In vitro formation of amyloid fibrils from intact beta 2-microglobulin

Prompted by the identification of hemodialysis-associated amyloid protein as beta 2-microglobulin, we attempted to create in vitro amyloid fibrils from the native protein. Beta 2-microglobulin in PBS was slowly dialyzed free of salt and then concentrated. The residue showed Congophilia with green birefringence by light microscopy and polarization, and non-branching fibrils of indeterminate length, measuring 8 to 10 nm in diameter by electron microscopy, thus meeting the morphologic criteria for amyloid. The present study demonstrates the first successful in vitro creation of amyloid fibrils with intact precursor protein molecules and provides supporting evidence that hemodialysis-associated amyloid is constituted from beta 2-microglobulin.     

Population dynamics of the philometrid nematode Margolisianum bulbosum in the southern flounder Paralichthys lethostigma (Pisces: Paralichthyidae) in South Carolina, USA

This is the first report of the philometrid nematode Margolisianum bulbosum Blaylock and Overstreet, 1999 from the southern flounder Paralichthys lethostigma on the east coast of the USA. Observation of adult female worms was used as an indication of the parasite's presence in the fish. Adult females were found only in P. lethostigma >50 mm total length. The overall prevalence was 74%, with a mean intensity of 5 female nematodes per parasitized fish. Infected flounders were found throughout the year with a statistically significant decrease in intensity in the winter months. Neither salinity, water temperature, fish gender nor fish age were found to influence either prevalence or intensity of infection in the flounder. While larvigerous (gravid) females were found throughout the year, the significant decrease in their occurrence during the summer through fall, in concert with an observed decrease in intensity of infection during the winter, indicated that the life cycle of this philometrid species is likely to be annual.     

Identification of novel Y chromosome encoded transcripts by testis transcriptome analysis of mice with deletions of the Y chromosome long arm

Background  

The male-specific region of the mouse Y chromosome long arm (MSYq) is comprised largely of repeated DNA, including multiple copies of the spermatid-expressed Ssty gene family. Large deletions of MSYq are associated with sperm head defects for which Ssty deficiency has been presumed to be responsible.     

A duplexed microsphere-based cellular adhesion assay

Interactions of integrin cellular adhesion molecules with matrix proteins play important roles in complex bidirectional signaling pathways. To investigate these interactions, a novel flow-cytometry-based cellular adhesion assay has been developed. Based on the concept of microcarrier cell culture, derivatized polystyrene microspheres (9.6 microm) are used as a substrate for the immobilization of type I collagen to which cells then adhere. Using cytometric detection, the extent of cellular adhesion can be precisely determined by comparison of adhered and nonadhered populations based on the side scatter properties of the microspheres. In combination with immunostaining, the novel format of this assay enables the correlation of adhesive function to other cellular characteristics such as surface expression. In this work, the protein kinase C activator 12-O-tetradecanoylphorbol-13-acetate (TPA) was used to stimulate increased adhesion in Chinese hamster ovary cells stably transfected with the collagen receptor integrin alpha2beta1. Multiple clones of varying expression distributions were analyzed, and correlations of adherent populations versus receptor distributions show a threefold increase in functional cellular adhesion to collagen upon treatment with TPA. Probability binning analysis of duplexed data revealed subtle changes in adhesion versus receptor distribution mediated by TPA which otherwise would not have been detectable.     

Thermodynamic stability of a kappaI immunoglobulin light chain: relevance to multiple myeloma

Immunoglobulin light chains have two similar domains, each with a hydrophobic core surrounded by beta-sheet layers, and a highly conserved disulfide bond. Differential scanning calorimetry and circular dichroism were used to study the folding and stability of MM-kappaI, an Ig LC of kappaI subtype purified from the urine of a multiple myeloma patient. The complete primary structure of MM-kappaI was determined by Edman sequence analysis and mass spectrometry. The protein was found to contain a cysteinyl post-translational modification at Cys(214). Protein stability and conformation of MM-kappaI as a function of temperature or denaturant conditions at pH 7.4 and 4.8 were investigated. At pH 4.8, calorimetry demonstrated that MM-kappaI undergoes an incomplete, cooperative, partially reversible thermal unfolding with increased unfolding temperature and calorimetric enthalpy as compared to pH 7.4. Secondary and tertiary structural analyses provided evidence to support the presence of unfolding intermediates. Chemical denaturation resulted in more extensive protein unfolding. The stability of MM-kappaI was reduced and protein unfolding was irreversible at pH 4.8, thus suggesting that different pathways are utilized in thermal and chemical unfolding.     

Model for the interaction of gammaherpesvirus 68 RING-CH finger protein mK3 with major histocompatibility complex class I and the peptide-loading complex

The mK3 protein of gammaherpesvirus 68 and the kK5 protein of Kaposi's sarcoma-associated herpesvirus are members of a family of structurally related viral immune evasion molecules that all possess a RING-CH domain with ubiquitin ligase activity. These proteins modulate the expression of major histocompatibility complex class I molecules (mK3 and kK5) as well as other molecules like ICAM-1 and B7.2 (kK5). Previously, mK3 was shown to ubiquitinate nascent class I molecules, resulting in their rapid degradation, and this process was found to be dependent on TAP and tapasin, endoplasmic reticulum molecules involved in class I assembly. Here, we demonstrate that in murine cells, kK5 does not affect class I expression but does downregulate human B7.2 molecules in a TAP/tapasin-independent manner. These differences in substrate specificity and TAP/tapasin dependence between mK3 and kK5 permitted us, using chimeric molecules, to map the sites of mK3 interaction with TAP/tapasin and to determine the requirements for substrate recognition by mK3. Our findings indicate that mK3 interacts with TAP1 and -2 via their C-terminal domains and with class I molecules via their N-terminal domains. Furthermore, by orienting the RING-CH domain of mK3 appropriately with respect to class I, mK3 binding to TAP/tapasin, rather than the presence of unique sequences in class I, appears to be the primary determinant of substrate specificity.     

T-cell subsets that harbor human immunodeficiency virus (HIV) in vivo: implications for HIV pathogenesis

Identification of T-cell subsets that are infected in vivo is essential to understanding the pathogenesis of human immunodeficiency virus (HIV) disease; however, this goal has been beset with technical challenges. Here, we used polychromatic flow cytometry to sort multiple T-cell subsets to 99.8% purity, followed by quantitative PCR to quantify HIV gag DNA directly ex vivo. We show that resting memory CD4(+) T cells are the predominantly infected cells but that terminally differentiated memory CD4(+) T cells contain 10-fold fewer copies of HIV DNA. Memory CD8(+) T cells can also be infected upon upregulation of CD4; however, this is infrequent and HIV-specific CD8(+) T cells are not infected preferentially. Na?ve CD4(+) T-cell infection is rare and principally confined to those peripheral T cells that have proliferated. Furthermore, the virus is essentially absent from na?ve CD8(+) T cells, suggesting that the thymus is not a major source of HIV-infected T cells in the periphery. These data illuminate the underlying mechanisms that distort T-cell homeostasis in HIV infection.     

The potassium channel Kir4.1 associates with the dystrophin-glycoprotein complex via alpha-syntrophin in glia

One of the major physiological roles of potassium channels in glial cells is to promote "potassium spatial buffering" in the central nervous system, a process necessary to maintain an optimal potassium concentration in the extracellular environment. This process requires the precise distribution of potassium channels accumulated at high density in discrete subdomains of glial cell membranes. To obtain a better understanding of how glial cells selectively target potassium channels to discrete membrane subdomains, we addressed the question of whether the glial inwardly rectifying potassium channel Kir4.1 associates with the dystrophin-glycoprotein complex (DGC). Immunoprecipitation experiments revealed that Kir4.1 is associated with the DGC in mouse brain and cultured cortical astrocytes. In vitro immunoprecipitation and pull-down assays demonstrated that Kir4.1 can bind directly to alpha-syntrophin, requiring the presence of the last three amino acids of the channel (SNV), a consensus PDZ domain-binding motif. Furthermore, Kir4.1 failed to associate with the DGC in brains from alpha-syntrophin knockout mice. These results suggest that Kir4.1 is localized in glial cells by its association with the DGC through a PDZ domain-mediated interaction with alpha-syntrophin and suggest an important role for the DGC in central nervous system physiology.     

Relationship between α-glycerophosphate dehydrogenase activity and metabolic rate during flight in Drosophila melanogaster

Measurements of wing-beat frequency (WBF) have been used to characterize flight muscle metabolic rate in Drosophila melanogaster during tethered flight. Progeny of crosses between 17 X-chromosome substitution lines and three null-activity stocks have been studied in order to determine the effect on flight metabolism of sharply reduced activity of -glycerophosphate dehydrogenase (GPDH). It was found that flies with an approximate 50% reduction in GPDH activity have a metabolic rate that is, in most cases, indistinguishable from that of wild-type flies and, in the most extreme cases, reduced by only 4%. These results demonstrate that Gpdh is not a major gene for flight metabolism, in the quantitative genetic sense of the term. These results are in agreement with the Kacser and Burns (1973, 1979, 1981) theory of flux, which postulates that the activity of an enzyme embedded in a multienzyme pathway can sometimes vary from wild-type to very low levels (perhaps 5–10% wild type) with no significant effect on flux through the total pathway.This research was supported by several grants to JWC: NSF Grant 8211667, a grant from the Graduate School, University of Minnesota, and a Research Career Development Award from the NIH.