TRIP8b splice forms act in concert to regulate the localization and expression of HCN1 channels in CA1 pyramidal neurons

HCN1 channel subunits, which contribute to the hyperpolarization-activated cation current (Ih), are selectively targeted to distal apical dendrites of hippocampal CA1 pyramidal neurons. Here, we addressed the importance of the brain-specific auxiliary subunit of HCN1, TRIP8b, in regulating HCN1 expression and localization. More than ten N-terminal splice variants of TRIP8b exist in brain and exert distinct effects on HCN1 trafficking when overexpressed. We found that isoform-wide disruption of the TRIP8b/HCN1 interaction caused HCN1 to be mistargeted throughout CA1 somatodendritic compartments. In contrast, HCN1 was targeted normally to CA1 distal dendrites in a TRIP8b knockout mouse that selectively lacked exons 1b and 2. Of the two remaining hippocampal TRIP8b isoforms, TRIP8b(1a-4) promoted HCN1 surface expression in dendrites, whereas TRIP8b(1a) suppressed HCN1 misexpression in axons. Thus, proper subcellular localization of HCN1 depends on its differential additive and subtractive sculpting by two isoforms of a single auxiliary subunit.     

Analysis of the interaction of membrane-active peptides with membranes: The case of melittin in surfactant assemblies

Some of the factors that govern the interaction between membrane-active peptides and membranes were analyzed using melittin as an example. The structural properties of melittin were studied in a variety of micellar and reverse micellar assemblies of varying sizes, shapes, and net charges. The roles of the polarity, packing, and the charge carried by the host assembly on the structural features of the incorporated melittin are delineated. Such an analysis is expected to be of relevance to other membrane-active peptides as well.     

The non-MHC quantitative trait locus Cia5 contains three major arthritis genes that differentially regulate disease severity, pannus formation, and joint damage in collagen- and pristane-induced arthritis

Cia5 is a locus on rat chromosome 10 which regulates the severity of collagen- and pristane-induced arthritis (CIA and PIA). To refine the region toward positional identification, Cia5 subcongenic strains were generated and studied in PIA and CIA. The protective effect of the telomeric locus Cia5a was confirmed in both models. A second arthritis severity locus (Cia5d) was identified within the most centromeric portion of Cia5. DA.F344(Cia5d) rats had a significantly lower median arthritis severity index in PIA, but not in CIA, compared with DA. On histologic analyses DA.F344(Cia5a) and DA.F344(Cia5d) congenics with PIA preserved a nearly normal joint architecture compared with DA, including significant reduction in synovial hyperplasia, pannus, angiogenesis, inflammatory infiltration, bone and cartilage erosions. Cia5 and Cia5a synovial levels of IL-1beta mRNA were reduced. Although both DA.F344(Cia5) and DA.F344(Cia5a) rats were protected in CIA, the arthritis scores of DA.F344(Cia5) were significantly higher than those of DA.F344(Cia5a), suggesting the existence of a third locus where F344-derived alleles centromeric from Cia5a contribute to increased arthritis severity. The existence of the third locus was further supported by higher levels of autoantibodies against rat type II collagen in DA.F344(Cia5) congenics compared with DA.F344(Cia5a). Our results determined that Cia5 contains three major arthritis severity regulatory loci regulating central events in the pathogenesis of arthritis, and differentially influencing CIA and PIA. These loci are syntenic to regions on human chromosomes 17q and 5q implicated in the susceptibility to rheumatoid arthritis, suggesting that the identification of these genes will be relevant to human disease.     

Identification of Francisella tularensis lipoproteins that stimulate the toll-like receptor (TLR) 2/TLR1 heterodimer

The innate immune response to Francisella tularensis is primarily mediated by TLR2, though the bacterial products that stimulate this receptor remain unknown. Here we report the identification of two Francisella lipoproteins, TUL4 and FTT1103, which activate TLR2. We demonstrate that TUL4 and FTT1103 stimulate chemokine production in human and mouse cells in a TLR2-dependent way. Using an assay that relies on chimeric TLR proteins, we show that TUL4 and FTT1103 stimulate exclusively the TLR2/TLR1 heterodimer. Our results also show that yet unidentified Francisella proteins, possibly unlipi-dated, have the ability to stimulate the TLR2/TLR6 heterodimer. Through domain-exchange analysis, we determined that an extended region that comprises LRR 9-17 in the extra-cellular portion of TLR1 mediates response to Francisella lipoproteins and triacylated lipopeptide. Substitution of the corresponding LRR of TLR6 with the LRR derived from TLR1 enables TLR6 to recognize TUL4, FTT1103, and triacylated lipopeptide. This study identifies for the first time specific Fran-cisella products capable of stimulating a proinflammatory response and the cellular receptors they trigger.     

Salt-stable association of simian virus 40 capsid with simian virus 40 DNA

In 8 M CsCl, a fraction of the wild-type previrions and tsB228 nucleoprotein complexes lose their core histones but retain their capsid. These histone-depleted complexes appear in the electron microscope as a protein shell attached to supercoiled DNA. Consistent with this result, we find that in 1 M NaCl, the wild-type previrions dissociate into two populations of nucleoprotein complexes. One population sediments between 50 and 140 S and morphologically resembles the shell-DNA complexes isolated in CsCl gradients. The other population is comprised primarily of nucleoproteins which sediment at 40 S.     

Cryptorchidism and Infertility in Rats with Targeted Disruption of the Adamts16 Locus

A Disintegrin And Metalloproteinase with ThromboSpondin motifs16 (ADAMTS-16) is a member of a family of metalloproteinases. Using a novel zinc-finger nuclease based gene-edited rat model harboring a targeted mutation of the Adamts16 locus, we previously reported this gene to be linked to blood pressure regulation. Here we document our observation with this model that Adamts16 is essential for normal development of the testis. Absence of Adamts16 in the homozygous Adamts16^mutant males resulted in cryptorchidism and male sterility. Heterozygous Adamts16^mutant males were normal, indicating that this is a recessive trait. Testes of homozygous Adamts16^mutant males were significantly smaller with significant histological changes associated with the lack of sperm production. Temporal histological assessments of the testis demonstrated that the seminiferous tubules did not support active spermatogenesis, but progressively lost germ cells, accumulated vacuoles and did not have any sperm. These observations, taken together with our previous report of renal abnormalities observed with the same Adamts16^mutant rats, suggest an important mechanistic link between Adamts16 and the functioning of the male genitourinary system.     

Correlation between dinucleotide periodicities and nucleosome positioning on mouse satellite DNA

Previous studies demonstrated 16 well-defined nucleosome locations (A-P) on a tandemly repeated prototype 234 base pair (bp) mouse satellite repeat unit. We have aligned the A-P fragments to search for DNA sequence elements that might contribute to nucleosome placement at these positions. Our results demonstrate a strikingly regular, uninterrupted, periodic pattern for the AA dinucleotide occurrences along the entire length of the aligned fragments. The periodicity of the AA occurrences is about 9.7 bp. The pattern exhibits a local minimum at position 74, near the nucleosome dyad axis of symmetry. Other dinucleotides--including AC: GT, CA: TG, and CC: GG--are also placed periodically, but their patterns of occurrence are less regular and less frequent than AA. The calculated spacings between consecutive preferred nucleosome locations on mouse satellite DNA are nearly identical, corresponding to multiples of 9.7 bp. The correlation between the periodicity of dinucleotide occurrences and the average spacing of nucleosome positions suggests that the preferred nucleosome locations recur at intervals that may correspond to the DNA helical repeat in the mouse satellite nucleosomes, and that the histone octamers sample (or slip along) the duplex in steps of 9.7 bp during nucleosome formation on mouse satellite DNA.     

Ultrafiltration and Microarray for Detection of Microbial Source Tracking Marker and Pathogen Genes in Riverine and Marine Systems

Pathogen identification and microbial source tracking (MST) to identify sources of fecal pollution improve evaluation of water quality. They contribute to improved assessment of human health risks and remediation of pollution sources. An MST microarray was used to simultaneously detect genes for multiple pathogens and indicators of fecal pollution in freshwater, marine water, sewage-contaminated freshwater and marine water, and treated wastewater. Dead-end ultrafiltration (DEUF) was used to concentrate organisms from water samples, yielding a recovery efficiency of >95% for Escherichia coli and human polyomavirus. Whole-genome amplification (WGA) increased gene copies from ultrafiltered samples and increased the sensitivity of the microarray. Viruses (adenovirus, bocavirus, hepatitis A virus, and human polyomaviruses) were detected in sewage-contaminated samples. Pathogens such as Legionella pneumophila, Shigella flexneri, and Campylobacter fetus were detected along with genes conferring resistance to aminoglycosides, beta-lactams, and tetracycline. Nonmetric dimensional analysis of MST marker genes grouped sewage-spiked freshwater and marine samples with sewage and apart from other fecal sources. The sensitivity (percent true positives) of the microarray probes for gene targets anticipated in sewage was 51 to 57% and was lower than the specificity (percent true negatives; 79 to 81%). A linear relationship between gene copies determined by quantitative PCR and microarray fluorescence was found, indicating the semiquantitative nature of the MST microarray. These results indicate that ultrafiltration coupled with WGA provides sufficient nucleic acids for detection of viruses, bacteria, protozoa, and antibiotic resistance genes by the microarray in applications ranging from beach monitoring to risk assessment.     

Leishmanicidal Triterpenes from Lantana camara

Two new natural triterpenes, lantaninilic acid and lantoic acid, along with the known triterpenes lantadene A, and oleanolic, ursolic, betulinic, lantanolic, and camaric acid, were obtained from the aerial parts of Lantana camara through bioassay‐guided isolation, monitoring the in vitro antileishmanial activity against promastigotes of Leishmania major. Oleanolic acid ( 3 ), ursolic acid ( 4 ), lantadene A ( 5 ), and lantanilic acid ( 7 ) showed significant leishmanicidal activities with IC₅₀ values of 53.0, 12.4, 20.4, and 21.3 μM , respectively. The IC₅₀ value of ursolic acid ( 4 ; 12.4 μM ) was found to be comparable with that of the standard drugs, pentamidine (IC₅₀ 15.0 μM ) and amphotericin B (IC₅₀ 0.31 μM ). The in vitro activities of L. camara and its constituents against promastigotes of Leishmania major are reported here for the first time.