Mechanism and significance of specific proteolytic cleavage of Reelin

Reelin is a secreted glycoprotein essential for normal brain development and function. In the extracellular milieu, Reelin is subject to specific cleavage at two (N-t and C-t) sites. The N-t cleavage of Reelin is implicated in psychiatric and Alzheimer’s diseases, but the molecular mechanism and physiological significance of this cleavage are not completely understood. Particularly, whether the N-t cleavage affects the signaling activity of Reelin remains controversial.Here, we show that the protease in charge of the N-t cleavage of Reelin requires the activity of certain proprotein convertase family for maturation and has strong affinity for heparin. By taking advantage of these observations, we for the first time succeeded in obtaining “Uncleaved” and “Completely Cleaved” Reelin proteins. The N-t cleavage splits Reelin into two distinct fragments and virtually abolishes its signaling activity. These findings provide an important biochemical basis for the function of Reelin proteolysis in brain development and function.     

Shank2 associates with and regulates Na+/H+ exchanger 3

Na+/H+ exchanger 3 (NHE3) plays a pivotal role in transepithelial Na+ and HCO3(-) absorption across a wide range of epithelia in the digestive and renal-genitourinary systems. Accumulating evidence suggests that PDZ-based adaptor proteins play an important role in regulating the trafficking and activity of NHE3. A search for NHE3-binding modular proteins using yeast two-hybrid assays led us to the PDZ-based adaptor Shank2. The interaction between Shank2 and NHE3 was further confirmed by immunoprecipitation and surface plasmon resonance studies. When expressed in PS120/NHE3 cells, Shank2 increased the membrane expression and basal activity of NHE3 and attenuated the cAMP-dependent inhibition of NHE3 activity. Furthermore, knock-down of native Shank2 expression in Caco-2 epithelial cells by RNA interference decreased NHE3 protein expression as well as activity but amplified the inhibitory effect of cAMP on NHE3. These results indicate that Shank2 is a novel NHE3 interacting protein that is involved in the fine regulation of transepithelial salt and water transport through affecting NHE3 expression and activity.     

Direct interaction of post-synaptic density-95/Dlg/ZO-1 domain-containing synaptic molecule Shank3 with GluR1 alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor

A class of scaffolding protein containing the post-synaptic density-95/Dlg/ZO-1 (PDZ) domain is thought to be involved in synaptic trafficking of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors during development. To clarify the molecular mechanism of AMPA receptor trafficking, we performed a yeast two-hybrid screening system using the cytoplasmic tail of the GluR1 subunit of AMPA receptor as a bait and identified a synaptic molecule, Shank3/ProSAP2, as a GluR1 subunit-interacting molecule. Shank3 is a PDZ domain-containing multidomain protein and is predominantly expressed in developing neurons. Using the glutathione S-transferase pull-down assay and immunoprecipitation technique we demonstrated that the GluR1 subunit directly binds to the PDZ domain of Shank3 via its carboxyl terminal PDZ-binding motif. We raised anti-Shank3 antibody to investigate the expression of Shank3 in cortical neurons. The pattern of Shank3 immunoreactivity was strikingly punctate, mainly observed in the spines, and closely matched the pattern of post-synaptic density-95 immunoreactivity, indicating that Shank3 is colocalized with post-synaptic density-95 in the same spines. When Shank3 and the GluR1 subunit were overexpressed in primary cortical neurons, they were also colocalized in the spines. Taken together with the biochemical interaction of Shank3 with the GluR1 subunit, these results suggest that Shank3 is an important molecule that interacts with GluR1 AMPA receptor at synaptic sites of developing neurons.     

The expression profile of de-N-acetyl polysialic acid (NeuPSA) in normal and diseased human tissue

Although sialic acids have a key role in many aspects of human biology, the expression of polysialic acid (PSA) in human tissues is thought to be relatively rare. We identified a derivative of PSA called neuraminic acid-containing PSA or NeuPSA that was highly expressed in primary human melanoma tumors and in several cancer cell lines. Moreover, anti-NeuPSA antibodies could induce apoptosis of cancer cells. However, little was known about NeuPSA expression in normal or diseased tissues. In this study we investigated the complete expression profile of NeuPSA in human tissues and a few primary tumors using the anti-NeuPSA monoclonal antibody, SEAM 3. Almost every human tissue tested spanning a representative sample of all organ types was positive for SEAM 3 binding. Specificity of SEAM 3 binding was established by inhibition with NeuPSA but not closely related meningococcal C polysaccharide and loss of SEAM 3 binding when specimens were treated with periodate at high pH, which specifically destroys NeuPSA. Only subsets of cells in each specimen stained positive, and the relative staining between tissues was variable. The distribution and amount of NeuPSA antigen in tissues was correlated with known levels of polysialyltransferase PST or STX expression. The majority of anti-NeuPSA binding occurred intracellularly in the cytoplasm of cells. Tumors generally exhibited considerably increased staining compared with corresponding normal tissues. Identifying the diverse tissue distribution and intracellular location of NeuPSA provides a foundation for investigating the functional role of NeuPSA in human health and disease.     

Dietary adaptations of temperate primates: comparisons of Japanese and Barbary macaques

Habitat, diet and leaf chemistry are compared between Japanese and Barbary macaques to reveal the similarities and differences in dietary adaptations of temperate primates living at the eastern and western extremes of the genus Macaca. Tree species diversity and proportion of fleshy-fruited species are much higher in Japan than in North Africa. Both species spend considerable annual feeding time on leaves. Japanese macaques prefer fruits and seeds over leaves, and Barbary macaques prefer seeds. These characteristics are adaptive in temperate regions where fruit availability varies considerably with season, since animals can survive during the lean period by relying on leaf and other vegetative foods. The two species are different with respect to the higher consumption of herbs by Barbary macaques, and the leaves consumed contain high condensed and hydrolysable tannin for Barbary but not for Japanese macaques. Barbary macaques supplement less diverse tree foods with herbs. Because of the low species diversity and high tannin content of the dominant tree species, Barbary macaques may have developed the capacity to cope with tannin. This supports the idea that digestion of leaves is indispensable to survive in temperate regions where fruit and seed foods are not available for a prolonged period during each year.     

Pih1d3 is required for cytoplasmic preassembly of axonemal dynein in mouse sperm

Axonemal dynein complexes are preassembled in the cytoplasm before their transport to cilia, but the mechanism of this process remains unclear. We now show that mice lacking Pih1d3, a PIH1 domain–containing protein, develop normally but manifest male sterility. Pih1d3^−/− sperm were immotile and fragile, with the axoneme of the flagellum lacking outer dynein arms (ODAs) and inner dynein arms (IDAs) and showing a disturbed 9+2 microtubule organization. Pih1d3 was expressed specifically in spermatogenic cells, with the mRNA being most abundant in pachytene spermatocytes. Pih1d3 localized to the cytoplasm of spermatogenic cells but was not detected in spermatids or mature sperm. The levels of ODA and IDA proteins were reduced in the mutant testis and sperm, and Pih1d3 was found to interact with an intermediate chain of ODA as well as with Hsp70 and Hsp90. Our results suggest that Pih1d3 contributes to cytoplasmic preassembly of dynein complexes in spermatogenic cells by stabilizing and promoting complex formation by ODA and IDA proteins.     

Biodegradation of volatile organic compounds by five fungal species

Five fungal species, Cladosporium resinae (ATCC 34066), Cladosporium sphaerospermum (ATCC 200384), Exophiala lecanii-corni (CBS 102400), Mucor rouxii (ATCC 44260), and Phanerochaete chrysosporium (ATCC 24725), were tested for their ability to degrade nine compounds commonly found in industrial off-gas emissions. Fungal cultures inoculated on ceramic support media were provided with volatile organic compounds (VOCs) via the vapor phase as their sole carbon and energy sources. Compounds tested included aromatic hydrocarbons (benzene, ethylbenzene, toluene, and styrene), ketones (methyl ethyl ketone, methyl isobutyl ketone, and methyl propyl ketone), and organic acids ( n-butyl acetate, ethyl 3-ethoxypropionate). Experiments were conducted using three pH values ranging from 3.5 to 6.5. Fungal ability to degrade each VOC was determined by observing the presence or absence of visible growth on the ceramic support medium during a 30-day test period. Results indicate that E. lecanii-corni and C. sphaerospermum can readily utilize each of the nine VOCs as a sole carbon and energy source. P. chrysosporium was able to degrade all VOCs tested except for styrene under the conditions imposed. C. resinae was able to degrade both organic acids, all of the ketones, and some of the aromatic compounds (ethylbenzene and toluene); however, it was not able to grow utilizing benzene or styrene under the conditions tested. With the VOCs tested, M. rouxiiproduced visible growth only when supplied with n-butyl acetate or ethyl 3-ethoxypropionate. Maximum growth for most fungi was observed at a pH of approximately 5.0. The experimental protocol utilized in these studies is a useful tool for assessing the ability of different fungal species to degrade gas-phase VOCs under conditions expected in a biofilter application.     

Endogenous gibberellin A1 levels control thermoperiodic stem elongation in Pisum sativum

Gibberellin (GA) is believed to be involved in thermoperiodic stem elongation. With this in mind, we studied the correlation between gibberellin A₁ (GA₁) levels and stem elongation affected by alternating day (DT) and night temperature (NT) in 5 genotypes of Pisum sativum differing in their degree of dwarfism. The endogenous GA content in the tissue of two of the genotypes was determined by combined gas chromatography and mass spectrometry. The wild genotype developed 40 to 50% shorter stems and internodes under a low DT and high NT combination (negative difference [DIF] between DT and NT, DT/NT 15.5/21.5 or 14/24°C) than under the opposite regime of high DT and low NT (positive DIF, DT/NT 22.5/16.5 or 24/14°C). The GA biosynthetic mutants ls and le, and the auxin and brassinosteroid mutant lkb responded in a similar way, but not as strongly as the wild type. The stem length of the GA-insensitive slender mutant (la cry^s) was reduced by only 8% under negative compared to positive DIF. In the wild type endogenous GA levels decreased by 60% from positive to negative DIF in the upper part of the stem. Further, there was a corresponding decrease in the levels of precursors to GA₁, i.e. GA₅₃, GA₄₄, GA₁₉ and GA₂₀, while 2β-hydroxylated GA₂₀ and GA₁, GA₂₉ and GA₈, respectively, were unaffected by DIF. A similar increase in the ratios of GA₂₉ to GA₂₀ and GA₈ to GA₁ from positive to negative DIF was seen in the stem tissue of the le mutant as in the wild type. The temperature regimes affected the levels of GA₁ and its precursors in combined leaf and petiole samples and in the shoot tip in a similar manner as in the stem tissue. However, the different temperature regimes did not affect the ratio of GA₈/GA₁ in the shoot tip. The results indicate that altered stem elongation of the pea plants in response to diurnal temperature alternations may be mediated by changes in endogenous levels of GA₁. The GA₁ levels may be controlled by an effect of DIF on both biosynthetic and inactivation steps.     

Protective efficacy of IgM monoclonal antibodies in experimental group B streptococcal infection is a function of antibody avidity

We have produced and characterized six mAb directed against group B streptococci (GBS). All antibodies are IgM. We have previously shown that some of these antibodies are highly protective in the treatment of experimental infections in neonatal rats, whereas others do not appear to have any protective efficacy. Using an ELISA, we demonstrate the specificity of both protective and nonprotective antibodies. Two antibodies, binding different epitopes, are directed against antigenic structures present on all GBS; two are specific for type III carbohydrate determinants; one binds to a protein Ag present on all type I and II GBS; and one appears to bind to type Ia GBS only. Quantitative absorption assays provide evidence that the difference between protective antibodies and nonprotective antibodies is the avidity that the antibody demonstrates for the epitope recognized on the surface of the bacteria; 10 to 15 times as much protective antibody binds to GBS as does nonprotective antibody. Direct binding experiments with radiolabeled antibody confirm this conclusion.     

A quantitative study of the epiphytic vegetation on pollarded trunks of Fraxinus excelsior at Havrå, Osterøy,western Norway

The epiphytic vegetation on 19 pollarded trees of Fraxinus excelsior at the farm Havrå, Osterøy, western Norway was investigated. Each trunk was divided into a basal zone, a middle zone and a top zone. In each zone the four different aspects were analysed by squares (12 sampling units from each trunk). Within a total of 225 sampling units, 173 taxa were recorded (84 lichens, 72 bryophytes, 17 vascular plants). The epiphytic vegetation was classified into seven TWINSPAN groups, and it is shown by detrended correspondence analysis (DCA) that these groups are mainly separated by the first two DCA axes. In an attempt to explain the floristic composition, several environmental variables were measured, and the floristic and environmental data were analysed by canonical correspondence analysis (CCA). A pollarded tree, depending on its age, represents a continuum of perhaps several hundred years. Some of the recorded epiphytic lichens are very rare in Norway, and the pollarded trees thus contribute greatly to the biodiversity of the old cultural landscape. Several epiphytic species take advantage of favourable light conditions on trunks in the open fields. At Havrå, woodland has developed by tree colonization of old meadows and open fields during the last 40 years after the cessation of earlier managements. Some floristic differences between the epiphytic vegetation on trunks in the open fields and in the woodland are found. This suggests a change in the epiphytic vegetation because of the creation of a more shady habitat in parts of the area during a few decades.