Human Alzheimer’s disease synaptic <Emphasis Type=Italic>O</Emphasis>-GlcNAc site mapping and iTRAQ expression proteomics with ion trap mass spectrometry

Neuronal synaptic functional deficits are linked to impaired learning and memory in Alzheimer’s disease (AD). We recently demonstrated that O-GlcNAc, a novel cytosolic and nuclear carbohydrate post-translational modification, is enriched at neuronal synapses and positively regulates synaptic plasticity linked to learning and memory in mice. Reduced levels of O-GlcNAc have been observed in AD, suggesting a possible link to deficits in synaptic plasticity. Using lectin enrichment and mass spectrometry, we mapped several human cortical synaptic O-GlcNAc modification sites. Overlap in patterns of O-GlcNAcation between mouse and human appears to be high, as previously mapped mouse synaptic O-GlcNAc sites in Bassoon, Piccolo, and tubulin polymerization promoting protein p25 were identified in human. Novel O-GlcNAc modification sites were identified on Mek2 and RPN13/ADRM1. Mek2 is a signaling component of the Erk 1/2 pathway involved in synaptic plasticity. RPN13 is a component of the proteasomal degradation pathway. The potential interplay of phosphorylation with mapped O-GlcNAc sites, and possible implication of those sites in synaptic plasticity in normal versus AD states is discussed. iTRAQ is a powerful differential isotopic quantitative approach in proteomics. Pulsed Q dissociation (PQD) is a recently introduced fragmentation strategy that enables detection of low mass iTRAQ reporter ions in ion trap mass spectrometry. We optimized LTQ ion trap settings for PQD-based iTRAQ quantitation and demonstrated its utility in O-GlcNAc site mapping. Using iTRAQ, abnormal synaptic expression levels of several proteins previously implicated in AD pathology were observed in addition to novel changes in synaptic specific protein expression including Synapsin II.     

Strongyloidiasis and Infective Dermatitis Alter Human T Lymphotropic Virus-1 Clonality in vivo

Human T-lymphotropic Virus-1 (HTLV-1) is a retrovirus that persists lifelong by driving clonal proliferation of infected T-cells. HTLV-1 causes a neuroinflammatory disease and adult T-cell leukemia/lymphoma. Strongyloidiasis, a gastrointestinal infection by the helminth Strongyloides stercoralis, and Infective Dermatitis associated with HTLV-1 (IDH), appear to be risk factors for the development of HTLV-1 related diseases. We used high-throughput sequencing to map and quantify the insertion sites of the provirus in order to monitor the clonality of the HTLV-1-infected T-cell population (i.e. the number of distinct clones and abundance of each clone). A newly developed biodiversity estimator called “DivE” was used to estimate the total number of clones in the blood. We found that the major determinant of proviral load in all subjects without leukemia/lymphoma was the total number of HTLV-1-infected clones. Nevertheless, the significantly higher proviral load in patients with strongyloidiasis or IDH was due to an increase in the mean clone abundance, not to an increase in the number of infected clones. These patients appear to be less capable of restricting clone abundance than those with HTLV-1 alone. In patients co-infected with Strongyloides there was an increased degree of oligoclonal expansion and a higher rate of turnover (i.e. appearance and disappearance) of HTLV-1-infected clones. In Strongyloides co-infected patients and those with IDH, proliferation of the most abundant HTLV-1⁺ T-cell clones is independent of the genomic environment of the provirus, in sharp contrast to patients with HTLV-1 infection alone. This implies that new selection forces are driving oligoclonal proliferation in Strongyloides co-infection and IDH. We conclude that strongyloidiasis and IDH increase the risk of development of HTLV-1-associated diseases by increasing the rate of infection of new clones and the abundance of existing HTLV-1⁺ clones.     

The sensitive giant: the role of titin-based stretch sensing complexes in the heart

Every heart beat is not equal. As physiological demands of the cardiovascular system change, cardiac myocytes modulate contractile parameters including the rate and force of contraction. Adaptive responses require the sensing of biomechanical signals involving the interface between the contractile cytoskeleton (myofibrils) and the sarcolemma at specialized cell-cell junctions (intercalated discs) and cell-substrate adhesion complexes (costameres). Recent studies have shed insight into how protein complexes within cardiac myocytes sense biomechanical signals, processes required for normal adaptive or pathological responses. This new evidence suggests that complexes associated with the giant, myofibrillar protein titin sense myocyte stretch. Here, we discuss evidence supporting titin being an ideal biomechanical sensor.     

Culture of Amelanchier x grandiflora in a programmable micropropagation apparatus

A micropropagation system was developed to test concepts for automation and microenvironment alteration. Amelanchier x grandiflora Rehd. Princess Diana (serviceberry) shoot cultures were grown in seven-liter polycarbonate containers. Through computer control, the apparatus intermittently applied culture medium to the plant material according to a selected schedule. Shoot growth in the programmable system was compared with four micropropagation treatments: gelled and liquid medium in baby food jars and gelled and non-cycling liquid medium in a seven-liter vessel. Plants cultured in continuous contact with liquid medium became increasingly vitrified. Significantly greater shoot number, shoot length, shoot weight, and culture weight occurred in the programmable system than in jars with gelled medium. The combination of liquid medium, 7-liter vessel, and intermittent contact with medium caused a significantly higher proliferation rate than any combination of jar/vessel and gelled/liquid medium tested.     

Uncovering principles that control septin-septin interactions

Septins comprise a conserved family of GTPases important in cytokinesis. These proteins polymerize into filaments from rod-shaped heteromeric septin complexes. Septins interact with one another at two interfaces (NC and G) that alternate within the complex. Here, we show that small mutations at the N terminus greatly enhance the formation of SEPT2 homopolymers. Taking advantage of this mutation to examine polymer formation using SEPT2 alone, we show that both NC and G interfaces are required for filament formation. However, co-expression of wild type SEPT2 with SEPT2 containing mutations at either NC or G interfaces revealed that only the NC mutant suppressed filament formation. NC mutants are able to interact with one another at putative G interfaces, whereas G mutants fail to interact at NC interfaces. In addition, all promiscuous septin pairwise interactions occur at the G interface. These findings suggest that G interface interactions must occur before NC interactions during polymer formation.     

(4-Piperidinylphenyl)aminoethyl amides as a novel class of non-covalent cathepsin K inhibitors

A series of (4-piperidinylphenyl)aminoethyl amides based on dipeptide anilines were synthesized and tested against cathepsin K, cathepsin L and cathepsin B. These new non-covalent inhibitors exhibited single-digit nM inhibition of the cysteine proteases. Compounds 3 and 7 demonstrated potency in both mouse and human osteoclast resorption assays.     

The new higher level classification of eukaryotes with emphasis on the taxonomy of protists

This revision of the classification of unicellular eukaryotes updates that of Levine et al. (1980) for the protozoa and expands it to include other protists. Whereas the previous revision was primarily to incorporate the results of ultrastructural studies, this revision incorporates results from both ultrastructural research since 1980 and molecular phylogenetic studies. We propose a scheme that is based on nameless ranked systematics. The vocabulary of the taxonomy is updated, particularly to clarify the naming of groups that have been repositioned. We recognize six clusters of eukaryotes that may represent the basic groupings similar to traditional "kingdoms." The multicellular lineages emerged from within monophyletic protist lineages: animals and fungi from Opisthokonta, plants from Archaeplastida, and brown algae from Stramenopiles.     

Phosphorylation of the septin cdc3 in g1 by the cdc28 kinase is essential for efficient septin ring disassembly

The septins constitute a family of filament-forming proteins ubiquitous in eukaryotic species. We demonstrate here that the Saccharomyces cerevisiae septin, Cdc3, is a substrate of the cell cycle regulatory cyclin-dependent kinase (Cdk), Cdc28. Two serines near the C-terminus of Cdc3 are phosphorylated in a Cdc28-dependent manner. Analysis of a mutant allele that cannot be phosphorylated at these sites revealed an effect of Cdc28 phosphorylation of Cdc3 at the time of budding. Immunofluorescence analysis of wild-type and mutant Cdc3 indicated that prevention of phosphorylation at Cdc28-dependent sites impairs the disassembly of the old septin ring, which is inherited at mitosis but which usually disappears immediately prior to assembly of a new ring. Furthermore, immuno-fluorescence analysis of septin ring dynamics in a G1 cyclin (Cln) mutant suggests that G1 cyclin function is required for efficient ring disassembly. Thus, phosphorylation of Cdc3 by the Cdc28 kinase at the end of G1 may facilitate initiation of a new cell cycle by promoting disassembly of the obsolete septin ring from the previous cell cycle.     

Abnormal functional development of sympathetic nerve terminal-smooth muscle complex in neonatal spontaneously hypertensive rats

In neonatal and young adult SH and WK rats, maturation of a functional unit consisting of sympathetic nerve terminals and smooth muscle (levator palpebrae) was assessed $\text{in vivo}$ by measuring the contractile response to tyramine, an agent which releases endogenous norepinephrine from sympathetic nerve terminals. Responses in SH are comparable to WK at 5–6 days postnatally, smaller from the 8th through 19th day and larger in young adults (41–46 days old). Results indicate that functional maturation of the nerve terminal-smooth muscle complex is retarded in SH relative to WK during the 2nd and 3rd postnatal weeks. It is suggested that retardation in the neonatal SH rat is an expression of a genetic defect in the growth of the complex and that the enhanced response in young adult SH is a consequence of the neonatal abnormality.