Interaction with GM130 during HERG ion channel trafficking. Disruption by type 2 congenital long QT syndrome mutations. Human Ether-à-go-go-Related Gene

Many mutations in the Human Ether-à-go-go-Related Gene (HERG) cause type 2 congenital long QT syndrome (LQT2) by disrupting trafficking of the HERG-encoded potassium channel. Beyond observations that some mutations trap channels in the endoplasmic reticulum, little is known about how trafficking fails. Even less is known about what checkpoints are encountered in normal trafficking. To identify protein partners encountered as HERG channels are transported among subcellular compartments, we screened a human heart library with the C terminus of HERG using yeast two-hybrid technology. Among the proteins isolated was GM130, a Golgi-associated protein involved in vesicular transport. The interaction mapped to two non-contiguous regions of HERG and to a region just upstream of the GRASP-65 interaction domain of GM130. GM130 did not interact with the N or C terminus of either KvLQT1 or Shaker channels. LQT2-causing mutations in the HERG C terminus selectively disrupted interactions with GM130 but not Tara, another HERG-interacting protein. Native GM130 and stably expressed HERG were co-immunoprecipitated from HEK-293 cells using GM130 antibodies. In rat cardiac myocytes and HEK-293 cells, confocal immunocytochemistry showed co-localization of GM130 and HERG to the Golgi apparatus. Overexpression of GM130 suppressed HERG current amplitude in Xenopus oocytes, as if by providing an excess of substrate at the Golgi checkpoint. These findings indicate that GM130 plays a previously undefined role in cargo transport. We propose that the cytoplasmic C terminus of HERG participates in the tethering or possibly targeting of HERG-containing vesicles within the Golgi via its interaction with GM130.     

Molecular analysis of Vibrio parahaemolyticus isolated from human patients and shellfish during US Pacific north-west outbreaks

AIMS: The objective of this study was to investigate the occurrence and distribution of haemolysin genes, plasmid profile, serogroup analysis and cellular urease activity for Vibrio parahaemolyticus isolates from infected human patients and oysters from the Pacific north-western United States between 1988 and 1997. METHODS AND RESULTS: All of the clinical and environmental isolates tested in this study exhibited the presence of the thermolabile haemolysin gene, tl, confirming that all of the isolates were V. parahaemolyticus. Furthermore, the V. parahaemolyticus isolates that contained either the thermostable direct haemolysin gene, tdh, or the thermostable direct haemolysin-related gene, trh, or both, were also positive for urease. Isolates from infected human patients belong to serogroups O1 and O4, whereas, the isolates from oysters belong to serogroups O1, O4 and O5. These results suggest that the presence of a V. parahaemolyticus serogroup O1 and O4 could indicate the presence of a virulent strain of this pathogen. In this study, the presence of the haemolysin genes, serogroup profiles and urease production in V. parahaemolyticus isolated from human patients correlated with the oysters collected during the outbreaks. However, no significant correlation of the plasmid profiles was detected, based on their distribution and molecular weights, between V. parahaemolyticus isolated from infected human patients and from oysters collected during this outbreak. CONCLUSIONS, SIGNIFICANCE AND IMPACT OF STUDY: It is apparent from this study that the identification of the haemolysin genes by multiplex PCR amplification, in conjunction with serogroup analysis and urease production, can be used to monitor shellfish for the presence of potentially pathogenic strains of V. parahaemolyticus.     

A single-chain class II MHC-IgG3 fusion protein inhibits autoimmune arthritis by induction of antigen-specific hyporesponsiveness

T cells play a central role in many autoimmune diseases. A method to specifically target the function of autoreactive T cell clones would avoid the global immunosuppression associated with current therapies. To develop a molecule capable of inhibiting autoreactive T cell responses in vivo, single-chain peptide-I-A-IgG3 fusion proteins were constructed and expressed in both mammalian and insect cells. The fusion proteins were designed with an IgG3 Fc moiety to make them divalent, allowing TCR cross-linking, while lacking FcR binding and costimulation. The fusion proteins stimulated T cell hybridomas in vitro in a peptide-specific, MHC-restricted manner but failed to do so in soluble form. In vivo administration of an I-A(q) fusion protein, containing an immunodominant collagen II peptide, significantly delayed the onset and reduced the severity of collagen-induced arthritis in DBA/1 mice by induction of Ag-specific hyporesponsiveness. Such fusion proteins may be useful to study novel therapeutic approaches for T cell-mediated autoimmune diseases.     

Two recessive gene inheritance for triallate resistance in Avena fatua L

Extensive use of the preemergence herbicide triallate over the last three decades has selected for resistant (R) Avena fatua L. populations in several areas of the United States and Canada. R plants are also cross-resistant to the unrelated pyrazolium herbicide difenzoquat. We made reciprocal crosses between inbred R and susceptible (S) lines to determine the genetic basis of triallate resistance. Seeds from parental lines and F(2) populations were treated with soil applications of 0.275, 0.55, or 1.1 kg/ha triallate in the greenhouse and plant heights recorded after 37 days. Surviving F(2) plants were selfed and the resulting F(3) families were screened with 1.1 kg/ha triallate. In the F(2) populations, assortment of S and R phenotypes fit a 15:1 segregation ratio, suggesting that resistance was controlled by the two independently segregating recessive genes TRR1 and TRR2. None of the 912 F(3) progeny from 51 R F(2) individuals was susceptible to triallate treatment, further supporting a two-gene mode of inheritance. There was a possible maternal effect on susceptibility at the highest triallate rate tested.     

Physical association of the K3 protein of gamma-2 herpesvirus 68 with major histocompatibility complex class I molecules with impaired peptide and beta(2)-microglobulin assembly

To persist in the presence of an active immune system, viruses encode proteins that decrease expression of major histocompatibility complex class I molecules by using a variety of mechanisms. For example, murine gamma-2 herpesvirus 68 expresses the K3 protein, which causes the rapid turnover of nascent class I molecules. In this report we show that certain mouse class I alleles are more susceptible than others to K3-mediated down regulation. Prior to their rapid degradation, class I molecules in K3-expressing cells exhibit impaired assembly with beta(2)-microglobulin. Furthermore, K3 is detected predominantly in association with class I molecules lacking assembly with high-affinity peptides, including class I molecules associated with the peptide loading complex TAP/tapasin/calreticulin. The detection of K3 with class I assembly intermediates raises the possibility that molecular chaperones involved in class I assembly are involved in K3-mediated class I regulation.     

Systems-level reconsolidation: reengagement of the hippocampus with memory reactivation

Certain types of memories are dependent on the hippocampus for a short period of time following training, after which they are no longer susceptible to hippocampal manipulations. Having completed this initial consolidation process, a memory may once again engage the hippocampus (undergo reconsolidation) when recalled. Two studies in the current issue of Neuron make important advances in our understanding of reconsolidation but reach different conclusions about the modifiability of old memories.     

Behavioral and neural analysis of extinction

The neural mechanisms by which fear is inhibited are poorly understood at the present time. Behaviorally, a conditioned fear response may be reduced in intensity through a number of means. Among the simplest of these is extinction, a form of learning characterized by a decrease in the amplitude and frequency of a conditioned response when the conditioned stimulus that elicits it is repeatedly nonreinforced. Because clinical interventions for patients suffering from fear dysregulation seek to inhibit abnormal, presumably learned fear responses, an understanding of fear extinction is likely to inform and increase the efficacy of these forms of treatment. This review considers the behavioral, cellular, and molecular literatures on extinction and presents the most recent advances in our understanding while identifying issues that require considerable further research.     

Indicators of AEI applied to the Delaware Estuary

We evaluated the impacts of entrainment and impingement at the Salem Generating Station on fish populations and communities in the Delaware Estuary. In the absence of an agreed-upon regulatory definition of "adverse environmental impact" (AEI), we developed three independent benchmarks of AEI based on observed or predicted changes that could threaten the sustainability of a population or the integrity of a community. Our benchmarks of AEI included: (1) disruption of the balanced indigenous community of fish in the vicinity of Salem (the "BIC" analysis); (2) a continued downward trend in the abundance of one or more susceptible fish species (the "Trends" analysis); and (3) occurrence of entrainment/impingement mortality sufficient, in combination with fishing mortality, to jeopardize the future sustainability of one or more populations (the "Stock Jeopardy" analysis). The BIC analysis utilized nearly 30 years of species presence/absence data collected in the immediate vicinity of Salem. The Trends analysis examined three independent data sets that document trends in the abundance of juvenile fish throughout the estuary over the past 20 years. The Stock Jeopardy analysis used two different assessment models to quantify potential long-term impacts of entrainment and impingement on susceptible fish populations. For one of these models, the compensatory capacities of the modeled species were quantified through meta-analysis of spawner-recruit data available for several hundred fish stocks. All three analyses indicated that the fish populations and communities of the Delaware Estuary are healthy and show no evidence of an adverse impact due to Salem. Although the specific models and analyses used at Salem are not applicable to every facility, we believe that a weight of evidence approach that evaluates multiple benchmarks of AEI using both retrospective and predictive methods is the best approach for assessing entrainment and impingement impacts at existing facilities.     

A model of the biogeographical journey from<Emphasis Type="Italic"> Proto-pan</Emphasis> to<Emphasis Type="Italic"> Pan paniscus</Emphasis>

Pan paniscus is unique in the group of African apes because of its range south of the Congo River. Examination of the bio-geographical journey of the genus Pan to the species P. paniscus is important when discussing the evolution of African apes. This paper is a review of the paleo-geographic events, the zoogeography, and faunal sorting which influenced P. paniscus divergence from the Proto-pan ancestor within the recent Miocene through Pliocene Epochs, approximately 10–2 MYA. Finally, by elucidating modern day evidence of food plant forms in the southern periphery exploited by P. paniscus in the forest/savanna mosaic habitat, we are able to conclude with those extrinsic events that most influenced the occurrence and distribution of P. paniscus. Electronic Publication     

Differential contributions of condensin I and condensin II to mitotic chromosome architecture in vertebrate cells

The canonical condensin complex (henceforth condensin I) plays an essential role in mitotic chromosome assembly and segregation from yeast to humans. We report here the identification of a second condensin complex (condensin II) from vertebrate cells. Condensins I and II share the same pair of structural maintenance of chromosomes (SMC) subunits but contain different sets of non-SMC subunits. siRNA-mediated depletion of condensin I- or condensin II-specific subunits in HeLa cells produces a distinct, highly characteristic defect in chromosome morphology. Simultaneous depletion of both complexes causes the severest defect. In Xenopus egg extracts, condensin I function is predominant, but lack of condensin II results in the formation of irregularly shaped chromosomes. Condensins I and II show different distributions along the axis of chromosomes assembled in vivo and in vitro. We propose that the two condensin complexes make distinct mechanistic contributions to mitotic chromosome architecture in vertebrate cells.