Identification of thaumatin-like protein and aspartyl protease as new major allergens in lettuce (Lactuca sativa)

Scope: Today, about 2–8% of the population of Western countries exhibits some type of food allergy whose impact ranges from localized symptoms confined to the oral mucosa to severe anaphylactic reactions. Consumed worldwide, lettuce is a Compositae family vegetable that can elicit allergic reactions. To date, however, only one lipid transfer protein has been described in allergic reaction to lettuce. The aim of this study was to identify potential new allergens involved in lettuce allergy. Methods and results: Sera from 42 Spanish lettuce-allergic patients were obtained from pa-tients recruited at the outpatient clinic. IgE-binding proteins were detected by SDS-PAGE and immunoblotting. Molecular characterization of IgE-binding bands was performed by MS. Thaumatin was purified using the Agilent 3100 OFFGEL system. The IgE-binding bands recognized in the sera of more than 50% of patients were identified as lipid transfer protein (9 kDa), a thaumatin-like protein (26 kDa), and an aspartyl protease (35 and 45 kDa). ELISA inhibition studies were performed to confirm the IgE reactivity of the purified allergen. Conclusion: Two new major lettuce allergens—a thaumatin-like protein and an aspartyl protease—have been identified and characterized. These allergens may be used to improve both diagnosis and treatment of lettuce-allergic patients.

The effect of a mono component protease and different AMEn levels in broiler diets on growth performance from 1 to 18 days of age.

The objective of the present study was to evaluate the effect of the inclusion of a mono component serine protease (RONOZYME ProAct, DSM Nutritional Products) in diets with two different AMEn contents on apparent ileal digestibility (AID) of amino acids (AA) and growth performance in broilers from 1 to 18 days of age.

The AtCathB3 gene, encoding a cathepsin B-like protease, is expressed during germination of Arabidopsis thaliana and transcriptionally repressed by the basic leucine zipper protein GBF1.

Protein hydrolysis plays an important role during seed germination and post-germination seedling establishment. In Arabidopsis thaliana, cathepsin B-like proteases are encoded by a gene family of three members, but only the AtCathB3 gene is highly induced upon seed germination and at the early post-germination stage. Seeds of a homozygous T-DNA insertion mutant in the AtCathB3 gene have, besides a reduced cathepsin B activity, a slower germination than the wild type. To explore the transcriptional regulation of this gene, we used a combined phylogenetic shadowing approach together with a yeast one-hybrid screening of an arrayed library of approximately 1200 transcription factor open reading frames from Arabidopsis thaliana. We identified a conserved CathB3-element in the promoters of orthologous CathB3 genes within the Brassicaceae species analysed, and, as its DNA-interacting protein, the G-Box Binding Factor1 (GBF1). Transient overexpression of GBF1 together with a PAtCathB3::uidA (β-glucuronidase) construct in tobacco plants revealed a negative effect of GBF1 on expression driven by the AtCathB3 promoter. In stable P35S::GBF1 lines, not only was the expression of the AtCathB3 gene drastically reduced, but a significant slower germination was also observed. In the homozygous knockout mutant for the GBF1 gene, the opposite effect was found. These data indicate that GBF1 is a transcriptional repressor of the AtCathB3 gene and affects the germination kinetics of Arabidopsis thaliana seeds. As AtCathB3 is also expressed during post-germination in the cotyledons, a role for the AtCathB3-like protease in reserve mobilization is also inferred.

C1A Cysteine protease-cystatin interactions in leaf senescence

Senescence-associated proteolysis in plants is a crucial process to relocalize nutrients from leaves to growing or storage tissues. The massive net degradation of proteins involves broad metabolic networks, different subcellular compartments, and several types of proteases and regulators. C1A cysteine proteases, grouped as cathepsin L-, B-, H-, and F-like according to their gene structures and phylogenetic relationships, are the most abundant enzymes responsible for the proteolytic activity during leaf senescence. Besides, cystatins as specific modulators of C1A peptidase activities exert a complex regulatory role in this physiological process. This overview article covers the most recent information on C1A proteases in leaf senescence in different plant species. Particularly, it is focussed on barley, as the unique species where the whole gene family members of C1A cysteine proteases and cystatins have been analysed.

Native bradyrhizobial symbionts of Lupinus mariae-josephae, a unique endemism thriving in alkaline soils in Eastern Spain

Lupinus mariae-josephae (Lmj) es una especie de lupino endémica de una pequeña y específica área de Comunidad Valenciana (Este de España), donde prospera en suelos alcalinoscalcáreos, un hábitat singular para los altramuces, que crecen preferentemente en suelos ácidos o neutros. Esto hace de Lmj una especie de lupino única. Cuando se inició este trabajo, la extensión conocida de este endemismo abarcaba unos 700 kilómetros cuadrados, confinados en la provincia de Valencia. En esta área, Lmj prospera en pequeñas poblaciones aisladas que contienen un número reducido de plantas por lo que se la consideró una especie en peligro de extinción. Todos los esfuerzos, utilizando estrategias clásicas dirigidas a ampliar el área de crecimiento de Lmj y garantizar su conservación, han tenido un éxito limitado. El trabajo que se presenta está dirigido a mejorar el conocimiento de la ecología de Lmj, en particular la interacción simbiótica que establece con bacterias del suelo denominadas rizobios y se centra en la caracterización fenotípica, filogenética y genómica de esos rizobios. También se investiga la posible contribución de la simbiosis en mejorar la conservación de Lmj. Para este fin, se han estudiado diferentes aspectos que se describen a continuación. El primero objetivo se centró en aislar y estudiar de la diversidad genética de las bacterias endosimbióticas de Lmj. . Se realizó un análisis filogenético de genes esenciales que mostró que las cepas de Lmj pertenecen al género Bradyrhizobium y que presentan una gran diversidad con características fenotípicas y simbióticas diferentes de cepas de Bradyrhizobium que nodulan otras especies de lupinos nativos de España (cepas ISLU). Las cepas estudiadas se dividieron en dos grupos (Clado I y Clado II). El Clado I, incluye a las cepas Lmj, definiendo un nuevo linaje, filogenéticamente relacionado con otras especies de Bradyrhizobium, como B. jicamae y B. elkanii. El Clado II contiene cepas ISLU relacionadas con cepas de B. canariense y B. japonicum que establecen simbiosis con lupinos de suelos ácidos. Otro análisis filogenético basado en genes simbióticos, distribuyó las cepas de Lmj en sólo dos grupos diferentes. La singularidad y gran diversidad de estas cepas en una pequeña área geográfica, hacen de este, un atractivo sistema para el estudio de la evolución y adaptación de las bacterias simbióticas a su respectiva planta huésped. Adicionalmente, se estudio la presencia de bacterias capaces de nodular Lmj en suelos básicos de Chiapas, México. Sorprendentemente, estos suelos contienen bacterias capaces establecer interacciones simbióticas eficientes con Lmj en ensayos de invernadero. A continuación se investigó la taxonomía de los endosimbiontes de Lmj analizando la secuencia de cuatro genes esenciales (16S rRNA, recA, glnII y atpD) y el promedio de identidad de nucleótidos de genomas completos de algunas cepas representativas de la diversidad (ANIm). Se identificaron nuevas especies de Bradyrhizobium dentro del Clado I y se definió una de ellas: 'Bradyrhizobium valentinum' sp. nov (cepa tipo LmjM3T = CECT 8364T, LMG 2761T). También se abordó cómo conservar Lmj en su hábitat natural mediante inoculación con alguna de las cepas aisladas. Se demostró la ausencia de bacterias capaces de nodular Lmj en suelos rojos alcalinos o ‘‘terra rossa’’ de la Península Ibérica y Baleares. Dos cepas, altamente eficientes en cuanto a la fijación de nitrógeno, LmjC y LmjM3T, fueron seleccionadas para ser empleadas como inoculantes. Dos experimentos de campo llevados a cabo en años consecutivos en áreas con características edafoclimáticas similares a las que presentan las poblaciones de Lmj, lograron la reproducción exitosa de la planta. Se concluyó que un ciclo reproductivo exitoso de Lmj es absolutamente dependiente de la inoculación con sus simbiontes naturales y que la simbiosis debe ser considerada un factor esencial en estrategias de conservación de leguminosas en peligro. La obtención de varias secuencias genómicas de cepas aisladas de Lmj y de otras cepas de Bradyrhizobium reveló una alta similitud entre los genomas de las cepas del Clado I, y permitió la identificación de cinco posibles nuevas especies. Además, se estudiaron tres agrupaciones de genes relacionados con la simbiosis (nod, nif y fix) definiendo un nuevo linaje para las cepas de Lmj, diferente del symbiovar “genistearum” de B. canariense y B. japonicum. La baja diversidad encontrada en el análisis filogenético de los genes simbióticos contrasta con la gran diversidad asociada a genes esenciales. La presencia de plásmidos en cepas del género Bradyrhizobium ha sido descrita en muy pocas ocasiones, sin embargo el análisis de la secuencia genómica de la cepa ISLU101, aislada de Lupinus angustifolius, reveló la presencia de un origen de replicación extracromosómico homólogo al operón repABC, presente en el plásmido de Bradyrhizobium sp BTAi1. Gracias a esta secuencia se identificaron genes homólogos en 19 de 72 cepas ISLU. Filogenéticamente, las secuencias de repABC se agruparon en un grupo monofilético con las de pBTAi1 y separadas de los rizobios de crecimiento rápido. Finalmente, se identificaron sistemas de secreción de proteínas de tipo III (T3SS) en nueve genomas de cepas de Lmj. Los T3SS pueden inyectar proteínas efectoras al interior de células vegetales. Su presencia en rizobios se ha relacionado con la gama de hospedador que pueden nodular y puede tener un efecto beneficioso, neutro o perjudicial en la simbiosis. Los T3SS de las cepas de Lmj codifican para una proteína efectora similar a NopE, un efector dependiente de T3SS descrito en B. diazoefficiens USDA 110T. La proteína NopE de la cepa LmjC se ha caracterizado bioquímicamente. ABSTRACT Lupinus mariae-josephae (Lmj) is a lupine species endemic of a unique small area in Valencia region (Eastern Spain) where the lupine plants thrive in alkaline-limed soils, which preferentially grow in acid or neutral soils. This is the type of soils native lupines of Spain. When this work was initiated, the extension of the endemic area of Lmj was of about 700 squared kilometers confined to the Valencia province. In this area, Lmj thrives in small, isolated patches containing a reduced number of plants, and points to an endemism that can easily became endangered or extinct. Consequently, the Valencia Community authorities gave a ‘‘microreserve” status for conservation of the species. All efforts, using classical strategies directed to extend the area of Lmj growth and ensure its conservation have been so far unsuccessful. The work presented here is directed to improve our knowledge of Lmj ecology and it is centered in the characterization of the rhizobial symbiosis by phenotypic, phylogenetic and genomic analysis as well as in investigate the potential contribution of the symbiosis to improve its conservation. To this end, five different topics have been studied, and results are briefly described here. Extensive details can be followed en the attached, published articles. The first topic deals with the indigenous rhizobial symbionts of the Lmj endemism, and its genetic diversity was investigated. The Lmj root symbionts belong to the Bradyrhizobium genus, and phylogenetic analysis based on core genes identified a large diversity of Bradyrhizobium strains with phenotypic and symbiotic characteristics different from rhizobia nodulating other Lupinus spp. native of Spain. The strains were split in two clades. Clade II contained strains close to classical B. canariense and B. japonicum lineages that establish symbioses with lupines in acid soils of the Mediterranean area. Clade I included Lmj strains that define a new lineage, close to other Bradyrhizobium species as B. jicamae and B. elkanii. The phylogenetic analysis based on symbiotic genes identified only two distinct clusters. The singularity and large diversity of these strains in such a small geographical area makes this an attractive system for studying the evolution and adaptation of the rhizobial symbiont to the plant host. Additionally, the presence of bacteria able to nodulate Lmj in basic soils from Chiapas, Mexico was investigated. Surprisingly, these soils contain bacteria able to effectively nodulate and fix nitrogen with Lmj plants in greenhouse assays. In the second topic, the taxonomic status of the endosymbiotic bacteria of Lmj from Valencia endemism and Chiapas was investigated. Results from phylogenetic analysis of core genes and Average Nucleotide Identity (ANIm) using draft genomic sequences identified new Bradyrhizobium species within strains of Clade I of Lmj endosymbiotic bacteria. Only one of these potentially new species has been defined, meanwhile the others are under process of characterization. The name ‘Bradyrhizobium valentinum’ sp. nov. was proposed for the defined species (type strain LmjM3T= CECT 8364T, LMG 2761T). The third topic was directed to conservation of endangered Lmj in its natural habitat. The relevant conclusion of this experimentation is that the symbiosis should be considered as a relevant factor in the conservation strategies for endangered legumes. First, we showed absence of bacteria able to nodulate Lmj in all the inspected ‘‘terra rossa’’ or alkaline red soils of the Iberian Peninsula and Balearic Islands. Then, two efficient nitrogen fixing strains with Lmj plants, LmjC and LmjM3T, were selected as inoculum for seed coating. Two planting experiments were carried out in consecutive years under natural conditions in areas with edapho-climatic characteristics identical to those sustaining natural Lmj populations, and successful reproduction of the plant was achieved. The relevant conclusion from these assays was that the successful reproductive cycle was absolutely dependent on seedling inoculation with effective bradyrhizobia The forth topic deep into the analysis of the genomic of Lmj representative strains. To this end, draft genomic sequences of selected Lmj strains and type strains of Bradyrhizobium spp. were assembled. The comparison analysis of the draft genomic sequences of Lmj strains and related Bradyrhizobium species grouped in Clade I, revealed a high genomic homology among them, and allowed the definition of five potentially new species of Lmj nodulating bacteria. Also, based on the available draft genomic sequences, only three clusters of nod, fix and nif genes from Lmj strains were identified and showed to define a new symbiotic lineage, distant from that of B. canariense and B. japonicum bv. genistearum. The low diversity exhibited by the phylogenetic analysis of symbiotic genes contrast with the large diversity of strains as regards the housekeeping genes analyzed. Besides, the genomic analysis of a Lupinus angustifolius strain ISLU101, revealed the presence of an extrachromosomal replication origin homologous to repABC cluster from plasmid present in Bradyrhizobium spp BTAi1. This repABC cluster gene sequence allowed the identification of extrachromosomic replication origin in 19 out of 72 Bradyrhizobium strains from Lupinus spp., a highly significant result since the absence of plasmids in the Bradyrhizobium genus was traditionally assumed. The repABC gene sequences of these strains grouped them in a unique monophyletic group, related to B. sp. BTAi1 plasmid, but differentiated from the repABC gene cluster of plasmids in fast growing rhizobium strains. The last topic was focused on characterization of type III secreted effectors present in Lmj endosymbiotic bacteria. Type III secretion systems (T3SS) are specialized protein export machineries which can deliver effector proteins into plant cells. The presence of T3SS in rhizobia has frequently been related to the symbiotic nodulation host-range and may have a beneficial or detrimental effect on the symbiosis with legumes. In this context, the presence of T3SS in genomes of nine Lmj strains was investigated, and it was shown the presence of clusters encoding NopE type III-secreted protein similar to the NopE1 and NopE2 of B. diazoefficiens USDA 110T. The putative NopE protein of LmjC strain is at present being characterized regarding its structure and function.

Cloning and characterization of human protease-activated receptor 4

Protease-activated receptors 1–3 (PAR1, PAR2, and PAR3) are members of a unique G protein-coupled receptor family. They are characterized by a tethered peptide ligand at the extracellular amino terminus that is generated by minor proteolysis. A partial cDNA sequence of a fourth member of this family (PAR4) was identified in an expressed sequence tag database, and the full-length cDNA clone has been isolated from a lymphoma Daudi cell cDNA library. The ORF codes for a seven transmembrane domain protein of 385 amino acids with 33% amino acid sequence identity with PAR1, PAR2, and PAR3. A putative protease cleavage site (Arg-47/Gly-48) was identified within the extracellular amino terminus. COS cells transiently transfected with PAR4 resulted in the formation of intracellular inositol triphosphate when treated with either thrombin or trypsin. A PAR4 mutant in which the Arg-47 was replaced with Ala did not respond to thrombin or trypsin. A hexapeptide (GYPGQV) representing the newly exposed tethered ligand from the amino terminus of PAR4 after proteolysis by thrombin activated COS cells transfected with either wild-type or the mutant PAR4. Northern blot showed that PAR4 mRNA was expressed in a number of human tissues, with high levels being present in lung, pancreas, thyroid, testis, and small intestine. By fluorescence in situ hybridization, the human PAR4 gene was mapped to chromosome 19p12.

The gastrulation defective gene of Drosophila melanogaster is a member of the serine protease superfamily

The establishment of dorsal–ventral polarity in the oocyte involves two sets of genes. One set belongs to the gurken-torpedo signaling pathway and affects the development of the egg chorion as well as the polarity of the embryo. The second set of genes affects only the dorsal–ventral polarity of the embryo but not the eggshell. gastrulation defective is one of the earliest acting of this second set of maternally required genes. We have cloned and characterized the gastrulation defective gene and determined that it encodes a protein structurally related to the serine protease superfamily, which also includes the Snake, Easter, and Nudel proteins. These data provide additional support for the involvement of a protease cascade in generating an asymmetric signal (i.e., asymmetric Spätzle activity) during establishment of dorsal–ventral polarity in the Drosophila embryo.

Inhibition of protease-resistant prion protein formation by porphyrins and phthalocyanines

A central aspect of pathogenesis in the transmissible spongiform encephalopathies or prion diseases is the conversion of normal protease-sensitive prion protein (PrP-sen) to the abnormal protease-resistant form, PrP-res. Here we identify porphyrins and phthalocyanines as inhibitors of PrP-res accumulation. The most potent of these tetrapyrroles had IC50 values of 0.5–1 μM in scrapie-infected mouse neuroblastoma (ScNB) cell cultures. Inhibition was observed without effects on protein biosynthesis in general or PrP-sen biosynthesis in particular. Tetrapyrroles also inhibited PrP-res formation in a cell-free reaction composed predominantly of hamster PrP-res and PrP-sen. Inhibitors were found among phthalocyanines, deuteroporphyrins IX, and meso-substituted porphines; examples included compounds containing anionic, neutral protic, and cationic peripheral substituents and various metals. We conclude that certain tetrapyrroles specifically inhibit the conversion of PrP-sen to PrP-res without apparent cytotoxic effects. The inhibition observed in the cell-free conversion reaction suggests that the mechanism involved direct interactions of the tetrapyrrole with PrP-res and/or PrP-sen. These findings introduce a new class of inhibitors of PrP-res formation that represents a potential source of therapeutic agents for transmissible spongiform encephalopathies.

Cytokine suppression of protease activation in wild-type p53-dependent and p53-independent apoptosis

M1 myeloid leukemic cells overexpressing wild-type p53 undergo apoptosis. This apoptosis can be suppressed by some cytokines, protease inhibitors, and antioxidants. We now show that induction of apoptosis by overexpressing wild-type p53 is associated with activation of interleukin-1β-converting enzyme (ICE)-like proteases, resulting in cleavage of poly(ADP- ribose) polymerase and the proenzyme of the ICE-like protease Nedd-2. Activation of these proteases and apoptosis were suppressed by the cytokine interleukin 6 or by a combination of the cytokine interferon γ and the antioxidant butylated hydroxyanisole, and activation of poly(ADP-ribose) polymerase and apoptosis were suppressed by some protease inhibitors. In a clone of M1 cells that did not express p53, vincristine or doxorubicin induced protease activation and apoptosis that were not suppressed by protease inhibitors, but were suppressed by interleukin 6. In another myeloid leukemia (7-M12) doxorubicin also induced protease activation and apoptosis that were not suppressed by protease inhibitors, but were suppressed by granulocyte–macrophage colony-stimulating factor. The results indicate that (i) overexpression of wild-type p53 by itself or treatment with cytotoxic compounds in wild-type p53-expressing or p53-nonexpressing myeloid leukemic cells is associated with activation of ICE-like proteases; (ii) cytokines exert apoptosis-suppressing functions upstream of protease activation; (iii) the cytotoxic compounds induce additional pathways in apoptosis; and (iv) cytokines can also suppress these other components of the apoptotic machinery.

Structural Characterization of the Loop at the Alpha-Subunit C-Terminus of the Mixed Lineage Leukemia Protein Activating Protease Taspase1.

Funding: The authors acknowledge the Fonds of Chemical Industry for funding JvdB by their Chemiefonds grant and the DFG for funding PB and CB (CRC 1093).

Activation of a caspase 3-related cysteine protease is required for glutamate-mediated apoptosis of cultured cerebellar granule neurons

Neurotoxicity induced by overstimulation of N-methyl-d-aspartate (NMDA) receptors is due, in part, to a sustained rise in intracellular Ca2+; however, little is known about the ensuing intracellular events that ultimately result in cell death. Here we show that overstimulation of NMDA receptors by relatively low concentrations of glutamate induces apoptosis of cultured cerebellar granule neurons (CGNs) and that CGNs do not require new RNA or protein synthesis. Glutamate-induced apoptosis of CGNs is, however, associated with a concentration- and time-dependent activation of the interleukin 1β-converting enzyme (ICE)/CED-3-related protease, CPP32/Yama/apopain (now designated caspase 3). Further, the time course of caspase 3 activation after glutamate exposure of CGNs parallels the development of apoptosis. Moreover, glutamate-induced apoptosis of CGNs is almost completely blocked by the selective cell permeable tetrapeptide inhibitor of caspase 3, Ac-DEVD-CHO but not by the ICE (caspase 1) inhibitor, Ac-YVAD-CHO. Western blots of cytosolic extracts from glutamate-exposed CGNs reveal both cleavage of the caspase 3 substrate, poly(ADP-ribose) polymerase, as well as proteolytic processing of pro-caspase 3 to active subunits. Our data demonstrate that glutamate-induced apoptosis of CGNs is mediated by a posttranslational activation of the ICE/CED-3-related cysteine protease caspase 3.

An inhibitor of HIV-1 protease modulates proteasome activity, antigen presentation, and T cell responses

Inhibitors of the protease of HIV-1 have been used successfully for the treatment of HIV-1-infected patients and AIDS disease. We tested whether these protease inhibitory drugs exerted effects in addition to their antiviral activity. Here, we show in mice infected with lymphocytic choriomeningitis virus and treated with the HIV-1 protease inhibitor ritonavir a marked inhibition of antiviral cytotoxic T lymphocyte (CTL) activity and impaired major histocompatibility complex class I-restricted epitope presentation in the absence of direct effects on lymphocytic choriomeningitis virus replication. A potential molecular target was found: ritonavir selectively inhibited the chymotrypsin-like activity of the 20S proteasome. In view of the possible role of T cell-mediated immunopathology in AIDS pathogenesis, the two mechanisms of action (i.e., reduction of HIV replication and impairment of CTL responses) may complement each other beneficially. Thus, the surprising ability of ritonavir to block the presentation of antigen to CTLs may possibly contribute to therapy of HIV infections but potentially also to the therapy of virally induced immunopathology, autoimmune diseases, and transplantation reactions.

Probing the assembly of transcription initiation complexes through changes in σN protease sensitivity

The alternative bacterial σN RNA polymerase holoenzyme binds promoters as a transcriptionally inactive complex that is activated by enhancer-binding proteins. Little is known about how sigma factors respond to their ligands or how the responses lead to transcription. To examine the liganded state of σN, the assembly of end-labeled Klebsiella pneumoniae σN into holoenzyme, closed promoter complexes, and initiated transcription complexes was analyzed by enzymatic protein footprinting. V8 protease-sensitive sites in free σN were identified in the acidic region II and bordering or within the minimal DNA binding domain. Interaction with core RNA polymerase prevented cleavage at noncontiguous sites in region II and at some DNA binding domain sites, probably resulting from conformational changes. Formation of closed complexes resulted in further protections within the DNA binding domain, suggesting close contact to promoter DNA. Interestingly, residue E36 becomes sensitive to proteolysis in initiated transcription complexes, indicating a conformational change in holoenzyme during initiation. Residue E36 is located adjacent to an element involved in nucleating strand separation and in inhibiting polymerase activity in the absence of activation. The sensitivity of E36 may reflect one or both of these functions. Changing patterns of protease sensitivity strongly indicate that σN can adjust conformation upon interaction with ligands, a property likely important in the dynamics of the protein during transcription initiation.

Evidence that the 42- and 40-amino acid forms of amyloid β protein are generated from the β-amyloid precursor protein by different protease activities

Cerebral deposition of the amyloid β protein (Aβ) is an early and invariant feature of Alzheimer disease (AD). Whereas the 40-amino acid form of Aβ (Aβ40) accounts for ≈90% of all Aβ normally released from cells, it appears to contribute only to later phases of the pathology. In contrast, the longer more amyloidogenic 42-residue form (Aβ42), accounting for only ≈10% of secreted Aβ, is deposited in the earliest phase of AD and remains the major constituent of most amyloid plaques throughout the disease. Moreover, its levels have been shown to be increased in all known forms of early-onset familial AD. Thus, inhibition of Aβ42 production is a prime therapeutic goal. The same protease, γ-secretase, is assumed to generate the C termini of both Aβ40 and Aβ42. Herein, we analyze the effect of the compound MDL 28170, previously suggested to inhibit γ-secretase, on β-amyloid precursor protein processing. By immunoprecipitating conditioned medium of different cell lines with various Aβ40- and Aβ42-specific antibodies, we demonstrate a much stronger inhibition of the γ-secretase cleavage at residue 40 than of that at residue 42. These data suggest that different proteases generate the Aβ40 and Aβ42 C termini. Further, they raise the possibility of identifying compounds that do not interfere with general β-amyloid precursor protein metabolism, including Aβ40 production, but specifically block the generation of the pathogenic Aβ42 peptide.

Stochastic processes strongly influence HIV-1 evolution during suboptimal protease-inhibitor therapy

It has long been assumed that HIV-1 evolution is best described by deterministic evolutionary models because of the large population size. Recently, however, it was suggested that the effective population size (Ne) may be rather small, thereby allowing chance to influence evolution, a situation best described by a stochastic evolutionary model. To gain experimental evidence supporting one of the evolutionary models, we investigated whether the development of resistance to the protease inhibitor ritonavir affected the evolution of the env gene. Sequential serum samples from five patients treated with ritonavir were used for analysis of the protease gene and the V3 domain of the env gene. Multiple reverse transcription–PCR products were cloned, sequenced, and used to construct phylogenetic trees and to calculate the genetic variation and Ne. Genotypic resistance to ritonavir developed in all five patients, but each patient displayed a unique combination of mutations, indicating a stochastic element in the development of ritonavir resistance. Furthermore, development of resistance induced clear bottleneck effects in the env gene. The mean intrasample genetic variation, which ranged from 1.2% to 5.7% before treatment, decreased significantly (P < 0.025) during treatment. In agreement with these findings, Ne was estimated to be very small (500–15,000) compared with the total HIV-1 RNA copy number. This study combines three independent observations, strong population bottlenecking, small Ne, and selection of different combinations of protease-resistance mutations, all of which indicate that HIV-1 evolution is best described by a stochastic evolutionary model.