Detection of cortical activation during averaged single trials of a cognitive task using functional magnetic resonance imaging

Functional neuroimaging studies in human subjects using positron emission tomography or functional magnetic resonance imaging (fMRI) are typically conducted by collecting data over extended time periods that contain many similar trials of a task. Here methods for acquiring fMRI data from single trials of a cognitive task are reported. In experiment one, whole brain fMRI was used to reliably detect single-trial responses in a prefrontal region within single subjects. In experiment two, higher temporal sampling of a more limited spatial field was used to measure temporal offsets between regions. Activation maps produced solely from the single-trial data were comparable to those produced from blocked runs. These findings suggest that single-trial paradigms will be able to exploit the high temporal resolution of fMRI. Such paradigms will provide experimental flexibility and time-resolved data for individual brain regions on a trial-by-trial basis.

A novel modifier gene for plasma von Willebrand factor level maps to distal mouse chromosome 11

Type 1 von Willebrand disease (VWD), characterized by reduced levels of plasma von Willebrand factor (VWF), is the most common inherited bleeding disorder in humans. Penetrance of VWD is incomplete, and expression of the bleeding phenotype is highly variable. In addition, plasma VWF levels vary widely among normal individuals. To identify genes that influence VWF level, we analyzed a genetic cross between RIIIS/J and CASA/Rk, two strains of mice that exhibit a 20-fold difference in plasma VWF level. DNA samples from F2 progeny demonstrating either extremely high or extremely low plasma VWF levels were pooled and genotyped for 41 markers spanning the autosomal genome. A novel locus accounting for 63% of the total variance in VWF level was mapped to distal mouse chromosome 11, which is distinct from the murine Vwf locus on chromosome 6. We designated this locus Mvwf for “modifier of VWF.” Additional genotyping of as many as 2407 meioses established a high resolution genetic map with gene order Cola1-Itg3a-Ngfr-Mvwf/Gip-Hoxb9-Hoxb1-Cbx·rs2-Cox5a-Gfap. The Mvwf candidate interval between Ngfr and Hoxb9 is ≈0.5 centimorgan (cM). These results demonstrate that a single dominant gene accounts for the low VWF phenotype of RIIIS/J mice in crosses with several other strains. The pattern of inheritance suggests a gain-of-function mutation in a unique component of VWF biosynthesis or processing. Characterization of the human homologue for Mvwf may have relevance for a subset of type 1 VWD cases and may define an important genetic factor modifying penetrance and expression of mutations at the VWF locus.

Consequences of placing an intramolecular crosslink in myosin S1

This paper describes the placement of a crosslinking agent (dibromobimane) between two thiols (Cys-522 and Cys-707) of a fragment, “S1,” of the motor protein, myosin. It turns out that fastening the first anchor of the crosslinker is easy and rapid, but fastening the second anchor (Cys-522) is very temperature dependent, taking 30 min at room temperature but about a week on ice. Moreover, crystallography taken at 4°C would seem to predict that the linkage is impossible, because the span of the crosslinking agent is much less than the interthiol distance. The simplest resolution of this seeming paradox is that structural fluctuations of the protein render the linkage increasingly likely as the temperature increases. Also, measurements of the affinity of MgADP for the protein, as well as the magnetic resonance of the P-atoms of the ADP once emplaced, suggest that binding the first reagent anchor to Cys-707 initiates an influence that travels to the rather distant ADP-binding site, and it is speculated what this “path of influence” might be.

RANK is the intrinsic hematopoietic cell surface receptor that controls osteoclastogenesis and regulation of bone mass and calcium metabolism

We have generated RANK (receptor activator of NF-κB) nullizygous mice to determine the molecular genetic interactions between osteoprotegerin, osteoprotegerin ligand, and RANK during bone resorption and remodeling processes. RANK−/− mice lack osteoclasts and have a profound defect in bone resorption and remodeling and in the development of the cartilaginous growth plates of endochondral bone. The osteopetrosis observed in these mice can be reversed by transplantation of bone marrow from rag1−/− (recombinase activating gene 1) mice, indicating that RANK−/− mice have an intrinsic defect in osteoclast function. Calciotropic hormones and proresorptive cytokines that are known to induce bone resorption in mice and human were administered to RANK−/− mice without inducing hypercalcemia, although tumor necrosis factor α treatment leads to the rare appearance of osteoclast-like cells near the site of injection. Osteoclastogenesis can be initiated in RANK−/− mice by transfer of the RANK cDNA back into hematopoietic precursors, suggesting a means to critically evaluate RANK structural features required for bone resorption. Together these data indicate that RANK is the intrinsic cell surface determinant that mediates osteoprotegerin ligand effects on bone resorption and remodeling as well as the physiological and pathological effects of calciotropic hormones and proresorptive cytokines.

Modulation of the baboon (Papio anubis) uterine endometrium by chorionic gonadotrophin during the period of uterine receptivity

This study was undertaken to determine the modulation of uterine function by chorionic gonadotrophin (CG) in a nonhuman primate. Infusion of recombinant human CG (hCG) between days 6 and 10 post ovulation initiated the endoreplication of the uterine surface epithelium to form distinct epithelial plaques. These plaque cells stained intensely for cytokeratin and the proliferating cell nuclear antigen. The stromal fibroblasts below the epithelial plaques stained positively for α-smooth muscle actin (αSMA). Expression of αSMA is associated with the initiation of decidualization in the baboon endometrium. Synthesis of the glandular secretory protein glycodelin, as assessed by Western blot analysis, was markedly up-regulated by hCG, and this increase was confirmed by immunocytochemistry, Northern blot analysis, and reverse transcriptase-PCR. To determine whether hCG directly modulated these uterine responses, we treated ovariectomized baboons sequentially with estradiol and progesterone to mimic the hormonal profile of the normal menstrual cycle. Infusion of hCG into the oviduct of steroid-hormone-treated ovariectomized baboons induced the expression of αSMA in the stromal cells and glycodelin in the glandular epithelium. The epithelial plaque reaction, however, was not readily evident. These studies demonstrate a physiological effect of CG on the uterine endometrium in vivo and suggest that the primate blastocyst signal, like the blastocyst signals of other species, modulates the uterine environment prior to implantation.

Dissociation of response conflict, attentional selection, and expectancy with functional magnetic resonance imaging

Two different attentional networks have been associated with visuospatial attention and conflict resolution. In most situations either one of the two networks is active or both are increased in activity together. By using functional magnetic resonance imaging and a flanker task, we show conditions in which one network (anterior attention system) is increased in activity whereas the other (visuospatial attention system) is reduced, showing that attentional conflict and selection are separate aspects of attention. Further, we distinguish between neural systems involved in different forms of conflict. Specifically, we dissociate patterns of activity in the basal ganglia and insula cortex during simple violations in expectancies (i.e., sudden changes in the frequency of an event) from patterns of activity in the anterior attention system specifically correlated with response conflict as evidenced by longer response latencies and more errors. These data provide a systems-level approach in understanding integrated attentional networks.

Identification of the vitamin K-dependent carboxylase active site: Cys-99 and Cys-450 are required for both epoxidation and carboxylation

The vitamin K-dependent carboxylase modifies and renders active vitamin K-dependent proteins involved in hemostasis, cell growth control, and calcium homeostasis. Using a novel mechanism, the carboxylase transduces the free energy of vitamin K hydroquinone (KH2) oxygenation to convert glutamate into a carbanion intermediate, which subsequently attacks CO2, generating the γ-carboxylated glutamate product. How the carboxylase effects this conversion is poorly understood because the active site has not been identified. Dowd and colleagues [Dowd, P., Hershline, R., Ham, S. W. & Naganathan, S. (1995) Science 269, 1684–1691] have proposed that a weak base (cysteine) produces a strong base (oxygenated KH2) capable of generating the carbanion. To define the active site and test this model, we identified the amino acids that participate in these reactions. N-ethyl maleimide inhibited epoxidation and carboxylation, and both activities were equally protected by KH2 preincubation. Amino acid analysis of 14C- N-ethyl maleimide-modified human carboxylase revealed 1.8–2.3 reactive residues and a specific activity of 7 × 108 cpm/hr per mg. Tryptic digestion and liquid chromatography electrospray mass spectrometry identified Cys-99 and Cys-450 as active site residues. Mutation to serine reduced both epoxidation and carboxylation, to 0.2% (Cys-99) or 1% (Cys-450), and increased the Kms for a glutamyl substrate 6- to 8-fold. Retention of some activity indicates a mechanism for enhancing cysteine/serine nucleophilicity, a property shared by many active site thiol enzymes. These studies, which represent a breakthrough in defining the carboxylase active site, suggest a revised model in which the glutamyl substrate indirectly coordinates at least one thiol, forming a catalytic complex that ionizes a thiol to initiate KH2 oxygenation.

Molecular phylogenetic analysis of evolutionary trends in stonefly wing structure and locomotor behavior

Insects in the order Plecoptera (stoneflies) use a form of two-dimensional aerodynamic locomotion called surface skimming to move across water surfaces. Because their weight is supported by water, skimmers can achieve effective aerodynamic locomotion even with small wings and weak flight muscles. These mechanical features stimulated the hypothesis that surface skimming may have been an intermediate stage in the evolution of insect flight, which has perhaps been retained in certain modern stoneflies. Here we present a phylogeny of Plecoptera based on nucleotide sequence data from the small subunit rRNA (18S) gene. By mapping locomotor behavior and wing structural data onto the phylogeny, we distinguish between the competing hypotheses that skimming is a retained ancestral trait or, alternatively, a relatively recent loss of flight. Our results show that basal stoneflies are surface skimmers, and that various forms of surface skimming are distributed widely across the plecopteran phylogeny. Stonefly wings show evolutionary trends in the number of cross veins and the thickness of the cuticle of the longitudinal veins that are consistent with elaboration and diversification of flight-related traits. These data support the hypothesis that the first stoneflies were surface skimmers, and that wing structures important for aerial flight have become elaborated and more diverse during the radiation of modern stoneflies.

A genome-wide search for chromosomal loci linked to mental health wellness in relatives at high risk for bipolar affective disorder among the Old Order Amish

Bipolar affective disorder (BPAD; manic-depressive illness) is characterized by episodes of mania and/or hypomania interspersed with periods of depression. Compelling evidence supports a significant genetic component in the susceptibility to develop BPAD. To date, however, linkage studies have attempted only to identify chromosomal loci that cause or increase the risk of developing BPAD. To determine whether there could be protective alleles that prevent or reduce the risk of developing BPAD, similar to what is observed in other genetic disorders, we used mental health wellness (absence of any psychiatric disorder) as the phenotype in our genome-wide linkage scan of several large multigeneration Old Order Amish pedigrees exhibiting an extremely high incidence of BPAD. We have found strong evidence for a locus on chromosome 4p at D4S2949 (maximum genehunter-plus nonparametric linkage score = 4.05, P = 5.22 × 10−4; sibpal Pempirical value <3 × 10−5) and suggestive evidence for a locus on chromosome 4q at D4S397 (maximum genehunter-plus nonparametric linkage score = 3.29, P = 2.57 × 10−3; sibpal Pempirical value <1 × 10−3) that are linked to mental health wellness. These findings are consistent with the hypothesis that certain alleles could prevent or modify the clinical manifestations of BPAD and perhaps other related affective disorders.

Metabolism of an insect diuretic hormone by Malpighian tubules studied by liquid chromatography coupled with electrospray ionization mass spectrometry

The larger of two diuretic hormones of the tobacco hornworm, Manduca sexta, (Mas-DH) is a peptide of 41 residues. It is one of a family of seven currently known insect diuretic hormones that are similar to the corticotropin-releasing factor–urotensin–sauvagine family of peptides. We investigated the possible inactivation of Mas-DH by incubating it in vitro with larval Malpighian tubules (Mt), the target organ of the hormone. The medium was analyzed, and degradation products were identified, using on-line microbore reversed-phase liquid chromatography coupled to electrospray ionization mass spectrometry (RPLC-ESI-MS). This sensitive technique allows identification of metabolites of Mas-DH (present at an initial level of ≈1 μM). An accurate Mr value for a metabolite is usually sufficient for unambiguous identification. Mas-DH is cleaved by Mt proteases initially at L29–R30 and R30–A31 under our assay conditions; some Mas-DH is also oxidized, apparently at M2 and M11. The proteolysis can be inhibited by 5 mM EDTA, suggesting that divalent metals are needed for peptide cleavage. The oxidation of the hormone can be inhibited by catalase or 1 mM methionine, indicating that H2O2 or related reactive oxygen species are responsible for the oxidative degradation observed. RPLC-ESI-MS is shown here to be an elegant and efficient method for studying peptide hormone metabolism resulting from unknown proteases and pathways.

Loss of HMW1 and HMW3 in noncytadhering mutants of Mycoplasma pneumoniae occurs post-translationally

The genomic sequence of Mycoplasma pneumoniae establish this cell-wall-less prokaryote as among the smallest known microorganisms capable of self-replication. However, this genomic simplicity and corresponding biosynthetic austerity are sharply contrasted by the complex terminal structure found in this species. This tip structure (attachment organelle) directs colonization of the human respiratory mucosa, leading to bronchitis and atypical pneumonia. Furthermore, formation of a second tip structure appears to precede cell division, implying temporal regulation. However, the organization, regulation, and assembly of the attachment organelle in M. pneumoniae are poorly understood, and no counterparts have been identified among the walled bacteria. M. pneumoniae possesses a cytoskeleton-like structure required to localize adhesin proteins to the attachment organelle. The cytadherence-associated proteins HMW1, HMW2, and HMW3 are components of the mycoplasma cytoskeleton, with HMW1 localizing strictly along the filamentous extensions from the cell body and HMW3 being a key structural element of the terminal organelle. Disruptions in hmw2 result in the loss of HMW1 and HMW3. However, the hmw1 and hmw3 genes were transcribed and translated at wild-type levels in hmw2 mutants. HMW1 and HMW3 were relatively stable in the wild-type background over 8 h but disappeared in the mutants over this time period. Evaluation of recombinant HMW1 levels in mycoplasma mutants suggested a requirement for the C-terminal domain of HMW1 for turnover. Finally, an apparent defect in the processing of the precursor for the adhesin protein P1 was noted in the HMW− mutants.

The HIV-1 regulatory proteins Tat and Rev are frequently targeted by cytotoxic T lymphocytes derived from HIV-1-infected individuals

The HIV-1 regulatory proteins Rev and Tat are expressed early in the virus life cycle and thus may be important targets for the immune control of HIV-1-infection and for effective vaccines. However, the extent to which these proteins are targeted in natural HIV-1 infection as well as precise epitopes targeted by human cytotoxic T lymphocytes (CTL) remain to be defined. In the present study, 57 HIV-1-infected individuals were screened for responses against Tat and Rev by using overlapping peptides spanning the entire Tat and Rev proteins. CD8+ T cell responses against Tat and Rev were found in up to 19 and 37% of HIV-1-infected individuals, respectively, indicating that these regulatory proteins are important targets for HIV-1-specific CTL. Despite the small size of these proteins, multiple CTL epitopes were identified in each. These data indicate that Tat and Rev are frequently targeted by CTL in natural HIV-1 infection and may be important targets for HIV vaccines.