Evolution of a minimal cell.

Possessing only essential genes, a minimal cell can reveal mechanisms and processes that are critical for the persistence and stability of life1,2. Here we report on how an engineered minimal cell3,4 contends with the forces of evolution compared with the Mycoplasma mycoides non-minimal cell from which it was synthetically derived. Mutation rates were the highest among all reported bacteria, but were not affected by genome minimization. Genome streamlining was costly, leading to a decrease in fitness of greater than 50%, but this deficit was regained during 2,000 generations of evolution. Despite selection acting on distinct genetic targets, increases in the maximum growth rate of the synthetic cells were comparable. Moreover, when performance was assessed by relative fitness, the minimal cell evolved 39% faster than the non-minimal cell. The only apparent constraint involved the evolution of cell size. The size of the non-minimal cell increased by 80%, whereas the minimal cell remained the same. This pattern reflected epistatic effects of mutations in ftsZ, which encodes a tubulin-homologue protein that regulates cell division and morphology5,6. Our findings demonstrate that natural selection can rapidly increase the fitness of one of the simplest autonomously growing organisms. Understanding how species with small genomes overcome evolutionary challenges provides critical insights into the persistence of host-associated endosymbionts, the stability of streamlined chassis for biotechnology and the targeted refinement of synthetically engineered cells2,7-9.

Adult Brain Serotonin Deficiency Causes Hyperactivity, Circadian Disruption, and Elimination of Siestas.

UNLABELLED: Serotonin (5-HT) is a crucial neuromodulator linked to many psychiatric disorders. However, after more than 60 years of study, its role in behavior remains poorly understood, in part because of a lack of methods to target 5-HT synthesis specifically in the adult brain. Here, we have developed a genetic approach that reproducibly achieves near-complete elimination of 5-HT synthesis from the adult ascending 5-HT system by stereotaxic injection of an adeno-associated virus expressing Cre recombinase (AAV-Cre) into the midbrain/pons of mice carrying a loxP-conditional tryptophan hydroxylase 2 (Tph2) allele. We investigated the behavioral effects of deficient brain 5-HT synthesis and discovered a unique composite phenotype. Surprisingly, adult 5-HT deficiency did not affect anxiety-like behavior, but resulted in a robust hyperactivity phenotype in novel and home cage environments. Moreover, loss of 5-HT led to an altered pattern of circadian behavior characterized by an advance in the onset and a delay in the offset of daily activity, thus revealing a requirement for adult 5-HT in the control of daily activity patterns. Notably, after normalizing for hyperactivity, we found that the normal prolonged break in nocturnal activity (siesta), a period of rapid eye movement (REM) and non-REM sleep, was absent in all animals in which 5-HT deficiency was verified. Our findings identify adult 5-HT as a requirement for siestas, implicate adult 5-HT in sleep-wake homeostasis, and highlight the importance of our adult-specific 5-HT-synthesis-targeting approach in understanding 5-HT's role in controlling behavior. SIGNIFICANCE STATEMENT: Serotonin (5-HT) is a crucial neuromodulator, yet its role in behavior remains poorly understood, in part because of a lack of methods to target specifically adult brain 5-HT synthesis. We developed an approach that reproducibly achieves near-complete elimination of 5-HT synthesis from the adult ascending 5-HT system. Using this technique, we discovered that adult 5-HT deficiency led to a novel compound phenotype consisting of hyperactivity, disrupted circadian behavior patterns, and elimination of siestas, a period of increased sleep during the active phase. These findings highlight the importance of our approach in understanding 5-HT's role in behavior, especially in controlling activity levels, circadian behavior, and sleep-wake homeostasis, behaviors that are disrupted in many psychiatric disorders such as attention deficit hyperactivity disorder.

Assessing vascular effects of adding bevacizumab to neoadjuvant chemotherapy in osteosarcoma using DCE-MRI.

BACKGROUND: The purpose of this study was to assess the impact of bevacizumab alone and in combination with cytotoxic therapy on tumour vasculature in osteosarcoma (OS) using DCE-MRI. METHODS: Six DCE-MRI and three (18)F-FDG PET examinations were scheduled in 42 subjects with newly diagnosed OS to monitor the response to antiangiogenic therapy alone and in combination with cytotoxic therapy before definitive surgery (week 10). Serial DCE-MRI parameters (K(trans), v(p), and v(e)) were examined for correlation with FDG-PET (SUV(max)) and association with drug exposure, and evaluated with clinical outcome. RESULTS: K(trans) (P=0.041) and v(p) (P=0.001) significantly dropped from baseline at 24 h after the first dose of bevacizumab alone, but returned to baseline by 72 h. Greater exposure to bevacizumab was correlated with larger decreases in v(p) at day 5 (P=0.04) and week 10 (P=0.02). A lower K(trans) at week 10 was associated with greater percent necrosis (P=0.024) and longer event-free survival (P=0.034). CONCLUSIONS: This is the first study to demonstrate significant changes of the plasma volume fraction and vascular leakage in OS with bevacizumab alone. The combination of demonstrated associations between drug exposure and imaging metrics, and imaging metrics and patient survival during neoadjuvant therapy, provides a compelling rationale for larger studies using DCE-MRI to assess vascular effects of therapy in OS.

The canonical twin-arginine translocase components are not required for secretion of folded green fluorescent protein from the ancestral strain of Bacillus subtilis.

Cellular processes, such as the digestion of macromolecules, phosphate acquisition, and cell motility, require bacterial secretion systems. In Bacillus subtilis, the predominant protein export pathways are Sec (generalized secretory pathway) and Tat (twin-arginine translocase). Unlike Sec, which secretes unfolded proteins, the Tat machinery secretes fully folded proteins across the plasma membrane and into the medium. Proteins are directed for Tat-dependent export by N-terminal signal peptides that contain a conserved twin-arginine motif. Thus, utilizing the Tat secretion system by fusing a Tat signal peptide is an attractive strategy for the production and export of heterologous proteins. As a proof of concept, we expressed green fluorescent protein (GFP) fused to the PhoD Tat signal peptide in the laboratory and ancestral strains of B. subtilis. Secretion of the Tat-GFP construct, as well as secretion of proteins in general, was substantially increased in the ancestral strain. Furthermore, our results show that secreted, fluorescent GFP could be purified directly from the extracellular medium. Nonetheless, export was not dependent on the known Tat secretion components or the signal peptide twin-arginine motif. We propose that the ancestral strain contains additional Tat components and/or secretion regulators that were abrogated following domestication.

The mammalian circadian clock in the suprachiasmatic nucleus exhibits rapid tolerance to ethanol in vivo and in vitro.

BACKGROUND: Ethanol (EtOH) triggers cellular adaptations that induce tolerance in many brain areas, including the suprachiasmatic nucleus (SCN), the site of the master circadian clock. EtOH inhibits light-induced phase shifts in the SCN in vivo and glutamate-induced phase shifts in vitro. The in vitro phase shifts develop acute tolerance to EtOH, occurring within minutes of initial exposure, while the in vivo phase shifts exhibit no evidence of chronic tolerance. An intermediate form, rapid tolerance, is not well studied but may predict subsequent chronic tolerance. Here, we investigated rapid tolerance in the SCN clock. METHODS: Adult C57BL/6 mice were provided 15% EtOH or water for one 12-hour lights-off period. For in vitro experiments, SCN-containing brain slices were prepared in the morning and treated for 10 minutes with glutamate +/- EtOH the following night. Single-cell neuronal firing rates were recorded extracellularly during the subsequent day to determine SCN clock phase. For in vivo experiments, mice receiving EtOH 24 hours previously were exposed to a 30-minute light pulse immediately preceded by intraperitoneal saline or 2 g/kg EtOH injection. Mice were then placed in constant darkness and their phase-shifting responses measured. RESULTS: In vitro, the SCN clock from EtOH-exposed mice exhibited rapid tolerance, with a 10-fold increase in EtOH needed to inhibit glutamate-induced phase shifts. Co-application of brain-derived neurotrophic factor prevented EtOH inhibition, consistent with experiments using EtOH-naïve mice. Rapid tolerance lasts 48 to 96 hours, depending on whether assessing in vitro phase advances or phase delays. Similarly, in vivo, prior EtOH consumption prevented EtOH's acute blockade of photic phase delays. Finally, immunoblot experiments showed no changes in SCN glutamate receptor subunit (NR2B) expression or phosphorylation in response to rapid tolerance induction. CONCLUSIONS: The SCN circadian clock develops rapid tolerance to EtOH as assessed both in vivo and in vitro, and the tolerance lasts for several days. These data demonstrate the utility of the circadian system as a model for investigating cellular mechanisms through which EtOH acts in the brain.

Selected Topics on the Synthesis, Properties and Applications of Multiwalled Carbon Nanotubes.

In summary, MWCNTs have been examined for a variety of electronic applications due to their unique structure and chemistry. Electrodes for field emission, energy and sensor applications hold particular interest. MWCNTs provide a very high surface area, relatively easy methods of surface modification, controllable and high concentration of reactive surface sites, and high specific capacitance. Combining MWCNTs with graphene structures, oxide and metal nanoparticles and certain polymers extends their performance and functionality. Such hybrid structures have been produced in situ during CNT growth and in two-step processes. Excellent progress on understanding the mechanisms of CNT growth has enabled numerous growth methods to all yield MWCNT structures in a variety of morphologies.

Indigenous peoples and local communities as partners in the sequencing of global eukaryotic biodiversity.

The aim to sequence, catalog, and characterize the genomes of all of Earth's eukaryotic biodiversity is the shared mission of many ongoing large-scale biodiversity genomics initiatives. Reference genomes of global flora and fauna have the potential to inform a broad range of major issues facing both biodiversity and humanity, such as the impact of climate change, the conservation of endangered species and ecosystems, public health crises, and the preservation and enhancement of ecosystem services. Biodiversity is dramatically declining: 28% of species being assessed by the IUCN are threatened with extinction, and recent reports suggest that a transformative change is needed to conserve and protect what remains. To provide a collective and global genomic response to the biodiversity crisis, many biodiversity genomics initiatives have come together, creating a network of networks under the Earth BioGenome Project. This network seeks to expedite the creation of an openly available, "public good" encyclopedia of high-quality eukaryotic reference genomes, in the hope that by advancing our basic understanding of nature, it can lead to the transformational scientific developments needed to conserve and protect global biodiversity. Key to completing this ambitious encyclopedia of reference genomes, is the ability to responsibly, ethically, legally, and equitably access and use samples from all of the eukaryotic species across the planet, including those that are under the custodianship of Indigenous Peoples and Local Communities. Here, the biodiversity genomics community is subject to the provisions codified in international, national, and local legislations and customary community norms, principles, and protocols. We propose a framework to support biodiversity genomic researchers, projects, and initiatives in building trustworthy and sustainable partnerships with communities, providing minimum recommendations on how to access, utilize, preserve, handle, share, analyze, and communicate samples, genomics data, and associated Traditional Knowledge obtained from, and in partnership with, Indigenous Peoples and Local Communities across the data-lifecycle.

Cocaine modulates pathways for photic and nonphotic entrainment of the mammalian SCN circadian clock.

Cocaine abuse is highly disruptive to circadian physiological and behavioral rhythms. The present study was undertaken to determine whether such effects are manifest through actions on critical photic and nonphotic regulatory pathways in the master circadian clock of the mouse suprachiasmatic nucleus (SCN). Impairment of SCN photic signaling by systemic (intraperitoneal) cocaine injection was evidenced by strong (60%) attenuation of light-induced phase-delay shifts of circadian locomotor activity during the early night. A nonphotic action of cocaine was apparent from its induction of 1-h circadian phase-advance shifts at midday. The serotonin receptor antagonist, metergoline, blocked shifting by 80%, implicating a serotonergic mechanism. Reverse microdialysis perfusion of the SCN with cocaine at midday induced 3.7 h phase-advance shifts. Control perfusions with lidocaine and artificial cerebrospinal fluid had little shifting effect. In complementary in vitro experiments, photic-like phase-delay shifts of the SCN circadian neuronal activity rhythm induced by glutamate application to the SCN were completely blocked by cocaine. Cocaine treatment of SCN slices alone at subjective midday, but not the subjective night, induced 3-h phase-advance shifts. Lidocaine had no shifting effect. Cocaine-induced phase shifts were completely blocked by metergoline, but not by the dopamine receptor antagonist, fluphenazine. Finally, pretreatment of SCN slices for 2 h with a low concentration of serotonin agonist (to block subsequent serotonergic phase resetting) abolished cocaine-induced phase shifts at subjective midday. These results reveal multiple effects of cocaine on adult circadian clock regulation that are registered within the SCN and involve enhanced serotonergic transmission.

Homozygous null mutations in ZMPSTE24 in restrictive dermopathy: evidence of genetic heterogeneity.

Restrictive dermopathy (RD) results in stillbirth or early neonatal death. RD is characterized by prematurity, intrauterine growth retardation, fixed facial expression, micrognathia, mouth in the 'o' position, rigid and tense skin with erosions and denudations and multiple joint contractures. Nearly all 25 previously reported neonates with RD had homozygous or compound heterozygous null mutations in the ZMPSTE24 gene. Here, we report three new cases of RD; all died within 3 weeks of birth. One of them had a previously reported homozygous c.1085dupT (p.Leu362PhefsX19) mutation, the second case had a novel homozygous c.1020G>A (p.Trp340X) null mutation in ZMPSTE24, but the third case, a stillborn with features of RD except for the presence of tapering rather than rounded, bulbous digits, harbored no disease-causing mutations in LMNA or ZMPSTE24. In the newborn with a novel ZMPSTE24 mutation, unique features included butterfly-shaped thoracic 5 vertebra and the bulbous appearance of the distal clavicles. Skin biopsies from both the stillborn fetus and the newborn with c.1020G>A ZMPSTE24 mutation showed absence of elastic fibers throughout the dermis. This report provides evidence of genetic heterogeneity among RD and concludes that there may be an additional locus for RD which remains to be identified.

Producer survey of bird-livestock interactions in commercial dairies.

The objective of this producer survey was to identify and estimate damage caused by bird-livestock interactions in commercial dairies. The interactions between birds and livestock have previously been implicated in causing economic damage while contributing to the environmental dissemination of microorganisms pathogenic to livestock and humans. Very little research exists to help producers understand what bird species use dairies, why they use dairies, or the scope and nature of damage created as a result of bird-livestock interactions. To better characterize these interactions, we surveyed dairy operators within Pennsylvania, New York, and Wisconsin. Survey results suggest that the most common and destructive bird species found on commercial dairies are invasive to North America, and their use of dairies is associated with the loss of cattle feed, increased operating costs, and an increase in dairies self-reporting Salmonella spp. and Mycobacterium avium ssp. paratuberculosis. Cattle feed loss estimates generated from this survey were used to parameterize an input-output (IO) economic model using data from 10 counties in the state of Pennsylvania (Bedford, Berks, Blair, Bradford, Chester, Cumberland, Franklin, Lancaster, Lebanon, and Somerset). This IO model allowed us to estimate direct, indirect, and induced economic effects of feed loss from bird damage to dairies within these counties. The IO model output suggests that feed loss costs Pennsylvania between $4.11 and $12.08 million (mean $10.6 million) in total economic damage, with approximately 43 to 128 jobs (mean 112) forgone statewide in 2009.

Fibromyalgia dyscognition: concepts and issues.

Fibromyalgia is characterized by widespread pain and tenderness; however, comorbid cognitive difficulties are a common complaint among patients. Known as fibro fog or dyscognition, this symptom comprises difficulties with complex cognitive processes including memory, executive function, concentration and attention. While the mechanisms that initiate and maintain these cognitive deficits are still largely unknown, recent research has increased the understanding of subjective symptoms and objectively-determined deficits in cognitive performance. Treatments have also improved to include complementary cognitive and physical strategies. This review focuses on issues of dyscognition in fibromyalgia. Details of objective testing methods are not within the scope of this paper.

Paternal Cocaine in Mice Alters Social Behavior and Brain Oxytocin Receptor Density in First Generation Offspring.

It is well established that the damaging effects of drugs of abuse, such as cocaine, can extend beyond the user to their offspring. While most preclinical models of the generational effects of cocaine abuse have focused on maternal effects, we, and others, report distinct effects on offspring sired by fathers treated with cocaine prior to breeding. However, little is known about the effects of paternal cocaine use on first generation (F1) offspring's social behaviors. Here, we expand upon our model of oral self-administered paternal cocaine use to address the idea that paternal cocaine alters first generation offspring social behaviors through modulation of the oxytocin system. F1 cocaine-sired males displayed unaltered social recognition vs. non-cocaine sired controls but showed increased investigation times that were not related to altered olfaction. Paternal cocaine did not alter F1 male-aggression behavior or depression-like behaviors, but cocaine-sired males did display decreased anxiety-like behaviors. Female F1 behavior was similarly examined, but there were no effects of paternal cocaine. Cocaine-sired male mice also exhibited localized oxytocin receptor expression differences vs. controls in several brain regions regulating social behavior. These results provide evidence for effects of paternal cocaine exposure on social behaviors in male offspring with associated alterations in central oxytocin transmission.

Cognitive Digital Biomarkers from Automated Transcription of Spoken Language.

BACKGROUND: Although patients with Alzheimer's disease and other cognitive-related neurodegenerative disorders may benefit from early detection, development of a reliable diagnostic test has remained elusive. The penetration of digital voice-recording technologies and multiple cognitive processes deployed when constructing spoken responses might offer an opportunity to predict cognitive status. OBJECTIVE: To determine whether cognitive status might be predicted from voice recordings of neuropsychological testing. DESIGN: Comparison of acoustic and (para)linguistic variables from low-quality automated transcriptions of neuropsychological testing (n = 200) versus variables from high-quality manual transcriptions (n = 127). We trained a logistic regression classifier to predict cognitive status, which was tested against actual diagnoses. SETTING: Observational cohort study. PARTICIPANTS: 146 participants in the Framingham Heart Study. MEASUREMENTS: Acoustic and either paralinguistic variables (e.g., speaking time) from automated transcriptions or linguistic variables (e.g., phrase complexity) from manual transcriptions. RESULTS: Models based on demographic features alone were not robust (area under the receiver-operator characteristic curve [AUROC] 0.60). Addition of clinical and standard acoustic features boosted the AUROC to 0.81. Additional inclusion of transcription-related features yielded an AUROC of 0.90. CONCLUSIONS: The use of voice-based digital biomarkers derived from automated processing methods, combined with standard patient screening, might constitute a scalable way to enable early detection of dementia.

Genome sequences of Mycoplasma alligatoris A21JP2T and Mycoplasma crocodyli MP145T.

Mycoplasma alligatoris and Mycoplasma crocodyli are closely related siblings, one being highly virulent and the other relatively attenuated. We compared their genomes to better understand the mechanisms and origins of M. alligatoris' remarkable virulence amid a clade of harmless or much less virulent species. Although its chromosome was refractory to closure, M. alligatoris differed most notably by its complement of sialidases and other genes of the N-acetylneuraminate scavenging and catabolism pathway.

Acamprosate-responsive brain sites for suppression of ethanol intake and preference.

Acamprosate suppresses alcohol intake and craving in recovering alcoholics; however, the central sites of its action are unclear. To approach this question, brain regions responsive to acamprosate were mapped using acamprosate microimplants targeted to brain reward and circadian areas implicated in alcohol dependence. mPer2 mutant mice with nonfunctional mPer2, a circadian clock gene that gates endogenous timekeeping, were included, owing to their high levels of ethanol intake and preference. Male wild-type (WT) and mPer2 mutant mice received free-choice (15%) ethanol/water for 3 wk. The ethanol was withdrawn for 3 wk and then reintroduced to facilitate relapse. Four days before ethanol reintroduction, mice received bilateral blank or acamprosate-containing microimplants releasing ∼50 ng/day into reward [ventral tegmental (VTA), peduculopontine tegmentum (PPT), and nucleus accumbens (NA)] and circadian [intergeniculate leaflet (IGL) and suprachiasmatic nucleus (SCN)] areas. The hippocampus was also targeted. Circadian locomotor activity was measured throughout. Ethanol intake and preference were greater in mPer2 mutants than in wild-type (WT) mice (27 g·kg(-1)·day(-1) vs. 13 g·kg(-1)·day(-1) and 70% vs. 50%, respectively; both, P < 0.05). In WTs, acamprosate in all areas, except hippocampus, suppressed ethanol intake and preference (by 40-60%) during ethanol reintroduction. In mPer2 mutants, acamprosate in the VTA, PPT, and SCN suppressed ethanol intake and preference by 20-30%. These data are evidence that acamprosate's suppression of ethanol intake and preference are manifest through actions within major reward and circadian sites.

Acute ethanol disrupts photic and serotonergic circadian clock phase-resetting in the mouse.

BACKGROUND: Alcohol dependence is associated with impaired circadian rhythms and sleep. Ethanol administration disrupts circadian clock phase-resetting, suggesting a mode for the disruptive effect of alcohol dependence on the circadian timing system. In this study, we extend previous work in C57BL/6J mice to: (i) characterize the suprachiasmatic nucleus (SCN) pharmacokinetics of acute systemic ethanol administration, (ii) explore the effects of acute ethanol on photic and nonphotic phase-resetting, and (iii) determine if the SCN is a direct target for photic effects. METHODS: First, microdialysis was used to characterize the pharmacokinetics of acute intraperitoneal (i.p.) injections of 3 doses of ethanol (0.5, 1.0, and 2.0 g/kg) in the mouse SCN circadian clock. Second, the effects of acute i.p. ethanol administration on photic phase delays and serotonergic ([+]8-OH-DPAT-induced) phase advances of the circadian activity rhythm were assessed. Third, the effects of reverse-microdialysis ethanol perfusion of the SCN on photic phase-resetting were characterized. RESULTS: Peak ethanol levels from the 3 doses of ethanol in the SCN occurred within 20 to 40 minutes postinjection with half-lives for clearance ranging from 0.6 to 1.8 hours. Systemic ethanol treatment dose-dependently attenuated photic and serotonergic phase-resetting. This treatment also did not affect basal SCN neuronal activity as assessed by Fos expression. Intra-SCN perfusion with ethanol markedly reduced photic phase delays. CONCLUSIONS: These results confirm that acute ethanol attenuates photic phase-delay shifts and serotonergic phase-advance shifts in the mouse. This dual effect could disrupt photic and nonphotic entrainment mechanisms governing circadian clock timing. It is also significant that the SCN clock is a direct target for disruptive effects of ethanol on photic shifting. Such actions by ethanol could underlie the disruptive effects of alcohol abuse on behavioral, physiological, and endocrine rhythms associated with alcoholism.

Mutational analysis reveals the FUS homolog TAF15 as a candidate gene for familial amyotrophic lateral sclerosis.

FUS, EWS, and TAF15 belong to the TET family of structurally similar DNA/RNA-binding proteins. Mutations in the FUS gene have recently been discovered as a cause of familial amyotrophic lateral sclerosis (FALS). Given the structural and functional similarities between the three genes, we screened TAF15 and EWS in 263 and 94 index FALS cases, respectively. No coding variants were found in EWS, while we identified six novel changes in TAF15. Of these, two 24 bp deletions and a R388H missense variant were also found in healthy controls. A D386N substitution was shown not to segregate with the disease in the affected pedigree. A single A31T and two R395Q changes were identified in FALS cases but not in over 1,100 controls. Interestingly, one of the R395Q FALS cases also harbors a TARDBP mutation (G384R). Altogether, these results suggest that additional studies are needed to determine whether mutations in the TAF15 gene represent a cause of FALS.

Relationship between cardiac cycle and the timing of actions during action execution and observation.

Previous research suggests that there may be a relationship between the timing of motor events and phases of the cardiac cycle. This relationship has thus far only been researched using simple isolated movements such as key-presses in reaction-time tasks and only in a single subject acting alone. Other research has shown both movement and cardiac coordination among interacting individuals. Here, we investigated how the cardiac cycle relates to ongoing self-paced movements in both action execution and observation using a novel dyadic paradigm. We recorded electrocardiography (ECG) in 26 subjects who formed 19 dyads containing an action executioner and observer as they performed a self-paced sequence of movements. We demonstrated that heartbeats are timed to movements during both action execution and observation. Specifically, movements were less likely to culminate synchronously with the heartbeat around the time of the R-peak of the ECG. The same pattern was observed for action observation, with the observer's heartbeats occurring off-phase with movement culmination. These findings demonstrate that there is coordination between an action executioner's cardiac cycle and the timing of their movements, and that the same relationship is mirrored in an observer. This suggests that previous findings of interpersonal coordination may be caused by the mirroring of a phasic relationship between movement and the heart.

Expression and characterization of a truncated murine Fc gamma receptor.

We have isolated a recombinant secreted Fc gamma R beta molecule by deletion of the transmembrane and cytoplasmic domains encoding sequence from a Fc gamma R beta 1 cDNA clone, and insertion of the truncated cDNA into a eukaryotic expression vector, pcEXV-3. To express and amplify the production of the truncated Fc gamma R beta molecule, we transfected the truncated cDNA plasmid into a dihydrofolate reductase-minus CHO cell line along with a dhfr minigene, and amplified the gene products with methotrexate. The resulting cell line secretes 2-3 micrograms/ml/24 h of truncated Fc gamma R beta, which can be readily purified by affinity chromatography on IgG-Sepharose. The truncated Fc gamma R beta has a Mr of 31-33,000 on SDS-PAGE and is glycosylated. N-glycosidase F cleavage reduces the Mr to 19,000, consistent with the size of the truncated product, 176 amino acid residues. There are two disulfide bonds in the protein. Binding of immune complexes formed between DNP20BSA and anti-DNP mAbs reveals better binding of IgG1 aggregates than that of IgG2b and IgG2a aggregates. The binding of the immune complexes was somewhat better at more acidic pH, in contrast to previous experiments with binding of purified Fc gamma R to immune complex-coated beads. We were surprised to observe that the truncated Fc gamma R beta did not react with the anti-Fc gamma R mAb 6B7C. Previous work had shown that 6B7C reacts with Fc gamma R on immunoblots, fails to bind to the surface of resting B cells and peritoneal macrophages, but does bind to macrophage cell lines and LPS-stimulated B cells. We show, by binding of mAb 6B7C to a peptide conjugate, that the 6B7C epitope lies within residues 169-183 of the intact Fc gamma R beta, which is just outside the plasma membrane. The availability of the truncated Fc gamma R beta in microgram quantities should facilitate further analysis of structure and function of these receptors.