The carboxyl-terminal domains of IgA and IgM direct isotype-specific polymerization and interaction with the polymeric immunoglobulin receptor

Mucosal surfaces are protected by polymeric immunoglobulins that are transported across the epithelium by the polymeric immunoglobulin receptor (pIgR). Only polymeric IgA and IgM containing a small polypeptide called the "joining" (J) chain can bind to the pIgR. J chain-positive IgA consists of dimers, and some larger polymers, whereas only IgM pentamers incorporate the J chain. We made domain swap chimeras between human IgA1 and IgM and found that the COOH-terminal domains of the heavy chains (Calpha3 and Cmu4, respectively) dictated the size of the polymers formed and also which polymers incorporated the J chain. We also showed that chimeric IgM molecules engineered to contain Calpha3 were able to bind the rabbit pIgR. Since the rabbit pIgR normally does not bind IgM, these results suggest that the COOH-terminal domain of the polymeric immunoglobulins is primarily responsible for interaction with the pIgR. Finally, we made a novel chimeric IgA immunoglobulin, containing the terminal domain from IgM. This recombinant molecule formed J chain-containing pentamers that could, like IgA, efficiently form covalent complexes with the human pIgR ectodomain, known as secretory component.     

Efficient delivery of T cell epitopes to APC by use of MHC class II-specific Troybodies

A major objective in vaccine development is the design of reagents that give strong, specific T cell responses. We have constructed a series of rAb with specificity for MHC class II (I-E). Each has one of four different class II-restricted T cell epitopes genetically introduced into the first C domain of the H chain. These four epitopes are: 91-101 lambda2(315), which is presented by I-E(d); 110-120 hemagglutinin (I-E(d)); 323-339 OVA (I-A(d)); and 46-61 hen egg lysozyme (I-A(k)). We denote such APC-specific, epitope-containing Ab "Troybodies." When mixed with APC, all four class II-specific Troybodies were approximately 1,000 times more efficient at inducing specific T cell activation in vitro compared with nontargeting peptide Ab. Furthermore, they were 1,000-10,000 times more efficient than synthetic peptide or native protein. Conventional intracellular processing of the Troybodies was required to load the epitopes onto MHC class II. Different types of professional APC, such as purified B cells, dendritic cells, and macrophages, were equally efficient at processing and presenting the Troybodies. In vivo, class II-specific Troybodies were at least 100 times more efficient at targeting APC and activating TCR-transgenic T cells than were the nontargeting peptide Ab. Furthermore, they were 100-100,000 times more efficient than synthetic peptide or native protein. The study shows that class II-specific Troybodies can deliver a variety of T cell epitopes to professional APC for efficient presentation, in vitro as well as in vivo. Thus, Troybodies may be useful as tools in vaccine development.     

Inductive patterning of the embryonic brain in Drosophila

In vertebrates (deuterostomes), brain patterning depends on signals from adjacent tissues. For example, holoprosencephaly, the most common brain anomaly in humans, results from defects in signaling between the embryonic prechordal plate (consisting of the dorsal foregut endoderm and mesoderm) and the brain. I have examined whether a similar mechanism of brain development occurs in the protostome Drosophila, and find that the foregut and mesoderm act to pattern the fly embryonic brain. When the foregut and mesoderm of Drosophila are ablated, brain patterning is disrupted. The loss of Hedgehog expressed in the foregut appears to mediate this effect, as it does in vertebrates. One mechanism whereby these defects occur is a disruption of normal apoptosis in the brain. These data argue that the last common ancestor of protostomes and deuterostomes had a prototype of the brains present in modern animals, and also suggest that the foregut and mesoderm contributed to the patterning of this 'proto-brain'. They also argue that the foreguts of protostomes and deuterostomes, which have traditionally been assigned to different germ layers, are actually homologous.     

Survival of Flavobacterium psychrophilum in rainbow trout (Oncorhynchus mykiss) serum in vitro

Virulent and non-virulent strains of Flavobacterium psychrophilum of different serotypes were examined for survival and growth in non-immune and immune rainbow trout serum, in vitro. A majority of the examined strains consumed complement of non-immune serum, but the complement cascade was not able to cause an immediate (after 3 h incubation) notable reduction in viability of the inoculated cells. After 24 h incubation a more pronounced reduction in the number of viable bacteria was observed in untreated serum as well as in serum heated at 45 degrees C. In serum heated at 56 degrees C this reduction in cell number was not observed, but an increase in cell number did not occur either. The serum survival of one of the examined strains was different from the others in showing cell multiplication after 24 h incubation in normal as well as heat-treated (45 and 56 degrees C) serum. In immune serum no immediate reduction in viability of inoculated cells, of all tested strains, was observed. The number of viable cells showed a slow decrease or remained almost unchanged for up to 72 h post-inoculation in untreated serum, at 5 degrees C as well as 15 degrees C. In heat-treated serum (45 degrees C) the number of viable cells decreased slowly at 5 degrees C and 15 degrees C for up to 72 h. The results suggest that the examined strains were unaffected by the alternative complement reaction present in fish serum as well as by antibodies against F. psychrophilum. However, some unknown component(s) in the fish sera, or lack of nutrients or essential growth factors, inhibited the growth of most of the examined strains in the tested fish sera.     

Evaluation of redox indicators and the use of digital scanners and spectrophotometer for quantification of microbial growth in microplates

The growth indicators 2,3,5-triphenyltetrazolium chloride (TTC), 2-[4-iodophenyl]-3-[4-dinitrophenyl]-5-phenyltetrazolium chloride (INT), 2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide inner salt (XTT), 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT), and resazurin were tested for their ability to indicate bacterial growth/growth inhibition. Two reading devices were evaluated and compared, a microplate spectrophotometer and a digital flatbed scanner. The bacteria used in the study were cultivated in 96-wells microplates and readings were made after 24 h. The scanned pictures were analysed with a software developed in-house to generate numerical values. It was found that resazurin was difficult to use since it shifts between three colours. MTT and TTC had a high correlation between the spectrophotometer data and the data from the scanned images. The reproducibility was similar for both reading devices. In no case was there a need to resuspend the pellets before reading. Both the XTT and INT showed lower correlations.It is concluded that bacterial growth/growth inhibition can be easily and reproducibly measured from microplate cultivations with a flatbed scanner or with a microplate spectrophotometer.     

A "dock, lock, and latch" structural model for a staphylococcal adhesin binding to fibrinogen

Gram-positive pathogens such as staphylococci contain multiple cell wall-anchored proteins that serve as an interface between the microbe and its environment. Some of these proteins act as adhesins and mediate bacterial attachment to host tissues. SdrG is a cell wall-anchored adhesin from Staphylococcus epidermidis that binds to the Bbeta chain of human fibrinogen (Fg) and is necessary and sufficient for bacterial attachment to Fg-coated biomaterials. Here, we present the crystal structures of the ligand binding region of SdrG as an apoprotein and in complex with a synthetic peptide analogous to its binding site in Fg. Analysis of the crystal structures, along with mutational studies of both the protein and of the peptide, reveals that SdrG binds to its ligand with a dynamic "dock, lock, and latch" mechanism. We propose that this mechanism represents a general mode of ligand binding for structurally related cell wall-anchored proteins of gram-positive bacteria.     

Radiation-mediated proteolysis of CDT1 by CUL4-ROC1 and CSN complexes constitutes a new checkpoint

Genomic integrity is maintained by checkpoints that guard against undesired replication after DNA damage. Here, we show that CDT1, a licensing factor of the pre-replication complex (preRC), is rapidly proteolysed after UV- or gamma-irradiation. The preRC assembles on replication origins at the end of mitosis and during G1 to license DNA for replication in S phase. Once the origin recognition complex (ORC) binds to origins, CDC6 and CDT1 associate with ORC and promote loading of the MCM2-7 proteins onto chromatin, generating the preRC. We show that radiation-mediated CDT1 proteolysis is independent of ATM and CHK2 and can occur in G1-phase cells. Loss of the COP9-signalosome (CSN) or CUL4-ROC1 complexes completely suppresses CDT1 proteolysis. CDT1 is specifically polyubiquitinated by CUL4 complexes and the interaction between CDT1 and CUL4 is regulated in part by gamma-irradiation. Our study reveals an evolutionarily conserved and uncharacterized G1 checkpoint that induces CDT1 proteolysis by the CUL4-ROC1 ubiquitin E3 ligase and CSN complexes in response to DNA damage.     

labial acts to initiate neuronal fate specification, but not axon pathfinding, in the embryonic brain of Drosophila

Drosophila embryos lacking the homeotic gene labial (lab) show two types of defects in brain development: (1) cells in the brain lab domain do not express neuronal markers or extend axons, and (2) axons originating from outside the lab domain stop at this region or project ectopically. A severe disruption of neuronal patterning and axon scaffolding is the net result. It is not clear how the absence of Lab can result in both neuronal fate defects and axon pathfinding defects. I have expressed Lab in short pulses in lab loss-of- function embryos, and this gave almost complete rescue; for example, the tritocerebral commissure was restored. Rescue only occurred when Lab was provided at the time when cells in the brain are adopting a neuronal fate. Lab expression later, when the first axons are seen in the lab domain, did not give rescue. I conclude that Lab expression helps to establish neuronal identity in the lab domain, and these neurons act as a permissive substrate for axon extension. However, Lab itself is not required at the time of axon pathfinding through this region. Received: 31 May 2000 / Accepted: 5 July 2000     

Molecular systematics and biogeography of the fanged frogs of Southeast Asia

Our analysis of parts of the mitochondrial ribosomal 12S and 16S genes from 39 populations of Southeast Asian ranid frogs confirms that the fanged frogs are a monophyletic clade. This group, properly called Limnonectes, appears to have arisen in the early Tertiary at a time when free faunal exchange was possible among Southeast Asia, Borneo, Sumatra, Java, and, probably, Sulawesi. Four species groups are tentatively identified within the clade. Part of group 1 includes species related to L. kuhlii that occur in Borneo. Another part of group 1 includes species from Malay Peninsula and Thailand that are related to L. pileata. Species group 2, L. leporina, occurs only in Borneo. Species group 3 is restricted to species distributed in Sulawesi and the Philippines. Species group 4 includes L. blythii and relatives. There is a lack of compatibility between phylogenetic hypotheses generated from molecular and morphological data sets. These differences are related, in large part, to whether some species of Limnonectes have secondarily lost fangs or whether lack of fangs represents the primitive condition.     

First evidence of uncoupling protein-2 (UCP-2) and -3 (UCP-3) gene expression in piglet skeletal muscle and adipose tissue

Uncoupling proteins (UCPs) facilitate proton transport inside the mitochondria and decrease the proton gradient, leading to heat production. Until now, the presence of UCP1 or other UCP homologs had not been detected in tissues of pig, a species where evidence for the presence of brown adipose tissue has only been provided in 2-3 month old animals. In the light of the improving knowledge on the UCPs family, we decided to examine both UCP2 and UCP3 mRNA expression in piglet skeletal muscle and adipose tissue. Using RT-PCR we have successfully cloned a partial UCP2 sequence and a complete UCP3 cDNA. UCP3's open reading frame (936bp) shares 90, 89 and 85% similarity with bovine, human and rat UCP3 nucleotide sequences, respectively. In 3-5 day old piglets, these genes are expressed in adipose tissue and in both longissimus thoracis (LT) and rhombo?deus (RH) muscles, without any effect of muscle metabolic type. This is in good agreement with the measurement of the same membrane potential in mitochondria isolated from both types of muscles. In triiodothyronine-treated piglets, UCP3 mRNA is more expressed in LT than in RH muscle. These genes may be involved in the control of the energy metabolism of the piglet.