The natural killer gene complex genetic locus Chok encodes Ly-49D, a target recognition receptor that activates natural killing

Previously, we established that natural killer (NK) cells from C57BL/6 (B6), but not BALB/c, mice lysed Chinese hamster ovary (CHO) cells, and we mapped the locus that determines this differential CHO-killing capacity to the NK gene complex on chromosome 6. The localization of Chok in the NK gene complex suggested that it may encode either an activating or an inhibitory receptor. Here, results from a lectin-facilitated lysis assay predicted that Chok is an activating B6 NK receptor. Therefore, we immunized BALB/c mice with NK cells from BALB.B6–Cmv1r congenic mice and generated a mAb, designated 4E4, that blocked B6-mediated CHO lysis. mAb 4E4 also redirected lysis of Daudi targets, indicating its reactivity with an activating NK cell receptor. Furthermore, only the 4E4+ B6 NK cell subset mediated CHO killing, and this lysis was abrogated by preincubation with mAb 4E4. Flow cytometric analysis indicated that mAb 4E4 specifically reacts with Ly-49D but not Ly-49A, B, C, E, G, H, or I transfectants. Finally, gene transfer of Ly-49DB6 into BALB/c NK cells conferred cytotoxic capacity against CHO cells, thus establishing that the Ly-49D receptor is sufficient to activate NK cells to lyse this target. Hence, Ly-49D is the Chok gene product and is a mouse NK cell receptor capable of directly triggering natural killing.

A computationally directed screen identifying interacting coiled coils from Saccharomyces cerevisiae

Computational methods can frequently identify protein-interaction motifs in otherwise uncharacterized open reading frames. However, the identification of candidate ligands for these motifs (e.g., so that partnering can be determined experimentally in a directed manner) is often beyond the scope of current computational capabilities. One exception is provided by the coiled-coil interaction motif, which consists of two or more α helices that wrap around each other: the ligands for coiled-coil sequences are generally other coiled-coil sequences, thereby greatly simplifying the motif/ligand recognition problem. Here, we describe a two-step approach to identifying protein–protein interactions mediated by two-stranded coiled coils that occur in Saccharomyces cerevisiae. Coiled coils from the yeast genome are first predicted computationally, by using the multicoil program, and associations between coiled coils are then determined experimentally by using the yeast two-hybrid assay. We report 213 unique interactions between 162 putative coiled-coil sequences. We evaluate the resulting interactions, focusing on associations identified between components of the spindle pole body (the yeast centrosome).

The role of prefrontal cortex and posterior parietal cortex in task switching

Human ability to switch from one cognitive task to another involves both endogenous preparation without an external stimulus and exogenous adjustment in response to the external stimulus. In an event-related functional MRI study, participants performed pairs of two tasks that are either the same (task repetition) or different (task switch) from each other. On half of the trials, foreknowledge about task repetition or task switch was available. On the other half, it was not. Endogenous preparation seems to involve lateral prefrontal cortex (BA 46/45) and posterior parietal cortex (BA 40). During preparation, higher activation increases in inferior lateral prefrontal cortex and superior posterior parietal cortex were associated with foreknowledge than with no foreknowledge. Exogenous adjustment seems to involve superior prefrontal cortex (BA 8) and posterior parietal cortex (BA 39/40) in general. During a task switch with no foreknowledge, activations in these areas were relatively higher than during a task repetition with no foreknowledge. These results suggest that endogenous preparation and exogenous adjustment for a task switch may be independent processes involving different brain areas.

Nociception in cyclooxygenase isozyme-deficient mice

Prostaglandins formed by cyclooxygenase-1 (COX-1) or COX-2 produce hyperalgesia in sensory nerve endings. To assess the relative roles of the two enzymes in pain processing, we compared responses of COX-1- or COX-2-deficient homozygous and heterozygous mice with wild-type controls in the hot plate and stretching tests for analgesia. Preliminary observational studies determined that there were no differences in gross parameters of behavior between the different groups. Surprisingly, on the hot plate (55°C), the COX-1-deficient heterozygous groups showed less nociception, because mean reaction time was longer than that for controls. All other groups showed similar reaction times. In the stretching test, there was less nociception in COX-1-null and COX-1-deficient heterozygotes and also, unexpectedly, in female COX-2-deficient heterozygotes, as shown by a decreased number of writhes. Measurements of mRNA levels by reverse transcription–PCR demonstrated a compensatory increase of COX-1 mRNA in spinal cords of COX-2-null mice but no increase in COX-2 mRNA in spinal cords of COX-1-null animals. Thus, compensation for the absence of COX-1 may not involve increased expression of COX-2, whereas up-regulation of COX-1 in the spinal cord may compensate for the absence of COX-2. The longer reaction times on the hot plate of COX-1-deficient heterozygotes are difficult to explain, because nonsteroid anti-inflammatory drugs have no analgesic action in this test. Reduction in the number of writhes of the COX-1-null and COX-1-deficient heterozygotes may be due to low levels of COX-1 at the site of stimulation with acetic acid. Thus, prostaglandins made by COX-1 mainly are involved in pain transmission in the stretching test in both male and female mice, whereas those made by COX-2 also may play a role in the stretching response in female mice.

Anomalies in coral reef community metabolism and their potential importance in the reef CO2 source-sink debate

It is not certain whether coral reefs are sources of or sinks for atmospheric CO2. Air–sea exchange of CO2 over reefs has been measured directly and inferred from changes in the seawater carbonate equilibrium. Such measurements have provided conflicting results. We provide community metabolic data that indicate that large changes in CO2 concentration can occur in coral reef waters via biogeochemical processes not directly associated with photosynthesis, respiration, calcification, and CaCO3 dissolution. These processes can significantly distort estimates of reef calcification and net productivity and obscure the contribution of coral reefs to global air–sea exchange of CO2. They may, nonetheless, explain apparent anomalies in the metabolic performance of reefs close to land and reconcile the differing experimental findings that have given rise to the CO2 debate.

Altering the anaerobic transcription factor FNR confers a hemolytic phenotype on Escherichia coli K12

The recent outbreaks of Escherichia coli 0157-associated food poisoning have focused attention on the virulence determinants of E. coli. Here, it is reported that single base substitutions in the fnr gene encoding the oxygen-responsive transcription regulator FNR (fumarate and nitrate reduction regulator) are sufficient to confer a hemolytic phenotype on E. coli K12, the widely used laboratory strain. The mechanism involves enhancing the expression of a normally dormant hemolysin gene (hlyE) located in the E. coli chromosome. The mutations direct single amino acid substitutions in the activating regions (AR1 and AR3) of FNR that contact RNA polymerase. It is concluded that altering a resident transcription regulator, or acquisition of a competent heterologous regulator, could generate a pool of hemolytic, and therefore more virulent, strains of E. coli in nature.

The interphotoreceptor retinoid binding protein gene in therian mammals: Implications for higher level relationships and evidence for loss of function in the marsupial mole

The subclass Theria of Mammalia includes marsupials (infraclass Metatheria) and placentals (infraclass Eutheria). Within each group, interordinal relationships remain unclear. One limitation of many studies is incomplete ordinal representation. Here, we analyze DNA sequences for part of exon 1 of the interphotoreceptor retinoid binding protein gene, including 10 that are newly reported, for representatives of all therian orders. Among placentals, the most robust clades are Cetartiodactyla, Paenungulata, and an expanded African clade that includes paenungulates, tubulidentates, and macroscelideans. Anagalida, Archonta, Altungulata, Hyracoidea + Perissodactyla, Ungulata, and the “flying primate” hypothesis are rejected by statistical tests. Among marsupials, the most robust clade includes all orders except Didelphimorphia. The phylogenetic placement of the monito del monte and the marsupial mole remains unclear. However, the marsupial mole sequence contains three frameshift indels and numerous stop codons in all three reading frames. Given that the interphotoreceptor retinoid binding protein gene is a single-copy gene that functions in the visual cycle and that the marsupial mole is blind with degenerate eyes, this finding suggests that phenotypic degeneration of the eyes is accompanied by parallel changes at the molecular level as a result of relaxed selective constraints.

A novel multiple affinity purification tag and its use in identification of proteins associated with a cyclin–CDK complex

A novel multiple affinity purification (MAFT) or tandem affinity purification (TAP) tag has been constructed. It consists of the calmodulin binding peptide, six histidine residues, and three copies of the hemagglutinin epitope. This ‘CHH’ MAFT tag allows two or three consecutive purification steps, giving high purity. Active Clb2–Cdc28 kinase complex was purified from yeast cells after inserting the CHH tag into Clb2. Associated proteins were identified using mass spectrometry. These included the known associated proteins Cdc28, Sic1 and Cks1. Several other proteins were found including the 70 kDa chaperone, Ssa1.

Mixed spermatogenic germ cell nuclear extracts exhibit high base excision repair activity

Spermatogenic cells exhibit a lower spontaneous mutation frequency than somatic tissues in a lacI transgene and many base excision repair (BER) genes display the highest observed level of expression in the testis. In this study, uracil-DNA glycosylase-initiated BER activity was measured in nuclear extracts prepared from tissues obtained from each of three mouse strains. Extracts from mixed spermatogenic germ cells displayed the greatest activity followed by liver then brain for all three strains, and the activity for a given tissue was consistent among the three strains. Levels of various BER proteins were examined by western blot analyses and found to be consistent with activity levels. Nuclear extracts prepared from purified Sertoli cells, a somatic component of the seminiferous epithelium, exhibited significantly lower activity than mixed spermatogenic cell-type nuclear extracts, thereby suggesting that the high BER activity observed in mixed germ cell nuclear extracts was not a characteristic of all testicular cell types. Nuclear extracts from thymocytes and small intestines were assayed to assess activity in a mitotically active cell type and tissue. Overall, the order of tissues/cells exhibiting the greatest to lowest activity was mixed germ cells > Sertoli cells > thymocytes > small intestine > liver > brain.

Arp2/3 complex and actin dynamics are required for actin-based mitochondrial motility in yeast

The Arp2/3 complex is implicated in actin polymerization-driven movement of Listeria monocytogenes. Here, we find that Arp2p and Arc15p, two subunits of this complex, show tight, actin-independent association with isolated yeast mitochondria. Arp2p colocalizes with mitochondria. Consistent with this result, we detect Arp2p-dependent formation of actin clouds around mitochondria in intact yeast. Cells bearing mutations in ARP2 or ARC15 genes show decreased velocities of mitochondrial movement, loss of all directed movement and defects in mitochondrial morphology. Finally, we observe a decrease in the velocity and extent of mitochondrial movement in yeast in which actin dynamics are reduced but actin cytoskeletal structure is intact. These results support the idea that the movement of mitochondria in yeast is actin polymerization driven and that this movement requires Arp2/3 complex.

Genome-wide expression analysis reveals dysregulation of myelination-related genes in chronic schizophrenia

Neuropathological and brain imaging studies suggest that schizophrenia may result from neurodevelopmental defects. Cytoarchitectural studies indicate cellular abnormalities suggestive of a disruption in neuronal connectivity in schizophrenia, particularly in the dorsolateral prefrontal cortex. Yet, the molecular mechanisms underlying these findings remain unclear. To identify molecular substrates associated with schizophrenia, DNA microarray analysis was used to assay gene expression levels in postmortem dorsolateral prefrontal cortex of schizophrenic and control patients. Genes determined to have altered expression levels in schizophrenics relative to controls are involved in a number of biological processes, including synaptic plasticity, neuronal development, neurotransmission, and signal transduction. Most notable was the differential expression of myelination-related genes suggesting a disruption in oligodendrocyte function in schizophrenia.

Roles of a Fimbrin and an α-Actinin-like Protein in Fission Yeast Cell Polarization and Cytokinesis

Eukaryotic cells contain many actin-interacting proteins, including the α-actinins and the fimbrins, both of which have actin cross-linking activity in vitro. We report here the identification and characterization of both an α-actinin-like protein (Ain1p) and a fimbrin (Fim1p) in the fission yeast Schizosaccharomyces pombe. Ain1p localizes to the actomyosin-containing medial ring in an F-actin–dependent manner, and the Ain1p ring contracts during cytokinesis. ain1 deletion cells have no obvious defects under normal growth conditions but display severe cytokinesis defects, associated with defects in medial-ring and septum formation, under certain stress conditions. Overexpression of Ain1p also causes cytokinesis defects, and the ain1 deletion shows synthetic effects with other mutations known to affect medial-ring positioning and/or organization. Fim1p localizes both to the cortical actin patches and to the medial ring in an F-actin–dependent manner, and several lines of evidence suggest that Fim1p is involved in polarization of the actin cytoskeleton. Although a fim1 deletion strain has no detectable defect in cytokinesis, overexpression of Fim1p causes a lethal cytokinesis defect associated with a failure to form the medial ring and concentrate actin patches at the cell middle. Moreover, an ain1 fim1 double mutant has a synthetical-lethal defect in medial-ring assembly and cell division. Thus, Ain1p and Fim1p appear to have an overlapping and essential function in fission yeast cytokinesis. In addition, protein-localization and mutant-phenotype data suggest that Fim1p, but not Ain1p, plays important roles in mating and in spore formation.