Production, specificity, and functionality of monoclonal antibodies to specific peptide–major histocompatibility complex class II complexes formed by processing of exogenous protein

Several unanswered questions in T cell immunobiology relating to intracellular processing or in vivo antigen presentation could be approached if convenient, specific, and sensitive reagents were available for detecting the peptide–major histocompatibility complex (MHC) class I or class II ligands recognized by αβ T cell receptors. For this reason, we have developed a method using homogeneously loaded peptide–MHC class II complexes to generate and select specific mAb reactive with these structures using hen egg lysozyme (HEL) and I-Ak as a model system. mAbs specific for either HEL-(46–61)–Ak or HEL-(116–129)–Ak have been isolated. They cross-react with a small subset of I-Ak molecules loaded with self peptides but can nonetheless be used for flow cytometry, immunoprecipitation, Western blotting, and intracellular immunofluorescence to detect specific HEL peptide–MHC class II complexes formed by either peptide exposure or natural processing of native HEL. An example of the utility of these reagents is provided herein by using one of the anti-HEL-(46–61)–Ak specific mAbs to visualize intracellular compartments where I-Ak is loaded with HEL-derived peptides early after antigen administration. Other uses, especially for in vivo tracking of specific ligand-bearing antigen-presenting cells, are discussed.

Syntax processing by auditory cortical neurons in the FM–FM area of the mustached bat Pteronotus parnellii

Syntax denotes a rule system that allows one to predict the sequencing of communication signals. Despite its significance for both human speech processing and animal acoustic communication, the representation of syntactic structure in the mammalian brain has not been studied electrophysiologically at the single-unit level. In the search for a neuronal correlate for syntax, we used playback of natural and temporally destructured complex species-specific communication calls—so-called composites—while recording extracellularly from neurons in a physiologically well defined area (the FM–FM area) of the mustached bat’s auditory cortex. Even though this area is known to be involved in the processing of target distance information for echolocation, we found that units in the FM–FM area were highly responsive to composites. The finding that neuronal responses were strongly affected by manipulation in the time domain of the natural composite structure lends support to the hypothesis that syntax processing in mammals occurs at least at the level of the nonprimary auditory cortex.

On the spurious endoproteolytic processing of the presenilin proteins in cultured cells and tissues

It has been widely reported that the presenilin proteins PS-1 and PS-2 in extracts derived from a variety of cultured cells and from tissues are fragmented extensively by endoproteolytic processing events. It generally has been presumed that this endoproteolysis is a physiologically normal intracellular event following presenilin expression, which might play an important role in the still unknown functions of these molecules in connection with Alzheimer disease. We demonstrate herein, however, that, if a variety of cultured cells and several mouse tissues are examined under conditions minimizing cell trauma, the presenilin molecules in the extracts are found to be intact but that, if the cells and tissues are prepared under somewhat more stressful conditions, the endoproteolytic fragments are then observed. We conclude that these particular endoproteolytic events are not the result of physiologically normal processing of the presenilins but are rather artifacts occurring during the common procedures of specimen preparation.

Enzymatic processing of DNA containing tandem dihydrouracil by endonucleases III and VIII

Endonuclease III from Escherichia coli, yeast (yNtg1p and yNtg2p) and human and E.coli endonuclease VIII have a wide substrate specificity, and recognize oxidation products of both thymine and cytosine. DNA containing single dihydrouracil (DHU) and tandem DHU lesions were used as substrates for these repair enzymes. It was found that yNtg1p prefers DHU/G and exhibits much weaker enzymatic activity towards DNA containing a DHU/A pair. However, yNtg2p, E.coli and human endonuclease III and E.coli endonuclease VIII activities were much less sensitive to the base opposite the lesion. Although these enzymes efficiently recognize single DHU lesions, they have limited capacity for completely removing this damaged base when DHU is present on duplex DNA as a tandem pair. Both E.coli endonuclease III and yeast yNtg1p are able to remove only one DHU in DNA containing tandem lesions, leaving behind a single DHU at either the 3′- or 5′-terminus of the cleaved fragment. On the other hand, yeast yNtg2p can remove DHU remaining on the 5′-terminus of the 3′ cleaved fragment, but is unable to remove DHU remaining on the 3′-terminus of the cleaved 5′ fragment. In contrast, both human endonuclease III and E.coli endonuclease VIII can remove DHU remaining on the 3′-terminus of a cleaved 5′ fragment, but are unable to remove DHU remaining on the 5′-terminus of a cleaved 3′ fragment. Tandem lesions are known to be generated by ionizing radiation and agents that generate reactive oxygen species. The fact that these repair glycosylases have only a limited ability to remove the DHU remaining at the terminus suggests that participation of other repair enzymes is required for the complete removal of tandem lesions before repair synthesis can be efficiently performed by DNA polymerase.

Localization of CD4+ T cell epitope hotspots to exposed strands of HIV envelope glycoprotein suggests structural influences on antigen processing

The spectrum of immunogenic epitopes presented by the H2-IAb MHC class II molecule to CD4+ T cells has been defined for two different (clade B and clade D) HIV envelope (gp140) glycoproteins. Hybridoma T cell lines were generated from mice immunized by a sequential prime and boost regime with DNA, recombinant vaccinia viruses, and protein. The epitopes recognized by reactive T cell hybridomas then were characterized with overlapping peptides synthesized to span the entire gp140 sequence. Evidence of clonality also was assessed with antibodies to T cell receptor Vα and Vβ chains. A total of 80 unique clonotypes were characterized from six individual mice. Immunogenic peptides were identified within only four regions of the HIV envelope. These epitope hotspots comprised relatively short sequences (≈20–80 aa in length) that were generally bordered by regions of heavy glycosylation. Analysis in the context of the gp120 crystal structure showed a pattern of uniform distribution to exposed, nonhelical strands of the protein. A likely explanation is that the physical location of the peptide within the native protein leads to differential antigen processing and consequent epitope selection.

The 3′→5′ exonuclease of DNA polymerase δ can substitute for the 5′ flap endonuclease Rad27/Fen1 in processing Okazaki fragments and preventing genome instability

Many DNA polymerases (Pol) have an intrinsic 3′→5′ exonuclease (Exo) activity which corrects polymerase errors and prevents mutations. We describe a role of the 3′→5′ Exo of Pol δ as a supplement or backup for the Rad27/Fen1 5′ flap endonuclease. A yeast rad27 null allele was lethal in combination with Pol δ mutations in Exo I, Exo II, and Exo III motifs that inactivate its exonuclease, but it was viable with mutations in other parts of Pol δ. The rad27-p allele, which has little phenotypic effect by itself, was also lethal in combination with mutations in the Pol δ Exo I and Exo II motifs. However, rad27-p Pol δ Exo III double mutants were viable. They exhibited strong synergistic increases in CAN1 duplication mutations, intrachromosomal and interchromosomal recombination, and required the wild-type double-strand break repair genes RAD50, RAD51, and RAD52 for viability. Observed effects were similar to those of the rad27-null mutant deficient in the removal of 5′ flaps in the lagging strand. These results suggest that the 3′→5′ Exo activity of Pol δ is redundant with Rad27/Fen1 for creating ligatable nicks between adjacent Okazaki fragments, possibly by reducing the amount of strand-displacement in the lagging strand.

Covert attention accelerates the rate of visual information processing

Whenever we open our eyes, we are confronted with an overwhelming amount of visual information. Covert attention allows us to select visual information at a cued location, without eye movements, and to grant such information priority in processing. Covert attention can be voluntarily allocated, to a given location according to goals, or involuntarily allocated, in a reflexive manner, to a cue that appears suddenly in the visual field. Covert attention improves discriminability in a wide variety of visual tasks. An important unresolved issue is whether covert attention can also speed the rate at which information is processed. To address this issue, it is necessary to obtain conjoint measures of the effects of covert attention on discriminability and rate of information processing. We used the response-signal speed-accuracy tradeoff (SAT) procedure to derive measures of how cueing a target location affects speed and accuracy in a visual search task. Here, we show that covert attention not only improves discriminability but also accelerates the rate of information processing.

The postnatal development of spinal sensory processing

The mechanisms by which infants and children process pain should be viewed within the context of a developing sensory nervous system. The study of the neurophysiological properties and connectivity of sensory neurons in the developing spinal cord dorsal horn of the intact postnatal rat has shed light on the way in which the newborn central nervous system analyzes cutaneous innocuous and noxious stimuli. The receptive field properties and evoked activity of newborn dorsal horn cells to single repetitive and persistent innocuous and noxious inputs are developmentally regulated and reflect the maturation of excitatory transmission within the spinal cord. These changes will have an important influence on pain processing in the postnatal period.

Subdivisions of auditory cortex and processing streams in primates

The auditory system of monkeys includes a large number of interconnected subcortical nuclei and cortical areas. At subcortical levels, the structural components of the auditory system of monkeys resemble those of nonprimates, but the organization at cortical levels is different. In monkeys, the ventral nucleus of the medial geniculate complex projects in parallel to a core of three primary-like auditory areas, AI, R, and RT, constituting the first stage of cortical processing. These areas interconnect and project to the homotopic and other locations in the opposite cerebral hemisphere and to a surrounding array of eight proposed belt areas as a second stage of cortical processing. The belt areas in turn project in overlapping patterns to a lateral parabelt region with at least rostral and caudal subdivisions as a third stage of cortical processing. The divisions of the parabelt distribute to adjoining auditory and multimodal regions of the temporal lobe and to four functionally distinct regions of the frontal lobe. Histochemically, chimpanzees and humans have an auditory core that closely resembles that of monkeys. The challenge for future researchers is to understand how this complex system in monkeys analyzes and utilizes auditory information.

Mechanisms and streams for processing of “what” and “where” in auditory cortex

The functional specialization and hierarchical organization of multiple areas in rhesus monkey auditory cortex were examined with various types of complex sounds. Neurons in the lateral belt areas of the superior temporal gyrus were tuned to the best center frequency and bandwidth of band-passed noise bursts. They were also selective for the rate and direction of linear frequency modulated sweeps. Many neurons showed a preference for a limited number of species-specific vocalizations (“monkey calls”). These response selectivities can be explained by nonlinear spectral and temporal integration mechanisms. In a separate series of experiments, monkey calls were presented at different spatial locations, and the tuning of lateral belt neurons to monkey calls and spatial location was determined. Of the three belt areas the anterolateral area shows the highest degree of specificity for monkey calls, whereas neurons in the caudolateral area display the greatest spatial selectivity. We conclude that the cortical auditory system of primates is divided into at least two processing streams, a spatial stream that originates in the caudal part of the superior temporal gyrus and projects to the parietal cortex, and a pattern or object stream originating in the more anterior portions of the lateral belt. A similar division of labor can be seen in human auditory cortex by using functional neuroimaging.

Spatial processing in the auditory cortex of the macaque monkey

The patterns of cortico-cortical and cortico-thalamic connections of auditory cortical areas in the rhesus monkey have led to the hypothesis that acoustic information is processed in series and in parallel in the primate auditory cortex. Recent physiological experiments in the behaving monkey indicate that the response properties of neurons in different cortical areas are both functionally distinct from each other, which is indicative of parallel processing, and functionally similar to each other, which is indicative of serial processing. Thus, auditory cortical processing may be similar to the serial and parallel “what” and “where” processing by the primate visual cortex. If “where” information is serially processed in the primate auditory cortex, neurons in cortical areas along this pathway should have progressively better spatial tuning properties. This prediction is supported by recent experiments that have shown that neurons in the caudomedial field have better spatial tuning properties than neurons in the primary auditory cortex. Neurons in the caudomedial field are also better than primary auditory cortex neurons at predicting the sound localization ability across different stimulus frequencies and bandwidths in both azimuth and elevation. These data support the hypothesis that the primate auditory cortex processes acoustic information in a serial and parallel manner and suggest that this may be a general cortical mechanism for sensory perception.

RNA editing activity is associated with splicing factors in lnRNP particles: The nuclear pre-mRNA processing machinery

Multiple members of the ADAR (adenosine deaminases acting on RNA) gene family are involved in A-to-I RNA editing. It has been speculated that they may form a large multicomponent protein complex. Possible candidates for such complexes are large nuclear ribonucleoprotein (lnRNP) particles. The lnRNP particles consist mainly of four spliceosomal subunits that assemble together with the pre-mRNA to form a large particle and thus are viewed as the naturally assembled pre-mRNA processing machinery. Here we investigated the presence of ADARs in lnRNP particles by Western blot analysis using anti-ADAR antibodies and by indirect immunoprecipitation. Both ADAR1 and ADAR2 were found associated with the spliceosomal components Sm and SR proteins within the lnRNP particles. The two ADARs, associated with lnRNP particles, were enzymatically active in site-selective A-to-I RNA editing. We demonstrate the association of ADAR RNA editing enzymes with physiological supramolecular complexes, the lnRNP particles.

Dribble, the Drosophila KRR1p Homologue, Is Involved in rRNA Processing

The Drosophila dribble (dbe) gene encodes a KH domain protein, homologous to yeast KRR1p. Expression of dbe transcripts is ubiquitous during embryogenesis. Overexpressed Dribble protein is localized in the nucleus and in some cell types in a subregion of the nucleolus. Homozygous dbe mutants die at first instar larval stage. Clonal analyses suggest that dbe+ is required for survival of dividing cells. In dbe mutants, a novel rRNA-processing defect is found and accumulation of an abnormal rRNA precursor is detected.