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Smalltooth sawfish Pristis pectinata use southwest Florida nurseries during at least their first 2 to 3 yr, and understanding region-specific habitat use patterns is important for effective management. Research occurred in 2 nurseries within the Charlotte Harbor estuarine system: the Caloosahatchee River, a highly human-altered nursery, and a more natural nursery, the Peace River. Between 2010 and 2013, a total of 148 juveniles ranging from 70.8 to 218.7 cm stretch total length were caught in gill nets, and 133 were acoustically tagged. The probability of encountering a sawfish during sampling was most influenced by season in both nurseries (highest during spring and summer), followed by dissolved oxygen in the Caloosahatchee River, and salinity and temperature in the Peace River. Temperature had lasting, atypical effects on movements when severe cold events occurred during the study. Outside of these disturbances, sawfish used all habitats available to them in both nurseries, but tended to reside in perennial high-use areas called nursery hotspots. Acoustic monitoring showed that juveniles gradually moved between 4 hotspots along a 20 river kilometer stretch of the Caloosahatchee River with seasonal changes in freshwater inflows or sometimes suddenly in response to large flow events. In contrast, sawfish in the Peace River remained associated with one nursery hotspot year-round. Reasons for these inter-nursery variations are attributed to differences in geomorphology and freshwater inflow regimes. This study illustrates that a full understanding of the life history of a species and the development of a conceptual model requires investigation of the species on a broad spatial scale. This consideration is important to avoid overgeneralization of habitat use patterns between nurseries.

Habitat use and movements of juvenile (

Growing concerns about the conservation of elasmobranchs have prompted a surge in research, because scientific studies that can support management actions are needed. Sawfishes are among the most threatened fishes worldwide and epitomize the challenge of conserving widely distributed, large-bodied marine fishes. We used a comparative approach to provide data on the trophic ecology of the smalltooth sawfish Pristis pectinata in the western Atlantic coastal waters of southwest Florida, USA. Specifically, we applied (1) stable isotope techniques to fin tissues of smalltooth sawfish and 2 sympatric elasmobranch species that have well-documented diets (i.e. bull shark Carcharhinus leucas and cownose ray Rhinoptera bonasus), and muscle tissue from a variety of known and potential prey species; and (2) an 18S rRNA gene sequencing technique to identify prey taxa in sawfish fecal samples. These analyses provided evidence that the smalltooth sawfish feeds primarily on teleost and elasmobranch fishes at all life stages even though sawfish move from estuarine to coastal habitats during their ontogeny. Although both sawfish and bull sharks occupy estuarine waters as juveniles and are piscivorous, the results also indicate that these species partition habitat. The cownose ray has been thought of as migratory throughout its range, but these data indicate that non-migratory, estuarine populations exist at lower latitudes. Collectively, these results will aid in the development of management decisions regarding these species and in improving long-term recovery planning for the smalltooth sawfish.

Background: Spawning migrations are a widespread phenomenon among fishes, often occurring in response to environmental conditions prompting movement into reproductive habitats (migratory cues). However, for many species, individual fish may choose not to migrate, and research suggests that conditions preceding the spawning season (migratory primers) may influence this decision. Few studies have provided empirical descriptions of these prior conditions, partly due to a lack of long-term data allowing for robust multi-year comparisons. To investigate how primers and cues interact to shape the spawning migrations of coastal fishes, we use acoustic telemetry data from Common Snook (Centropomus undecimalis) in Everglades National Park, Florida, USA. A contingent of Snook migrate between rivers and coastal spawning sites, varying annually in both the proportion of the population that migrates and the timing of migration within the spawning season. However, the specific environmental factors that serve as migratory primers and cues remain unknown., Methods: We used eight years of acoustic telemetry data (2012-2019) from 173 tagged Common Snook to investigate how primers and cues influence migratory patterns at different temporal scales. We hypothesize that (1) interannual differences in hydrologic conditions preceding the spawning season contribute to the number of individuals migrating each year, and (2) specific environmental cues trigger the timing of migrations during the spawning season. We used GLMMs to model both the annual and seasonal migratory response in relation to flow characteristics (water level, rate of change in water level), other hydrologic/abiotic conditions (temperature, salinity), fish size, and phenological cues independent of riverine conditions (photoperiod, lunar cycle)., Results: We found that the extent of minimum marsh water level prior to migration and fish size influence the proportion of Snook migrating each year, and that high river water level and daily rates of change serve as primary cues triggering migration timing., Conclusion: Our findings illustrate how spawning migrations are shaped by environmental factors acting at different temporal scales and emphasize the importance of long-term movement data in understanding these patterns. Research providing mechanistic descriptions of conditions that promote migration and reproduction can help inform management decisions aimed at conserving ecologically and economically important species., (© 2022. The Author(s).)

Microtubule-associated proteins (MAPs) modulate the motility of kinesin and dynein along microtubules to control the transport of vesicles and organelles. The neuronal MAP tau inhibits kinesin-dependent transport. Phosphorylation of tau at Tyr-18 by fyn kinase results in weakened inhibition of kinesin-1. We examined the motility of early endosomes and lysosomes in cells expressing wild-type (WT) tau and phosphomimetic Y18E tau. We quantified the effects on motility as a function of the tau expression level. Lysosome motility is strongly inhibited by tau. Y18E tau preferentially inhibits lysosomes in the cell periphery, while centrally located lysosomes are less affected. Early endosomes are more sensitive to tau than lysosomes and are inhibited by both WT and Y18E tau. Our results show that different cargoes have disparate responses to tau, likely governed by the types of kinesin motors driving their transport. In support of this model, kinesin-1 and -3 are strongly inhibited by tau while kinesin-2 and dynein are less affected. In contrast to kinesin-1, we find that kinesin-3 is strongly inhibited by phosphorylated tau.

Climate change is transforming ecosystems and affecting ecosystem goods and services. Along the Gulf of Mexico and Atlantic coasts of the southeastern United States, the frequency and intensity of extreme freeze events greatly influence whether coastal wetlands are dominated by freeze-sensitive woody plants (mangrove forests) or freeze-tolerant grass-like plants (salt marshes). In response to warming winters, mangroves have been expanding and displacing salt marshes at varying degrees of severity in parts of north Florida, Louisiana, and Texas. As winter warming accelerates, mangrove range expansion is expected to increasingly modify wetland ecosystem structure and function. Because there are differences in the ecological and societal benefits that salt marshes and mangroves provide, coastal environmental managers are challenged to anticipate the effects of mangrove expansion on critical wetland ecosystem services, including those related to carbon sequestration, wildlife habitat, storm protection, erosion reduction, water purification, fisheries support, and recreation. Mangrove range expansion may also affect wetland stability in the face of extreme climatic events and rising sea levels. Here, we review the current understanding of the effects of mangrove range expansion and displacement of salt marshes on wetland ecosystem services in the southeastern United States. We also identify critical knowledge gaps and emerging research needs regarding the ecological and societal implications of salt marsh displacement by expanding mangrove forests. One consistent theme throughout our review is that there are ecological trade-offs for consideration by coastal managers. Mangrove expansion and marsh displacement can produce beneficial changes in some ecosystem services, while simultaneously producing detrimental changes in other services. Thus, there can be local-scale differences in perceptions of the impacts of mangrove expansion into salt marshes. For very specific local reasons, some individuals may see mangrove expansion as a positive change to be embraced, while others may see mangrove expansion as a negative change to be constrained., (© 2022 John Wiley & Sons Ltd. This article has been contributed to by US Government employees and their work is in the public domain in the USA.)

Tropicalization is a term used to describe the transformation of temperate ecosystems by poleward-moving tropical organisms in response to warming temperatures. In North America, decreases in the frequency and intensity of extreme winter cold events are expected to allow the poleward range expansion of many cold-sensitive tropical organisms, sometimes at the expense of temperate organisms. Although ecologists have long noted the critical ecological role of winter cold temperature extremes in tropical-temperate transition zones, the ecological effects of extreme cold events have been understudied, and the influence of warming winter temperatures has too often been left out of climate change vulnerability assessments. Here, we examine the influence of extreme cold events on the northward range limits of a diverse group of tropical organisms, including terrestrial plants, coastal wetland plants, coastal fishes, sea turtles, terrestrial reptiles, amphibians, manatees, and insects. For these organisms, extreme cold events can lead to major physiological damage or landscape-scale mass mortality. Conversely, the absence of extreme cold events can foster population growth, range expansion, and ecological regime shifts. We discuss the effects of warming winters on species and ecosystems in tropical-temperate transition zones. In the 21st century, climate change-induced decreases in the frequency and intensity of extreme cold events are expected to facilitate the poleward range expansion of many tropical species. Our review highlights critical knowledge gaps for advancing understanding of the ecological implications of the tropicalization of temperate ecosystems in North America., (© 2021 John Wiley & Sons Ltd.)

Increasing coastal populations and urban development have led to the loss of estuarine habitats for fish and wildlife. Specifically, a decline in complexity and heterogeneity of tidal marshes and creeks is thought to negatively impact fish communities by altering the function of nursery grounds, including predator refuge and prey resources. To offset these impacts, numerous agencies are restoring degraded habitats while also creating new ones where habitat has been lost. To improve understanding of what contributes to a successful restoration, six quarterly sampling events using two gear types to collect small- and large-bodied fishes were conducted to compare the fish community structure and habitat characteristics at three natural, three restored, and three impacted (i.e. ditched) areas along the coast of Tampa Bay, Florida. Overall, impacted sites had significantly lower small-bodied and juvenile fish diversity than natural and restored areas, while restored sites harbored a greater number of fish species than impacted sites for both large- and small-bodied fish. Habitat features such as shoreline slope differentiated impacted and restored from natural areas. Although we did not find a direct correlation, habitat heterogeneity likely played a role in structuring fish communities. These findings provide guidance for future coastal restoration or modification of existing projects. Specifically, the habitat mosaic approach of creating a geographically compact network of heterogenous habitat characteristics is likely to support fish diversity, while decreasing shoreline slope in a greater amount of area within coastal wetland restorations would more closely mimic natural areas., Competing Interests: The authors have declared that no competing interests exist.

To aid recovery efforts of smalltooth sawfish (Pristis pectinata) populations in U.S. waters a research project was developed to assess how changes in environmental conditions within estuarine areas affected the presence, movements, and activity space of this endangered species. Forty juvenile P. pectinata were fitted with acoustic tags and monitored within the lower 27 km of the Caloosahatchee River estuary, Florida, between 2005 and 2007. Sawfish were monitored within the study site from 1 to 473 days, and the number of consecutive days present ranged from 1 to 125. Residency index values for individuals varied considerably, with annual means highest in 2005 (0.95) and lowest in 2007 (0.73) when several P. pectinata moved upriver beyond detection range during drier conditions. Mean daily activity space was 1.42 km of river distance. The distance between 30-minute centers of activity was typically <0.1 km, suggesting limited movement over short time scales. Salinity electivity analysis demonstrated an affinity for salinities between 18 and at least 24 psu, suggesting movements are likely made in part, to remain within this range. Thus, freshwater flow from Lake Okeechobee (and its effect on salinity) affects the location of individuals within the estuary, although it remains unclear whether or not these movements are threatening recovery.

Here we report proof-of-principle for a microsphere-based genotyping assay that detects single nucleotide polymorphisms (SNPs) directly from human genomic DNA samples. This assay is based on a structure-specific cleavage reaction that achieves single base discrimination with a 5'-nuclease which recognizes a tripartite substrate formed upon hybridization of target DNA with probe and upstream oligonucleotides. The assay is simple with two easy steps: a cleavage reaction, which generates fluorescent signal on microsphere surfaces, followed by flow cytometry analysis of the microspheres. Genomic DNA samples were genotyped for the SNP in the Apolipoprotein E gene at amino acid position 158. The assay successfully scored wild type, heterozygous and homozygous mutants. To our knowledge, this is the first report of a solid-support assay for detection of SNPs directly from genomic DNA without PCR amplification of the target.

Combining positive attributes of planar arrays and suspension arrays, immobilized particle arrays offer a new format in which immobilized submicrometer particles are arrayed on hydrogel-coated slides, providing 100+ assay replicates within each spot. This research describes how to prepare immobilized protein arrays and how to assay the binding of labeled target molecules to the arrayed capture probes. The assay system exhibits an intrinsic dynamic range of two to three decades, with coefficients of variation from 5 to 10%. For antibody-antigen binding, target capture appears to be reaction rate limited. For labeled antibody binding to antigen on the immobilized particles, the detection limit is approximately 0.5 ng/mL. When antibodies on the immobilized particles exhibit multivalent binding of target molecules, the detection limit is approximately 0.01 ng/mL. For protein arrays, potential advantages of this format are improved coating of the capture reagent, an increased number of options for protein presentation, reduced mass transport effects, and higher density multiplexing.

An automated imaging system was developed to quantify fluorescence signals from particles immobilized on hydrogel-coated slides. Arrays of submicrometer-diameter particles were printed with up to 600 particles/spot. The slides were read under 20x magnification without cover slips. Software was written to image individual spots and measure the median particle fluorescence in each spot. To locate array spots, an alignment program made use of two fiducial grids of fluorescent reference particles at either end of the slide. Focusing was adjusted locally using spots of reference particles located at the centers of focusing neighborhoods. The response was linear across a two-decade range, and the precision of readings was better than 5% down to approximately 1000 fluors/particle. Exposure times varied with signal intensity, reaching 1 s at the lowest levels of fluorescence. Data demonstrate feasibility for measuring fluorescence from immobilized particle arrays on an automated microscope with accuracy and precision similar to fluorescence measurements of microparticles with a flow cytometer. This work provides automation of imaging and analysis procedures necessary for development of immobilized particle arrays as an analytical platform that combines advantageous features of both planar and suspension arrays.

The structure-specific invasive cleavage reaction is a useful means for sensitive and specific detection of single nucleotide polymorphisms, or SNPs, directly from genomic DNA without a need for prior target amplification. A new approach integrating this invasive cleavage assay and surface DNA array technology has been developed for potentially large-scale SNP scoring in a parallel format. Two surface invasive cleavage reaction strategies were designed and implemented for a model SNP system in codon 158 of the human ApoE gene. The upstream oligonucleotide, which is required for the invasive cleavage reaction, is either co-immobilized on the surface along with the probe oligonucleotide or alternatively added in solution. The ability of this approach to unambiguously discriminate a single base difference was demonstrated using PCR-amplified human genomic DNA. A theoretical model relating the surface fluorescence intensity to the progress of the invasive cleavage reaction was developed and agreed well with experimental results.

The structure-specific invasive cleavage of single-stranded DNA by 5' nucleases is a useful means for sensitive detection of single-nucleotide polymorphisms or SNPs. The solution-phase invasive cleavage reaction has sufficient sensitivity for direct detection of as few as 600 target molecules with no prior target amplification. One approach to the parallelization of SNP analysis is to adapt the invasive cleavage reaction to an addressed array format. Two surface invasive cleavage reaction strategies were designed and tested using the polymorphic site in codon 158 of the human ApoE gene as a model system, with a synthetic oligonucleotide as target. The upstream oligonucleotide, which is required for the invasive cleavage reaction, was either added in solution (strategy 1), or co-immobilized on the surface along with the probe oligonucleotide (strategy 2). Both strategies showed target-concentration and time-dependent amplification of signal. Parameters that govern the rate of the surface-invasive cleavage reactions are discussed., (Copyright 2002 Wiley-Liss, Inc.)

Expression of the human apoptosis modulator protein Bax in Escherichia coli is highly toxic, resulting in cell lysis at very low concentrations (Asoh, S., et al., J. Biol. Chem. 273, 11384-11391, 1998). Attempts to express a truncated form of murine Bax in the periplasm by using an expression vector that attached the OmpA signal sequence to the protein failed to alleviate this toxicity. In contrast, attachment of a peptide based on a portion of the E. coli cochaperone GroES reduced Bax's toxicity significantly and allowed good expression. The peptide, which was attached to the N-terminus, included the amino acid sequence of the mobile loop of GroES that has been demonstrated to interact with the chaperonin, GroEL. Under normal growth conditions, expression of this construct was still toxic, but generated a small amount of detectable recombinant Bax. However, when cells were grown in the presence of 2% ethanol, which stimulated overproduction of the molecular chaperones GroEL and DnaK, toxicity was reduced and good overexpression occurred. Two-dimensional gel electrophoresis analysis showed that approximately 15-fold more GroES-loop-Bax was produced under these conditions than under standard conditions and that GroEL and DnaK were elevated approximately 3-fold., (Copyright 2001 Academic Press.)

Background: The self-assembly properties of beta domains are important features of diverse classes of proteins that include cell-adhesion molecules, surface receptors and the immunoglobulin superfamily. Immunoglobulin light-chain variable domains are well suited to the study of structural factors that determine dimerization, including how residues at the interface influence the preferred dimer arrangement., Results: Single-site mutants of light-chain variable domain Len, designated LenQ38E and LenK30T, formed 'flipped' dimers in which one domain was rotated by about 180 degrees compared with the native protein. The dimer in the native protein is similar to that found between variable domains in Fab immunoglobulin fragments. When compared to the native dimer, more surface area is buried, and more hydrogen bonds and salt bridges are formed between the monomers in the flipped conformation., Conclusions: Immunoglobulin light-chain variable domains can form a minimum of two distinct quaternary structures. Single-site mutations resulting from changes of one base, such as the exchange of Gln38 to Glu or Lys30 to Thr, change the 'conventional' dimer of protein Len to a flipped arrangement. Native Len is not found in the flipped-domain dimer conformation because it would have excess positive electrostatic potential at the dimer interface that is not compensated by other forces. Excess negative or positive electrostatic potential at the dimer interface can have a determining effect on the mode of dimerization.

The formation of the antibody variable domain binding unit (Fv) is the net result of three competing assembly reactions. The affinities of concurrent homologous interactions of heavy and light chain variable domains limits the heterologous interaction leading to productive formation of the Fv. To address the possible role of light chain dimerization in this phenomenon, the Gln38 residue at the dimer interface of an immunoglobulin light chain variable domain (VL) was replaced by charged amino acids. The effects of these mutations on VL homodimer formation were monitored by small-zone size exclusion HPLC and the affinities of interaction were determined by computer simulation. Reduced VL homodimerization was observed in three of the four mutants, Q38R, Q38D and Q38K. The association constants for the Q38R and Q38D homodimers were 1.2 x 10(4) and 3.2 x 10(3) M(-1), respectively. This corresponded to a 20-75-fold reduction in the homodimer association constant relative to the wild-type VL, which had an association constant of 2.4 x 10(5) M(-1). Surprisingly, the fourth charge mutant, Q38E, had a higher association constant than the wild-type VL. The potential for charged residues to facilitate heterodimeric assembly of immunoglobulin domains was also tested. Heterodimerization was observed between the Q38D and Q38R V(L)s, but with an association constant of 4.7 x 10(4) M(-1), approximately fivefold lower than that obtained for homodimerization of the native V(L). In addition, replacement of the neutral, solvent-accessible Gln38 residue with either Asp or Arg was found to be significantly destabilizing. These results suggest that charged residues could be introduced at immunoglobulin domain interfaces to guide heterodimer formation and to minimize unfavorable competing homologous associations. Nonetheless, these apparently simple modifications may also result in unintended consequences that are likely to depend upon structural features of particular variable domains.

Malate dehydrogenase and lactate dehydrogenase are members of the structurally and functionally homologous family of 2-ketoacid dehydrogenases. Both enzymes display high specificity for their respective keto substrates, oxaloacetate and pyruvate. Closer analysis of their specificity, however, reveals that the specificity of malate dehydrogenase is much stricter and less malleable than that of lactate dehydrogenase. Site-specific mutagenesis of the two enzymes in an attempt to reverse their specificity has met with contrary results. Conversion of a specific active-site glutamine to arginine in lactate dehydrogenase from Bacillus stearothermophilus generated an enzyme that displayed activity toward oxaloacetate equal to that of the native enzyme toward pyruvate (H. M. Wilks et al. (1988) Science 242, 1541-1544). We have constructed a series of mutants in the mobile, active site loop of the Escherichia coli malate dehydrogenase that incorporate the complementary change, conversion of arginine 81 to glutamine, to evaluate the role of charge distribution and conformational flexibility within this loop in defining the substrate specificity of these enzymes. Mutants incorporating the change R81Q all had reversed specificity, displaying much higher activity toward pyruvate than to the natural substrate, oxaloacetate. In contrast to the mutated lactate dehydrogenase, these reversed-specificity mutants were much less active than the native enzyme. Secondary mutations within the loop of the E. coli enzyme (A80N, A80P, A80P/M85E/D86T) had either no or only moderately beneficial effects on the activity of the mutant enzyme toward pyruvate. The mutation A80P, which can be expected to reduce the overall flexibility of the loop, modestly improved activity toward pyruvate. The possible physiological relevance of the stringent specificity of malate dehydrogenase was investigated. In normal strains of E. coli, fermentative metabolism was not affected by expression of the mutant malate dehydrogenase. However, when expressed in a strain of E. coli unable to ferment glucose, the mutant enzyme restored growth and produced lactic acid as the sole fermentation product.

Originally developed as a method for estimating the solid-phase concentration of an adsorbed protein, sequential enzyme-linked immunosorbent assay (ELISA) analysis (P. A. Underwood and J. G. Steele (1991) J. Immunol. Methods 142, 83-94) is also useful for assessing other aspects of protein-surface interactions without radioactively labeled reagents. The method provides a way to compare protein adsorption on various microwell surfaces and to track adhesion of the protein throughout an assay's wash and incubation steps. In cases where adsorption complies with Langmuir-type binding and protein desorption is minimal, sequential ELISA data can be analyzed with a mathematical model to quantitatively estimate the microwell's protein-binding capacity. Even for protein-surface interactions where the mathematical model is not appropriate, characteristics of sequential ELISA binding data qualitatively differentiate between initial adsorption with subsequent desorption and poor initial adsorption of the protein. Using this method, we analyzed the binding of three proteins (rabbit immunoglobulin G, bovine serum albumin, and horse spleen ferritin) to four types of polystyrene microwell surfaces (Immulons 1, 2, 3, and 4). In addition, we examined differences in protein adhesion to the four surfaces when Tween 20 was omitted from the assay's wash buffer or when fewer washes were used.

The primary structural features that render human monoclonal light chains amyloidogenic are presently unknown. To gain further insight into the physical and biochemical factors that result in the pathologic deposition of these proteins as amyloid fibrils, we have selected for detailed study three closely homologous protein products of the light-chain variable-region single-gene family VkIV. Two of these proteins, REC and SMA, formed amyloid fibrils in vivo. The third protein, LEN, was excreted by the patient at levels of 50 g/day with no indication of amyloid deposits. Sequences of amyloidogenic proteins REC and SMA differed from the sequence of the nonpathogenic protein LEN at 14 and 8 amino acid positions, respectively, and these amino acid differences have been analyzed in terms of the three-dimensional structure of the LEN dimer. To provide a replenishable source of these human proteins, we constructed synthetic genes coding for the REC, SMA, and LEN variable domains and expressed these genes in Escherichia coli. Immunochemical and biophysical comparisons demonstrated that the recombinant VkIV products have tertiary structural features comparable to those of the patient-derived proteins. This well-defined set of three clinically characterized human kIV light chains, together with the capability to produce these kIV proteins recombinantly, provide a system for biophysical and structural comparisons of two different amyloidogenic light-chain proteins and a nonamyloidogenic protein of the same subgroup. This work lays the foundation for future investigations of the structural basis of light-chain amyloidogenicity.

A non-linear mathematical method for estimating the protein binding capacity of a microwell is developed by applying Langmuir-type binding principles to data obtained from sequential ELISAs (Underwood and Steele (1991) J. Immunol. Methods 142, 83). Experimental data from sequential ELISAs measuring the binding of rabbit and mouse immunoglobulin G and of BSA to Immulon 2 microwells agree well with the binding curves predicted by our simple mathematical model. Estimates of Immulon 2 microwell capacity for these proteins when coated in a 100 microliters/well volume are between 250 and 325 ng/well. The method provides a convenient, quantitative method for estimating microwell capacity without the use of radioisotopes. A spreadsheet form of the method is also presented, so that the mathematical calculations for any set of experimental data may be performed readily using the spreadsheet's SOLVER function. The method is not applicable to data from experiments where significant desorption occurs or where binding to the solid phase deviates significantly from the simple Langmuir model.

Gamma and kappa chain cDNAs from four mouse monoclonal antibodies (mAbs) which bind three different sites on the core antigen (p24) of HIV-1 have been cloned and their V-region sequences determined. These mAbs are part of a larger group of seven anti-p24 mAbs analyzed in simultaneous competition assays with HIV-1 lysate as antigen and in protein blotting experiments using 10 carboxy-terminal truncations of a p24 fusion protein. One mAb, BB128, recognizes the p24 loop sequence EAAEWDRVHP and enhances the binding of two other mAbs (BI1777 and BI1279) when tested pairwise in simultaneous competition assays. The two monoclonals enhanced by BB128 recognize different antigenic sites on p24, with BI1777 binding to carboxy-terminal sequences and BI1279 to amino-terminal residues. In the pairwise assays, mAb BI1279 also acts as enhancing antibody for BI1777, as does mAb BB328, which recognizes residues in the central region of p24. Since aggregated p24 monomers form the HIV-1 capsid, p24 is a multivalent antigen in HIV-1 lysate. It seems likely, therefore, that synergistic binding of mAb pairs to p24 is effected by bivalent binding of the enhancing mAb stabilizing a conformation favorable for bivalent binding of the enhanced mAb.

A second-stage mutant of Escherichia coli K12 designated as strain 806 grew faster on D-lyxose than the mutant strain 805 previously described. Both mutants produced constitutively a novel enzyme, D-mannose isomerase, but strain 806 produced twice as much as strain 805. The enzyme could fortuitously convert D-lyxose to D-xylulose, which is a normal intermediate in the D-xylose catabolic pathway. The purified enzyme consisted of four subunits each with a molecular weight of about 40 000. In 0.14 M-Na2SO4, the tetramer dissociated completely into dimers. While the tetramer Km values for D-mannose and D-lyxose were 80 mM and 300 mM, respectively, the dimer Km values for these two sugars were both 300 mM. The amino acid composition of the enzyme was also determined.

The nucleotide sequences of two chicken histone genes encoding replacement variant H3.3 polypeptides are described. Unlike the replication variant genes of chickens (and almost all other organisms), these genes contain intervening sequences; introns are present in both genes in the 5' noncoding and coding sequences. Furthermore, the replacement variant histone mRNAs are post-transcriptionally polyadenylated. The locations, but not the sizes, of the two introns within the coding segments of the two genes have been exactly conserved, whereas the intron positions in their respective 5' flanking regions differ. Although both H3.3 genes predict the identical histone polypeptide sequence, they are as different from one another as each of them is from a more common replication variant H3.2 gene in silent base substitutions within the coding sequences. Thus, the H3.3 polypeptide sequence has been precisely maintained over a great evolutionary period, suggesting that this class of histones performs a strongly selected biological function. Although replacement variant histones can account for more than 50% of the total H3 protein in the nuclei of specific chicken tissues, the steady-state level of H3.3 mRNA is nearly the same (and is quite low) in all tissues and ages of animals examined. These properties suggest novel mechanisms for the control of the basal histone biosynthesis which takes place outside of the S phase of the cell cycle.

Although the genomes of many species contain multiple copies of ferritin heavy (H)- and light (L)-chain sequences, the chicken genome contains only a single copy of the H-subunit gene. The primary transcription unit of this gene is 4.6 kilobase pairs and contains four exons which are posttranscriptionally spliced to generate a mature transcript of 869 nucleotides. Chicken and human ferritin H-subunit genomic loci are organized with similar exon-intron boundaries. They exhibit approximately 85% nucleotide identity in coding regions, which yield proteins 93% identical in amino acid sequence. We have identified a sequence of 22 highly conserved nucleotides in the 5' untranslated sequences of chicken, human, and tadpole ferritin H-subunit genes and propose that this conserved sequence may regulate iron-modulated translation of ferritin H-subunit mRNAs.