we found that in contrast to internalization of engaged TCR, comodulation was highly dependent on protein kinase C activity and the CD3 gamma di-leucine-based motif. Based on these observations, a physiological role of comodulation is proposed and the plausibility of the TCR serial triggering model is discussed.     

Generation of a recombinant cytomegalovirus for expression of a hantavirus glycoprotein

A cytomegalovirus (CMV) was isolated from its natural host, Peromyscus maniculatus, and was designated Peromyscus CMV (PCMV). A recombinant PCMV was constructed that contained Sin Nombre virus glycoprotein G1 (SNV-G1) fused in frame to the enhanced green fluorescent protein (EGFP) gene inserted into a site homologous to the human CMV UL33 (P33) gene. The recombinant CMV was used for expression and immunization of deer mice against SNV-G1. The results of the study indicate that P. maniculatus could be infected with as few as 10 virus particles of recombinant virus. Challenge of P. maniculatus with either recombinant or wild-type PCMV produced no overt pathology in infected animals. P. maniculatus immunized with recombinant virus developed an antibody response to SNV and EGFP. When rechallenged with recombinant virus, animals exhibited an anamnestic response against SNV. Interestingly, a preexisting immune response against PCMV did not prevent reinfection with recombinant PCMV.