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Signaling levels within sensory neurons must be tightly regulated to allow cells to integrate information from multiple signaling inputs and to respond to new stimuli. Herein we report a new role for the cGMP-dependent protein kinase EGL-4 in the negative

Bacterial meningitis is a life-threatening infection of the central nervous system (CNS) that occurs when bacteria are able to cross the blood-brain barrier (BBB) or the meningeal-cerebrospinal fluid barrier (mBCSFB). The BBB and mBCSFB comprise highly specialized brain endothelial cells (BECs) that typically restrict pathogen entry. Group B Streptococcus (GBS or Streptococcus agalactiae ) is the leading cause of neonatal meningitis. Until recently, identification of GBS virulence factors has relied on genetic screening approaches. Instead, we here conducted RNA-seq analysis on GBS when interacting with induced pluripotent stem cell-derived BECs (iBECs) to pinpoint virulence-associated genes. Of the 2,068 annotated protein-coding genes of GBS, 430 transcripts displayed significant changes in expression after interacting with BECs. Notably, we found that the majority of differentially expressed GBS transcripts were downregulated (360 genes) during infection of iBECs. Interestingly, codY , encoding a pleiotropic transcriptional repressor in low-G + C Gram-positive bacteria, was identified as being highly downregulated. We conducted qPCR to confirm the codY downregulation observed via RNA-seq during the GBS-iBEC interaction and obtained codY mutants in three different GBS background parental strains. As anticipated from the RNA-seq results, the [Formula: see text] codY strains were more adherent and invasive in two in vitro BEC models. Together, this demonstrates the utility of RNA-seq during the BEC interaction to identify GBS virulence modulators., Importance: Group B Streptococcus (GBS) meningitis remains the leading cause of neonatal meningitis. Research work has identified surface factors and two-component systems that contribute to GBS disruption of the blood-brain barrier (BBB). These discoveries often relied on genetic screening approaches. Here, we provide transcriptomic data describing how GBS changes its transcriptome when interacting with brain endothelial cells. Additionally, we have phenotypically validated these data by obtaining mutants of a select regulator that is highly down-regulated during infection and testing on our BBB model. This work provides the research field with a validated data set that can provide an insight into potential pathways that GBS requires to interact with the BBB and open the door to new discoveries., Competing Interests: The authors declare no conflict of interest.

In this article we describe and analyze five videos created through an arts-informed research project, Precarious Inclusion: Studying Ontarian 2SLGBTQI+ parents' experiences childrearing in a post-legal parity framework. Precarious Inclusion used interviews and digital storytelling to investigate Ontario 2SLGBTQI+ parents' current experiences of inclusion and exclusion when navigating institutional and social interactions in everyday life in a post-legal parity context. The study centrally explored how intersecting identities with regards to sexuality, gender, geography, disability, class, race, Indigeneity, and ethnicity intersect with structural forces to influence 2SLGBTQI+ parents' inclusion and exclusion experiences. We examine research creation activities that supported 2SLGBTQI+ parents in making short videos about their experiences of parenting. Our analysis of the five videos created by Indigenous, racialized, trans, nonbinary, Two-Spirit, and disabled parents show how consistent experiences of exclusion mark 2SLGBTQI+ parents' everyday lives. We deepen theorizations of the material and psychological impacts of exclusion for 2SLGBTQI+ families through foregrounding three themes: 1) the operations of racism, white supremacy, and colonialism in makers' lives; 2) misrecognition and its psychic effects of bifurcation and disjuncture; and 3) love, joy, and multi-species kinship as powerful sites of healing and belonging. We further demonstrate how parents used their videos as self-advocacy for resisting precarious inclusion.

Radiofrequency reflectometry can provide fast and sensitive electrical read-out of charge and spin qubits in quantum dot devices coupled to resonant circuits. In situ frequency tuning and impedance matching of the resonator circuit using voltage-tunable capacitors (varactors) is needed to optimize read-out sensitivity, but the performance of conventional semiconductor- and ferroelectric-based varactors degrades substantially in the millikelvin temperature range relevant for solid-state quantum devices. Here we show that strontium titanate and potassium tantalate, materials which can exhibit quantum paraelectric behaviour with large field-tunable permittivity at low temperatures, can be used to make varactors with perfect impedance matching and resonator frequency tuning at 6 mK. We characterize the varactors at 6 mK in terms of their capacitance tunability, dissipative losses and magnetic field insensitivity. We use the quantum paraelectric varactors to optimize the radiofrequency read-out of carbon nanotube quantum dot devices, achieving a charge sensitivity of 4.8 μ e  Hz -1/2 and a capacitance sensitivity of 0.04 aF Hz -1/2 ., Competing Interests: Competing interestsUniversity College London has filed patent applications related to this technology: EP3997722 (Europe) and US20220351911A1 (US) by P.A., B.J.V. and M.R.B. The other authors declare no competing interests., (© The Author(s) 2024.)

The emergence of SARS-CoV-2 variants of concern (VOC) requires adequate coverage of vaccine protection. We evaluated whether a SARS-CoV-2 spike ferritin nanoparticle vaccine (SpFN), adjuvanted with the Army Liposomal Formulation QS21 (ALFQ), conferred protection against the Alpha (B.1.1.7), and Beta (B.1.351) VOCs in Syrian golden hamsters. SpFN-ALFQ was administered as either single or double-vaccination (0 and 4 week) regimens, using a high (10 μg) or low (0.2 μg) dose. Animals were intranasally challenged at week 11. Binding antibody responses were comparable between high- and low-dose groups. Neutralizing antibody titers were equivalent against WA1, B.1.1.7, and B.1.351 variants following two high dose vaccinations. Dose-dependent SpFN-ALFQ vaccination protected against SARS-CoV-2-induced disease and viral replication following intranasal B.1.1.7 or B.1.351 challenge, as evidenced by reduced weight loss, lung pathology, and lung and nasal turbinate viral burden. These data support the development of SpFN-ALFQ as a broadly protective, next-generation SARS-CoV-2 vaccine., (© 2021. This is a U.S. government work and not under copyright protection in the U.S.; foreign copyright protection may apply.)

Many neuronal identity regulators are expressed in distinct populations of cells in the nervous system, but their function is often analyzed only in specific isolated cellular contexts, thereby potentially leaving overarching themes in gene function undiscovered. We show here that the Caenorhabditis elegans Prop1-like homeobox gene unc-42 is expressed in 15 distinct sensory, inter- and motor neuron classes throughout the entire C. elegans nervous system. Strikingly, all 15 neuron classes expressing unc-42 are synaptically interconnected, prompting us to investigate whether unc-42 controls the functional properties of this circuit and perhaps also the assembly of these neurons into functional circuitry. We found that unc-42 defines the routes of communication between these interconnected neurons by controlling the expression of neurotransmitter pathway genes, neurotransmitter receptors, neuropeptides, and neuropeptide receptors. Anatomical analysis of unc-42 mutant animals reveals defects in axon pathfinding and synaptic connectivity, paralleled by expression defects of molecules involved in axon pathfinding, cell-cell recognition, and synaptic connectivity. We conclude that unc-42 establishes functional circuitry by acting as a terminal selector of functionally connected neuron types. We identify a number of additional transcription factors that are also expressed in synaptically connected neurons and propose that terminal selectors may also function as 'circuit organizer transcription factors' to control the assembly of functional circuitry throughout the nervous system. We hypothesize that such organizational properties of transcription factors may be reflective of not only ontogenetic, but perhaps also phylogenetic trajectories of neuronal circuit establishment., Competing Interests: EB, LG, AB, HS, EV, NM, AA, YF, KN, SC, JW, NM, CC, YR, DF, DH No competing interests declared, OH Reviewing editor, eLife, (© 2021, Berghoff et al.)

The emergence of SARS-CoV-2 variants of concern (VOC) requires adequate coverage of vaccine protection. We evaluated whether a spike ferritin nanoparticle vaccine (SpFN), adjuvanted with the Army Liposomal Formulation QS21 (ALFQ), conferred protection against the B.1.1.7 and B.1.351 VOCs in Syrian golden hamsters. SpFN-ALFQ was administered as either single or double-vaccination (0 and 4 week) regimens, using a high (10 μg) or low (0.2 μg) immunogen dose. Animals were intranasally challenged at week 11. Binding antibody responses were comparable between high- and low-dose groups. Neutralizing antibody titers were equivalent against WA1, B.1.1.7, and B.1.351 variants following two high dose two vaccinations. SpFN-ALFQ vaccination protected against SARS-CoV-2-induced disease and viral replication following intranasal B.1.1.7 or B.1.351 challenge, as evidenced by reduced weight loss, lung pathology, and lung and nasal turbinate viral burden. These data support the development of SpFN-ALFQ as a broadly protective, next-generation SARS-CoV-2 vaccine.

An impurity in the color additives D&C Red No. 30 (R30) and D&C Red No. 30 lakes (R30L) was newly identified and characterized as 7-chloro-5-methyl-2H-1,4-benzothiazin-3(4H)-one (BTZ), and its extent and level in certified batches of these color additives was determined. BTZ was extracted from the dye with ethanol, resulting in a crude extract enriched to a concentration of over 60%. BTZ was then separated from a portion of the enriched extract by high-speed counter-current chromatography using a spiral-tube assembly column with intermittently pressed tubing of 60 ml capacity. It was the first reported use of such a column to separate a small, moderately hydrophobic compound. The two-phase solvent system was also moderately hydrophobic, consisting of hexane-ethyl acetate-methanol-water (5:2:5:2), and the retention of the organic stationary phase measured after the separation was 83.3%. The separation yielded BTZ of two purity grades, the higher of which (~95.5%) was used as a standard to quantify the impurity in 37 batches of R30 and R30L using an HPLC method developed and validated for that purpose. Analyses revealed a wide range of BTZ levels across batches, <0.05 - 0.84%, and suggested that BTZ contamination could be reduced by appropriate adjustments in the manufacturing process. An explanation of the likely source of BTZ - as a side-reaction product in a particular step of the manufacturing process - was also presented., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Published by Elsevier B.V.)

All animals rely on their ability to sense and respond to their environment to survive. However, the suitability of a behavioral response is context-dependent, and must reflect both an animal's life history and its present internal state. Based on the integration of these variables, an animal's needs can be prioritized to optimize survival strategies. Nociceptive sensory systems detect harmful stimuli and allow for the initiation of protective behavioral responses. The polymodal ASH sensory neurons are the primary nociceptors in C. elegans. We show here that the guanylyl cyclase ODR-1 functions non-cell-autonomously to downregulate ASH-mediated aversive behaviors and that ectopic cGMP generation in ASH is sufficient to dampen ASH sensitivity. We define a gap junction neural network that regulates nociception and propose that decentralized regulation of ASH signaling can allow for rapid correlation between an animal's internal state and its behavioral output, lending modulatory flexibility to this hard-wired nociceptive neural circuit.

Protein arginine methylation regulates diverse functions of eukaryotic cells, including gene expression, the DNA damage response, and circadian rhythms. We showed that arginine residues within the third intracellular loop of the human D2 dopamine receptor, which are conserved in the DOP-3 receptor in the nematode Caenorhabditis elegans, were methylated by protein arginine methyltransferase 5 (PRMT5). By mutating these arginine residues, we further showed that their methylation enhanced the D2 receptor-mediated inhibition of cyclic adenosine monophosphate (cAMP) signaling in cultured human embryonic kidney (HEK) 293T cells. Analysis of prmt-5-deficient worms indicated that methylation promoted the dopamine-mediated modulation of chemosensory and locomotory behaviors in C. elegans through the DOP-3 receptor. In addition to delineating a previously uncharacterized means of regulating GPCR (heterotrimeric guanine nucleotide-binding protein-coupled receptor) signaling, these findings may lead to the development of a new class of pharmacological therapies that modulate GPCR signaling by changing the methylation status of these key proteins., (Copyright © 2015, American Association for the Advancement of Science.)

Hydrolysis of acetylcholine by acetylcholinesterase (AChE) is extremely rapid, with a second-order hydrolysis rate constant k(E) (often denoted k(cat)/K(M)) that approaches 10(8) M(-1) s(-1). AChE contains a deep active site gorge with two sites of ligand binding, an acylation site (or A-site) containing the catalytic triad at the base of the gorge and a peripheral site (or P-site) near the gorge entrance. The P-site is known to contribute to catalytic efficiency with acetylthiocholine (AcSCh) by transiently trapping the substrate in a low affinity complex on its way to the A-site, where a short-lived acyl enzyme intermediate is produced. Here we ask whether the P-site does more than simply trap the substrate but in fact selectively gates entry to the A-site to provide specificity for AcSCh (and acetylcholine) relative to the close structural analogs acetyl(homo)thiocholine (Ac-hSCh, which adds one additional methylene group to thiocholine) and acetyl(nor)thiocholine (Ac-nSCh, which deletes one methylene group from thiocholine). We synthesized Ac-hSCh and Ac-nSCh and overcame technical difficulties associated with instability of the northiocholine hydrolysis product. We then compared the catalytic parameters of these substrates with AChE to those of AcSCh. Values of k(E) for Ac-hSCh and Ac-nSCh were about 2% of that for AcSCh. The k(E) for AcSCh is close to the theoretical diffusion-controlled limit for the substrate association rate constant, but kE values for Ac-hSCh or Ac-nSCh are too low to be limited by diffusion control. However, analyses of kinetic solvent isotope effects and inhibition patterns for P-site inhibitors indicate that these two analogs also do not equilibrate with the A-site prior to the initial acylation step of catalysis. We propose that kE for these substrates is partially rate-limited by a gating step that involves the movement of bound substrate from the P-site to the A-site., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

Although the affinity optimization of protein binders is straightforward, engineering epitope specificity is more challenging. Targeting a specific surface patch is important because the biological relevance of protein binders depends on how they interact with the target. They are particularly useful to test hypotheses motivated by biochemical and structural studies. We used yeast display to engineer monobodies that bind a defined surface patch on the mitogen activated protein kinase (MAPK) Erk-2. The targeted area ("CD" domain) is known to control the specificity and catalytic efficiency of phosphorylation by the kinase by binding a linear peptide ("D" peptide) on substrates and regulators. An inhibitor of the interaction should thus be useful for regulating Erk-2 signaling in vivo. Although the CD domain constitutes only a small percentage of the surface area of the enzyme (~5%), sorting a yeast displayed monobody library with wild type (wt) Erk-2 and a rationally designed mutant led to isolation of high affinity clones with desired epitope specificity. The engineered binders inhibited the activity of Erk-2 in vitro and in mammalian cells. Furthermore, they specifically inhibited the activity of Erk-2 orthologs in yeast and suppressed a mutant phenotype in round worms caused by overactive MAPK signaling. The study therefore shows that positive and negative screening can be used to bias the evolution of epitope specificity and predictably design inhibitors of biologically relevant protein-protein interaction.

Signaling levels within sensory neurons must be tightly regulated to allow cells to integrate information from multiple signaling inputs and to respond to new stimuli. Herein we report a new role for the cGMP-dependent protein kinase EGL-4 in the negative regulation of G protein-coupled nociceptive chemosensory signaling. C. elegans lacking EGL-4 function are hypersensitive in their behavioral response to low concentrations of the bitter tastant quinine and exhibit an elevated calcium flux in the ASH sensory neurons in response to quinine. We provide the first direct evidence for cGMP/PKG function in ASH and propose that ODR-1, GCY-27, GCY-33 and GCY-34 act in a non-cell-autonomous manner to provide cGMP for EGL-4 function in ASH. Our data suggest that activated EGL-4 dampens quinine sensitivity via phosphorylation and activation of the regulator of G protein signaling (RGS) proteins RGS-2 and RGS-3, which in turn downregulate Gα signaling and behavioral sensitivity., Competing Interests: The authors have declared that no competing interests exist.

G protein-coupled receptor kinases (GRKs) are key regulators of signal transduction that specifically phosphorylate activated G protein-coupled receptors (GPCRs) to terminate signaling. Biochemical and crystallographic studies have provided great insight into mammalian GRK2/3 interactions and structure. However, despite extensive in vitro characterization, little is known about the in vivo contribution of these described GRK structural domains and interactions to proper GRK function in signal regulation. We took advantage of the disrupted chemosensory behavior characteristic of Caenorhabditis elegans grk-2 mutants to discern the interactions required for proper in vivo Ce-GRK-2 function. Informed by mammalian crystallographic and biochemical data, we introduced amino acid substitutions into the Ce-grk-2 coding sequence that are predicted to selectively disrupt GPCR phosphorylation, Gα(q/11) binding, Gβγ binding, or phospholipid binding. Changing the most amino-terminal residues, which have been shown in mammalian systems to be required specifically for GPCR phosphorylation but not phosphorylation of alternative substrates or recruitment to activated GPCRs, eliminated the ability of Ce-GRK-2 to restore chemosensory signaling. Disrupting interaction between the predicted Ce-GRK-2 amino-terminal α-helix and kinase domain, posited to stabilize GRKs in their active ATP- and GPCR-bound conformation, also eliminated Ce-GRK-2 chemosensory function. Finally, although changing residues within the RH domain, predicted to disrupt interaction with Gα(q/11), did not affect Ce-GRK-2 chemosensory function, disruption of the predicted PH domain-mediated interactions with Gβγ and phospholipids revealed that both contribute to Ce-GRK-2 function in vivo. Combined, we have demonstrated functional roles for broadly conserved GRK2/3 structural domains in the in vivo regulation of organismal behavior.

The global eradication of malaria will require the development of vaccines to prevent infection cause by Plasmodium vivax in addition to Plasmodium falciparum. In an attempt to contribute to this effort we have previously reported the cloning and expression of a vaccine based on the circumsporozoite protein of P. vivax. The synthetic vaccine encodes for a full-length molecule encompassing the N-terminal and C-terminal regions flanking a chimeric repeat region representing VK210 and VK247, the two major alleles of P. vivax CSP. The vaccine, designated vivax malaria protein 001 (VMP001), was purified to >95% homogeneity using a three-column purification scheme and had low endotoxin levels and passed the rabbit pyrogenicity assay. The protein is recognized by monoclonal antibodies directed against the two repeat motifs, as well as polyclonal antibodies. Immunization with VMP001 induced high titer antibodies in mice using Montanide ISA 720. We currently have more than 10,000 doses of purified bulk and 1800 vials of formulated bulk vaccine available for clinical testing and VMP001 is currently undergoing further development as a candidate vaccine to prevent malaria in humans.

Men's health has emerged as an important public concern that may require new kinds of healthcare interventions and increased resources. Considerable uncertainty and confusion surround prevailing understandings of men's health, particularly those generated by media debate and public policy, and health research has often operated on oversimplified assumptions about men and masculinity. A more useful way of understanding men's health is to adopt a gender-relations approach. This means examining health concerns in the context of men's and women's interactions with each other, and their positions in the larger, multidimensional structure of gender relations. Such an approach raises the issue of differences among men, which is a key issue in recent research on masculinity and an important health issue. The gender-relations approach offers new ways of addressing practical issues of healthcare for men in college environments.

The crystal complex of fluorescein bound to the high-affinity anti-fluorescein 4-4-20 Fab (Ka = 10(10) M-1 at 2 degrees C) has been determined at 1.85 A. Isomorphous crystals of two isoelectric forms (pI = 7.5 and 7.9) of the anti-fluorescein 4-4-20 Fab, an IgG2A [Gibson et al. (1988) Proteins: Struct. Funct. Genet., 3, 155-160], have been grown. Both complexes crystallize with one molecule in the asymmetric unit in space group P1, with a = 42.75 A, b = 43.87 A, c = 58.17 A, alpha = 95.15 degrees, beta = 86.85 degrees and gamma = 98.01 degrees. The final structure has an R value of 0.188 at 1.85 A resolution. Interactions between bound fluorescein, the complementarity-determining regions (CDRs) of the Fab and the active-site mutants of the 4-4-20 single-chain Fv will be discussed. Differences were found between the structure reported here and the previously reported 2.7 A 4-4-20 Fab structure [Herron et al. (1989) Proteins: Struct. Funct. Genet., 5, 271-280]. Our structure determination was based on 26,328 unique reflections--four times the amount of data used in the previous report. Differences in the two structures could be explained by differences in interpreting the electron density maps at the various resolutions. The r.m.s. deviations between the variable and constant domains of the two structures were 0.77 and 1.54 A, respectively. Four regions of the light chain and four regions of the heavy chain had r.m.s. backbone deviations of > 4 A. The most significant of these was the conformation of the light chain CDR 1.

Single-chain Fv proteins are known to aggregate and form multimeric species. We report here that these molecules represent a new class of molecular assembly, which we have termed multivalent Fvs. Each binding site in a multivalent Fv comprises the variable light-chain (VL) domain from a single-chain Fv, and the variable heavy-chain (VH) domain from a second single-chain Fv. Each single-chain Fv in a multivalent Fv is part of two binding sites. We have characterized the multivalent forms of the 4-4-20, CC49 and B6.2 sFvs. The degree of multivalent Fv formation is linker-dependent. Multivalent Fvs cannot form in the absence of an intact linker. Multivalent Fvs can be stabilized by their antigen. The conversion between different forms of the multivalent Fvs can be catalyzed by disassociating agents such as 0.5 M guanidine hydrochloride with 20% ethanol. Multivalent Fvs have significantly different stabilities depending on the specific variable domains from which they are constructed. Two models have been proposed for the structure of a multivalent Fv. We have tested each model by attempting to produce a heterodimer from the anti-fluorescein 4-4-20 and anti-tumor CC49 variable regions. We successfully produced a 4-4-20/CC49 heterodimer that comprises two mixed sFvs. The first mixed sFv is composed of the 4-4-20 VL domain, a 12 residue linker and the CC49 Vh domain. The second mixed sFv is composed of a CC49 VL domain, a 12 residue linker and the 4-4-20 VH domain. The 4-4-20/CC49 heterodimer bound both fluorescein and the tumor-associated glycoprotein-72 antigen. These results support a VH/VL 'rearrangement' model in which each variable domain of a multivalent Fv binding site comes from a different polypeptide chain.

Single-chain Fv (sFv) proteins consist of the variable heavy chain (VH) and variable light chain (VL) domains of an antibody, covalently joined by an engineered polypeptide linker. We report the crystallization of single-chain Fv's with specificities for fluorescein (4-4-20 sFv) and the TAG-72 pan-carcinoma glycoprotein antigen (CC49 sFv). Concentration of these proteins, preliminary to crystallization, results in a monomer-multimer equilibrium, causing aggregation which interferes with crystallization. Aggregation has been observed to depend primarily on an intact linker between VL and VH domains, although other factors are likely to modulate this phenomenon as well, including the specific identity of Fv and ligand, presence or absence of the ligand, linker length and possibly sequence. We have found two methods to overcome sFv aggregation, both of which yield X-ray diffraction quality crystals. The first, discovered serendipitously, is by introducing a proteolytic clip into the linker region (effectively yielding an Fv fragment). The second is the purification of the sFv dimer form, with linker regions intact, from an equilibrium mixture of aggregates. The sFv molecular association in a dimer is believed to be unusual in that each VL/VH interface may not be formed by the two linker-connected VL and VH domains, but rather by interaction of VL and VH domains from two distinct sFv monomers. Structure determination of the CC49 sFv dimer, with the 14-residue linker designated 212, is underway to test this model. Increasing linker length, to relieve steric strain on the monomer, and inclusion of the appropriate antigen, to slow transitions between monomeric and multimeric forms, may prove valuable strategies with sFv proteins less amenable to crystallization.

The effects of linker length on binding affinity and degree of aggregation have been examined in the antifluorescein 4-4-20 and anticarcinoma CC49 single-chain Fvs. Longer linkers in the antifluorescein sFvs have higher affinities for fluorescein and aggregate less. A proteolytically susceptible site between Lys8 and Ser9, in the previously reported 212 linker has been identified. A new linker sequence, 218 (GSTSGSGKPGSGEGSTKG) was designed in which a proline was placed at the C-terminal side of the proteolytic clip site in the 212 linker. The CC49 sFv containing the 218 linker showed reduced aggregation and was found to be more stable to proteolysis in vitro, when compared to the CC49/212 sFv. The CC49 sFv with the longer 218 linker had higher affinity than CC49/212 sFv. An aggregated CC49/212 sFv sample had higher affinity than CC49/218 sFv. The CC49/218 and CC49/212 sFvs had similar blood clearances in mice, while the aggregated CC49/212 sFv remained in circulation significantly longer. In mice bearing LS-174T human colon carcinoma xenografts, the CC49/218 sFv showed higher tumor uptake than the CC49/212 sFv and lower tumor uptake than the aggregated CC49/212 sFv. The higher tumor uptake of the CC49/218 is most likely a result of its higher resistance to proteolysis. The higher affinity and higher tumor uptake of the aggregated CC49/212 sFv are most likely due to the repetitive nature of the TAG-72 antigen and the higher avidity of multivalent aggregates. When the sFvs were radiolabeled with a lutetium-chelate the CC49/218 sFv showed a lower accumulation in the liver and spleen compared to the aggregated CC49/212 sFv.

In previous studies, we have compared the immunochemical properties, the in vivo pharmacokinetics, and the tumor penetrance of a radioiodinated single-chain Fv (sFv) in comparison with other immunoglobulin (Ig) forms (intact IgG, F(ab')2, and Fab') (Cancer Res., 51: 6363-6371, 1991). Biodistribution studies demonstrated a higher percent injected dose/g in the liver and spleen for the intact IgG and F(ab')2. Renal uptake was observed with the Fab' and F(ab')2, whereas the sFv demonstrated no specific localization in either of these organs. The 125I-labeled sFv also demonstrated a more even distribution throughout the tumor xenografts as compared to the other Ig forms (Cancer Res., 52: 3402-3408, 1992). Subsequent studies utilizing the sFv conjugated with a radiometal (177Lu) demonstrated that the sFv was being metabolized by the kidney, and a significantly higher percent injected dose/g was obtained with a 177Lu-labeled sFv as compared to a 125I-labeled sFv (Cancer Res., 52: 6413-6417, 1992). These previous studies indicated the potential utility of radioiodinated sFv and other Ig fragments for use in radioimmunoguided surgery with a hand-held probe, diagnostic imaging, and possibly therapy. The present study compares the distribution in normal tissues of the 4 Ig forms of monoclonal antibody (MAb) CC49, which is directed against a pancarcinoma antigen (tumor-associated glycoprotein-72). 125I-labeled sFv, Fab', F(ab')2, and IgG of MAb CC49 were administered to athymic mice either bearing or not bearing the tumor-associated glycoprotein-72 positive human colon carcinoma xenograft (LS-174T). At various intervals following the i.v. injection of the Ig forms, the liver, spleen, kidneys, and lungs were removed for autoradiographic analyses. Dramatic differences were observed in the kidney; the IgG was found only in the renal vasculature, whereas the Fab', F(ab')2, and sFv showed a high density of grains in the cortical tubules. In the liver, the IgG and F(ab')2 were found in association with hepatocytes, Kupffer cells, and in the sinusoids; the Fab' and sFv were primarily associated with the Kupffer cells. In the spleen, the Ig forms localized to the marginal zones surrounding the lymphoid follicles. No specific accumulation of grains for any of the Ig forms was observed in the lung. In each of the tissues, the clearance rates were related to the size of the Ig form. The localization in the liver and spleen was determined to be antigen-mediated.(ABSTRACT TRUNCATED AT 400 WORDS)

Genetically engineered single-chain Fvs (sFv) are defined as recombinant proteins composed of a variable light chain amino acid sequence of an immunoglobulin tethered to a variable heavy chain sequence by a designed peptide. Previous studies using iodine-labeled sFv, derived from the anticarcinoma monoclonal antibody CC49, showed that the 125I-sFv could efficiently target antigen-positive tumors in a human tumor xenograft model while demonstrating rapid plasma clearance and minimal uptake in normal organs. One of the issues we raised in the analysis of the iodinated sFv metabolic studies was whether similar metabolic patterns would be observed if the sFv were labeled with a radiometal. In the studies reported here, 125I-CC49 sFv and 177Lu-CC49 sFv were co-injected in mice bearing antigen-positive carcinoma xenografts. Both sFv forms showed similar tumor targeting and plasma clearance pharmacokinetics. The 177Lu-sFv, however, showed a greater uptake in liver and spleen and a much higher uptake in kidney. These studies thus demonstrate that despite their small size (M(r) 27,000), the metal-chelated sFv shows a metabolic pattern very different than that of the iodinated sFv, which is most likely due to retention of the metal by organs metabolizing the sFv.

CC49 is a "second generation" monoclonal antibody to B72.3, which reacts with the pancarcinoma antigen TAG-72. CC49 has been shown to efficiently target human colon carcinoma xenografts and is currently being evaluated in both diagnostic and therapeutic clinical trials. We describe here the construction and characterization of a recombinant single-chain Fv (sFv) of CC49. The sFv was shown to be a Mr 27,000 homogeneous entity which could be efficiently radiolabeled with 125I or 131I. Comparative direct binding studies and competition radioimmunoassays using CC49 intact IgG, F(ab')2, Fab', and sFv revealed that the monomeric CC49 Fab' and sFv had relative binding affinities 8-fold lower than the dimeric F(ab')2 and intact IgG. Nonetheless, the 131I-labeled sFv was shown to bind biopsies of TAG-72-expressing tumors. Metabolism studies in mice, using radiolabeled CC49 IgG, F(ab')2, Fab', and sFv, demonstrated an extremely rapid plasma and whole body clearance for the sFv. CC49 sFv plasma pharmacokinetic studies in rhesus monkeys also showed a very rapid plasma clearance (T1/2 alpha of 3.9 min and T1/2 beta of 4.2 h). Tumor targeting studies with all four radiolabeled Ig CC49 forms, using the LS-174T human colon carcinoma xenograft model, revealed a much lower percentage injected dose/g tumor binding for the CC49 monomeric sFv and Fab' as compared to the dimeric F(ab')2 and intact IgG. However, tumor:normal tissue ratios (radiolocalization indices) for the sFv were comparable to or greater than those of the other Ig forms. High kidney uptake with 125I-labeled Fab' and F(ab')2 was not seen with 125I-sFv. Gamma scanning studies also showed that 131I-CC49 sFv could efficiently localize tumors. The CC49 sFv may thus have utility in diagnostic and perhaps therapeutic applications for a range of human carcinomas.

A fluorescein-binding single-chain Fv (scFv) was chosen as a model for the study of the physicochemical parameters associated with synthetic IgG fragments. Three such scFv proteins were designed from the primary sequences of one anti-fluorescyl monoclonal antibody (Mab 4.4.20). These were constructed with varying-length interdomain peptide linkers of between 12 and 25 residues, expressed in Escherichia coli, and the protein folding, stability, and antigen-binding characteristics were assessed. Efficient renaturation could be accomplished in vitro to yield approximately 26 mg of active scFv/L of fermentation. Scatchard analysis for fluorescein ligand binding revealed that the scFv designs come within 2-fold of the Ka = 1.99 (+/- 0.18) x 10(9) observed for the parental 4.4.20 Fab and have identical stoichiometries (n approximately 0.99). Reversible solvent denaturation studies demonstrated that the unfolding/refolding equilibria for the scFv proteins can be fit to a simple two-state model and that two of the scFv designs were found to be slightly more stable than single IgG domains (VL and CL) when assessed in terms of the free energy of unfolding, delta Gon-u, or nearly identical to other multiple domain immunoglobulin proteins such as light chains and Fab's when relative transition midpoints, Cm, are compared. Linkers which conferred conformational flexibility beyond the minimally required length of 12 residues were found to have a stabilizing effect. By these criteria of ligand-binding function and protein stability, the scFv proteins were found to be bona fide minimal replicas of their parental IgG molecules.

Introduction of a disulfide bond by site-directed mutagenesis was found to enhance the stability of subtilisin BPN' (EC 3.4.21.14) under a variety of conditions. The location of the new disulfide bond was selected with the aid of a computer program, which scored various sites according to the amount of distortion that an introduced disulfide linkage would create in a 1.3-A X-ray model of native subtilisin BPN'. Of the several amino acid pairs identified by this program as suitable candidates, Thr-22 and Ser-87 were selected by using the additional requirement that the individual cysteine substitutions occur at positions that exhibit some degree of variability in related subtilisin amino acid sequences. A subtilisin variant containing cysteine residues at positions 22 and 87 was created by site-directed mutagenesis and was shown to have an activity essentially equivalent to that of the wild-type enzyme. Differential scanning calorimetry experiments demonstrated the variant protein to have a melting temperature 3.1 degrees C higher than that of the wild-type protein and 5.8 degrees C higher than that of the reduced form (-SH HS-) of the variant protein. Kinetic experiments performed under a variety of conditions, including 8 M urea, showed that the Cys-22/Cys-87 disulfide variant undergoes thermal inactivation at half the rate of that of the wild-type enzyme. The increased thermal stability of this disulfide variant is consistent with a decrease in entropy for the unfolded state relative to the unfolded state that contains no cross-link, as would be predicted from the statistical thermodynamics of polymers.

Six individual amino acid substitutions at separate positions in the tertiary structure of subtilisin BPN' (EC 3.4.21.14) were found to increase the stability of this enzyme, as judged by differential scanning calorimetry and decreased rates of thermal inactivation. These stabilizing changes, N218S, G169A, Y217K, M50F, Q206C, and N76D, were discovered through the use of five different investigative approaches: (1) random mutagenesis; (2) design of buried hydrophobic side groups; (3) design of electrostatic interactions at Ca2+ binding sites; (4) sequence homology consensus; and (5) serendipity. Individually, the six amino acid substitutions increase the delta G of unfolding between 0.3 and 1.3 kcal/mol at 58.5 degrees C. The combination of these six individual stabilizing mutations together into one subtilisin BPN' molecule was found to result in approximately independent and additive increases in the delta G of unfolding to give a net increase of 3.8 kcal/mol (58.5 degrees C). Thermodynamic stability was also shown to be related to resistance to irreversible inactivation, which included elevated temperatures (65 degrees C) or extreme alkalinity (pH 12.0). Under these denaturing conditions, the rate of inactivation of the combination variant is approximately 300 times slower than that of the wild-type subtilisin BPN'. A comparison of the 1.8-A-resolution crystal structures of mutant and wild-type enzymes revealed only independent and localized structural changes around the site of the amino acid side group substitutions.(ABSTRACT TRUNCATED AT 250 WORDS)

Strontium plasma clearance is an important factor determining the absorbed dose to metastases and bone marrow in patients receiving 89Sr radionuclide therapy for metastatic bone disease. Amongst male patients with disseminated prostatic carcinoma, the renal component of strontium clearance is frequently greatly reduced compared with values reported for healthy middle aged men. We report a study of renal and gut strontium plasma clearance, renal function, calcium urinary excretion, parathyroid function and extent of skeletal osteoblastic metastatic disease in patients referred for radiostrontium therapy for metastasised prostatic malignancy. The wide variation in net strontium clearance was principally due to variation in the renal component. Low values of strontium renal clearance were found to correlate with the elevation of serum PTH and nephrogenous cyclic AMP, which in turn correlated with extent of skeletal metastatic disease. This suggests that the osteosclerotic metastases characteristic of prostatic carcinoma induce secondary hyperparathyroidism due to the high avidity of the skeleton for calcium. The resulting reduction in strontium excretion may be beneficial to the objectives of radiostrontium therapy.

The growth and feed conversion of rabbits fed either grass silage or whole grains and supplementary pelleted concentrate of cassava/cottonseed diets were investigated. Poor quality grass silage (pH 4.9) was almost completely rejected by young rabbits initially fed either 17.8 g or 35.5 g DM/day of a supplementary concentrate. Rabbits on the lower level of concentrate provision lost 0.35 g liveweight per day. Rabbits initially weighing 1.77 kg fed complete pelleted diets containing 667 g/kg maize or 667 g/kg sorghum showed improved daily liveweight gains (22.6 g) over rabbits fed whole grains and pelleted supplements (19.4 g) in an experiment lasting 40 days. In a second experiment there were no significant effects of pelleting or type of cereal on liveweight gain or feed conversion ratio. Pelleting significantly improved crude protein digestibility of diets whilst maize diets were superior in DM, organic matter and crude protein digestibilities. The inclusion of cottonseed meal containing 700 mg/kg free gossypol in diets at levels of 150 and 300 g/kg did not affect growth rate or feed conversion in rabbits weighing 0.92 kg initially. These diets contained up to 364 g/kg cassava suggesting that this ingredient can be used in rabbit diets as an energy source in replacement for whole grains.

A weak Ca2+ binding site in the bacterial serine protease subtilisin BPN' (EC 3.4.21.14) was chosen as a model to explore the feasibility of stabilizing a protein by increasing the binding affinity at a metal ion binding site. The existence of this weak Ca2+ binding site was first discovered through a study of the rate of thermal inactivation of wild-type subtilisin BPN' at 65 degrees C as a function of the free [Ca2+]. Increasing the [Ca2+] in the range 0.10-100 mM caused a 100-fold decrease in the rate of thermal inactivation. The data were found to closely fit a theoretical titration curve for a single Ca2+ specific binding site with an apparent log Ka = 1.49. A series of refined X-ray crystal structures (R less than or equal to 0.15, 1.7 A) of subtilisin in the presence of 0.0, 25.0, and 40.0 mM CaCl2 has allowed a detailed structural characterization of this Ca2+ binding site. Negatively charged side chains were introduced in the vicinity of the bound Ca2+ by changing Pro 172 and Gly 131 to Asp residues through site-directed and random mutagenesis techniques, respectively. These changes were found to increase the affinity of the Ca2+ binding site by 3.4- and 2-fold, respectively, when compared with the wild-type protein (ionic strength = 0.10). X-ray studies of these new variants of subtilisin revealed the carboxylate side chains to be 6.8 and 13.2 A, respectively, from the bound Ca2+. These distances and the degree of enhanced binding are consistent with simple electrostatic theory.(ABSTRACT TRUNCATED AT 250 WORDS)

Although cell surface molecules are thought to be involved in macrophage (MO)-tumor-cell recognition, the nature of these molecules remains unknown. In this study we have shown that the glycoprotein laminin may facilitate macrophage-tumor-cell binding. Macrophage binding to tumor cells was assessed by measuring the adherence of radiolabelled 3-MCA2 induced malignant fibrosarcoma cells to syngeneic peritoneal MOs. Addition of exogenous laminin promoted the binding of a weakly metastatic subline of these tumor cells by 31-68%. These weakly metastatic tumor cells express negligible endogenous cell-surface laminin but display specific cell-surface receptors for binding soluble laminin. Exogenous laminin promoted MO binding of these tumor cells whether it was present during the assay or whether the tumor cells were pretreated with the laminin. This increase in binding was blocked by anti-laminin antibody. In contrast, MO binding of a strongly metastatic variant of the same tumor was not enhanced by the addition of exogenous laminin. This highly malignant fibrosarcoma line already expressed endogenous cell-surface laminin. Since the MOs were found to specifically bind 125I-laminin, the interaction between laminin-bearing tumor cells and MOs may be mediated via a specific MO plasma membrane receptor. Thus, the expression of cell-surface laminin and its receptors on both tumor cells and MOs may provide a mechanism for promoting MO-tumor-cell binding.

Male plasma testosterone (T) and thyroxine (T4) were monitored over several annual cycles in a captive breeding colony of green sea turtles, Chelonia mydas. Daily and annual water temperatures varied by only approximately 1 and 3 degrees, respectively. A pronounced season cycle in plasma T was evident in the population as a whole and in individual animals: plasma T was at a nadir (approximately 3 ng/ml) in September-November and then increased progressively to a peak (27-39 ng/ml) in April; levels began declining immediately thereafter, coincident with the onset of copulatory behavior. By contrast, plasma T4 remained uniform (approximately 9 ng/ml) throughout the year and, thus, could not readily account for the decline in androgen levels. Plasma hormones were relatively stable over a 24-hr period at three times a year, and there was a correlation for individual plasma T levels sampled in April and May. Thus, limited sampling should allow identification of seasonal rhythms and individual variability in plasma T levels. Testis mass and spermatogenic activity were significantly greater in January than in September; i.e., spermatogenesis and androgen secretion were not "uncoupled." Copulatory activity began in April but did not peak until May-June, after plasma T had significantly declined. However, there was a significant (but weak) correlation between individual peak levels of plasma T (i.e., in April) and the quantitative level of mating activity (time spent mounting and number of mates) measured for the entire subsequent season. Thus, green turtles do not exhibit the "postnuptial" type of testis cycle typical of many temperate-zone turtles, and the levels of plasma androgen may be important for initiating and maintaining sex behavior, although they are not tightly linked during the mating season.