Females of the sea urchin Strongylocentrotus purpuratus differ in the structures of their egg jelly sulfated fucans

The egg jelly coats of sea urchins contain sulfated fucans which bind to a sperm surface receptor glycoprotein to initiate the signal transduction events resulting in the sperm acrosome reaction. The acrosome reaction is an ion channel regulated exocytosis which is an obligatory event for sperm binding to, and fusion with, the egg. Approximately 90% of individual females of the sea urchin Strongylocentrotus purpuratus spawned eggs having only one of two possible sulfated fucan electrophoretic isotypes, a slow migrating (sulfated fucan I), or a fast migrating (sulfated fucan II) isotype. The remaining 10% of females spawned eggs having both sulfated fucan isotypes. The two sulfated fucan isotypes were purified from egg jelly coats and their structures determined by NMR spectroscopy and methylation analysis. Both sulfated fucans are linear polysaccharides composed of 1-->3-linked alpha-L-fucopyranosyl units. Sulfated fucan I is entirely sulfated at the O -2 position but with a heterogeneous sulfation pattern at O -4 position. Sulfated fucan II is composed of a regular repeating sequence of 3 residues, as follows: [3-alpha-L-Fuc p - 2,4(OSO3)-1-->3-alpha-L-Fuc p -4(OSO3)-1-->3-alpha-L-Fuc p -4(OSO3)- 1]n. Both purified sulfated fucans have approximately equal potency in inducing the sperm acrosome reaction. The significance of two structurally different sulfated fucans in the egg jelly coat of this species could relate to the finding that the sperm receptor protein which binds sulfated fucan contains two carbohydrate recognition modules of the C-type lectin variety which differ by 50% in their primary structure.      

The oscillatory system responsible for the oculomotor activity during the bursts of REM.

1. In precollicular decerebrate cats the electrical activity of single pontine neurons was recorded before, during and after the episodes of postural atonia produced by i.v. injection of 0.03-0.1 mg/kg of eserine sulphate. These episodes were characterized by the regular occurrence of horizontal conjugate eye movements, which were mainly grouped in bursts of REM; moreover, a burst of REM in one direction was generally followed by a burst of REM in the opposite direction. 2. Among the recorded units, 32 showed an increase in their discharge rate during these cataplectic episodes. However, while these units fired at regular frequency when postural rigidity was present, they showed periodic changes in their discharge rate as soon as the bursts of REM appeared in the electrooculogram. In particular a nearly sinusoidal increase in the discharge rate was related to the appearance of an ocular burst in one direction, while a decrease in the unit discharge occurred during an ocular burst in the opposite direction. In some instances neighbouring pontine units located within each side of the brain stem showed reciprocal rate profiles during REM bursts oriented in a given direction, making it likely that the cyclic alternation of their activity depended upon their reciprocal interaction. 3. The alternative hypothesis, i.e., that these periodic changes in unit discharge depend upon the proprioceptive feedback due to the eye movements was excluded by the fact that these changes started before the occurrence of the bursts of REM and began to decline before the end of the burst. Moreover no variation in their firing rate was observed during the positional nystagmus induced by tilting the animal in the control period, i.e., when postural rigidity had reappeared following the end of the cataplectic episode. 4. Most of the neurons showing periodic changes in their discharge frequency during the bursts of REM were located in the pontine reticular formation. Scattered units were also found within the region of the locus coeruleus and the raphe system, close to the surrounding reticular structures. 5. In addition to these neurons, 60 pontine units were recorded, which did not show any changes in their discharge rate during transition from the control period to the cataplectic episode. However, phsiic increases or phasic decreases in their discharge rate appeared synchronously with the individual eye movements. Since in most instances these phasic changes in unit activity coincided with the appearance of the individual monophasic potentials recorded from the ascending MLB, which immediately preceded the rapid eye movements, these units could be attributed either to the premotor neurons responsible for these REM or to the closely related structures which generate their rhythmic discharge. In only a few instances did the discharge of these units not precede but follow the individual eye movements, indicating that they resulted from a proprioceptive feedback originating during the eye movements. 6...     

Temporal distribution of rapid eye movements and related monophasic potentials in the brain stem following injection of an anticholinesterase.

The temporal distribution of the horizontal rapid eye movements and the related monophasic potentials recorded from the ascending MLF following intravenous injection of o.i mg/kg of anticholinesterase has been investigated in precollicular decerebrate animals. In particular the intervals between individual MLF potentials occurring during successive REM episodes have been evaluated over a total period of 2000 sec on each experiment. 2. There was a bimodal distribution of intervals due to the fact that all the rapid eye movements and the related MLF potentials were grouped in bursts which occurred at quite regular intervals. 3. During the cholinergically induced episodes of REM, there were usually bursts of REM in one direction followed by bursts of REM in the opposite direction. The mean number of individual eye movements within each burst was 4.67 +/- 0.84, S.D., while the average interval between the individual eye movements corresponded to 167 +/- 36 msec, S.D. 4. There was a great regularity in the periodic occurrence of the bursts of REM. In particular the mean interval between the beginning of a burst of REM in one direction (i.e., towards the left side) and that of the next train oriented in the opposite direction (i.e., towards the right side) was 1.97 +/- 0.47 sec, S.D., while the mean interval between the beginning of this last train and that of the successive train oriented in the former direction corresponded to 2.97 +/- 0.48 sec, S.D. Moreover, the duration of the whole period corresponding to the interval between two successive bursts of REM oriented in the same direction (i.e., towards left or towards right) corresponded on average to 4.94 +/- 0.55 sec S.D. and 4.99 +/- 0.52 sec, S.D. respectively. 5. In addition to these "simple bursts" of rapid eye movements oriented in one direction, there were "complex bursts" in which an alternation of the individual eye movements within each burst was observed. In these instances the mean number of spikes was greater (5.35 +/- 1.20, S.D.) and the mean interval shorter 119 +/- 44 msec, S.D.) than those observed in the "simple bursts", About 10-15% of the bursts occurring during the cholinergically induced REM episodes were of the complex type. 6. These findings obtained from an individual experiment were confirmed in all the decerebrate animals treated with the same dose of anticholinesterase; only slight quantitative differences were detected from case to case. 7. Since the bursts of REM induced by the anticholinesterase depend upon the activity of the vestibular nuclei, it is postulated that cholinergic reticular neurons activate structures which show waxing and waning in their activity before acting on the vestibulo-oculomotor system. This system probably contains the inhibitory interneurons which transform the regularly modulated input into a rhythmic vestibular output...     

DENEDDYLASE1 Deconjugates NEDD8 from Non-Cullin Protein Substrates in Arabidopsis thaliana

The evolutionarily conserved 8-kD protein NEDD8 (NEURAL PRECURSOR CELL EXPRESSED, DEVELOPMENTALLY DOWN-REGULATED8) belongs to the family of ubiquitin-like modifiers. Like ubiquitin, NEDD8 is conjugated to and deconjugated from target proteins. Many targets and functions of ubiquitylation have been described; by contrast, few targets of NEDD8 have been identified. In plants as well as in non-plant organisms, the cullin subunits of cullin-RING E3 ligases are NEDD8 conjugates with a demonstrated functional role for the NEDD8 modification. The existence of other non-cullin NEDD8 targets has generally been questioned. NEDD8 is translated as a precursor protein and proteolytic processing exposes a C-terminal glycine required for NEDD8 conjugation. In animals and yeast, DENEDDYLASE1 (DEN1) processes NEDD8. Here, we show that mutants of a DEN1 homolog from Arabidopsis thaliana have no detectable defects in NEDD8 processing but do accumulate a broad range of NEDD8 conjugates; this provides direct evidence for the existence of non-cullin NEDD8 conjugates. We further identify AUXIN RESISTANT1 (AXR1), a subunit of the heterodimeric NEDD8 E1 activating enzyme, as a NEDD8-modified protein in den1 mutants and wild type and provide evidence that AXR1 function may be compromised in the absence of DEN1 activity. Thus, in plants, neddylation may serve as a regulatory mechanism for cullin and non-cullin proteins.     

The FAt Spondyloarthritis Spine Score (FASSS): development and validation of a new scoring method for the evaluation of fat lesions in the spine of patients with axial spondyloarthritis

Introduction

Studies have shown that fat lesions follow resolution of inflammation in the spine of patients with axial spondyloarthritis (SpA). Fat lesions at vertebral corners have also been shown to predict development of new syndesmophytes. Therefore, scoring of fat lesions in the spine may constitute both an important measure of treatment efficacy as well as a surrogate marker for new bone formation. The aim of this study was to develop and validate a new scoring method for fat lesions in the spine, the Fat SpA Spine Score (FASSS), which in contrast to the existing scoring method addresses the localization and phenotypic diversity of fat lesions in patients with axial SpA.

Methods

Fat lesions at pre-specified anatomical locations at each vertebral endplate (C2 lower-S1 upper) were assessed dichotomously (present/absent) on spine MRIs. Two readers independently evaluated MRIs obtained at two time points for 58 patients (Exercise 1), followed by optimization of scoring methodology and reader calibration. Thereafter, the same readers read 135 pairs of MRI scans (Exercise 2; including the 58 pairs from exercise 1 randomly mixed with 77 new pairs).

Results

In Exercise 2, the mean (SD) baseline FASSS score for the two readers was 22.5(29.6) and 21.1(28.0), respectively, and the FASSS change score was 4.2(10.6) and 6.0(12.2). Inter-reader reliability assessed as intra-class correlation coefficients (ICCs) for status and change scores were excellent (0.96 (95% CI (0.94 to 0.97)) and very good (0.86 (0.80 to 0.90)), respectively. The smallest detectable change (SDC) was 3.7 for the 135 patients. Good reliability of change scores was also observed for MRI scans conducted one year apart (ICC 0.74 (95% CI 0.44 to 0.89) and SDC 4.5). For the 58 MRI-pairs assessed in both exercises, inter-reader reproducibility for the total FASSS status score improved from very good (ICCs: 0.89 (95% CI: 0.81 to 0.93) in exercise 1 to excellent in exercise 2 (0.96 (0.93 to 0.98)), and improved substantially for the total change score (from 0.67 (0.51 to 0.80) to 0.83 (0.73 to 0.90).

Conclusions

FASSS meets essential validation criteria for quantification of a common structural abnormality in clinical trials of axial spondyloarthritis.     

ImmunoPET and biodistribution with human epidermal growth factor receptor 3 targeting antibody 89Zr-RG7116

The humanized monoclonal antibody with high affinity for the human epidermal growth factor receptor (HER) 3, RG7116, is a glycoengineered, IgG1 class antibody. By labeling RG7116 with zirconium-89 (⁸⁹Zr) we aimed to visualize in vivo HER3 expression and study the biodistribution of this antibody in human tumor-bearing mice. Biodistribution of ⁸⁹Zr-RG7116 was studied in subcutaneously xenografted FaDu tumor cells (HER3-positive). Dose-dependency of ⁸⁹Zr-RG7116 organ distribution and specific tumor uptake was assessed by administering doses ranging from 0.05 to 10 mg/kg RG7116 to SCID/Beige mice. Biodistribution was analyzed at 24 and 144 h after injection. MicroPET imaging was performed at 1, 3, and 6 days after injection of 1.0 mg/kg ⁸⁹Zr-RG7116 in the FaDu, H441, QG-56 and Calu-1 xenografts with varying HER3 expression. The excised tumors were analyzed for HER3 expression. Biodistribution analyses showed a dose- and time-dependent ⁸⁹Zr-RG7116 tumor uptake in FaDu tumors. The highest tumor uptake of ⁸⁹Zr-RG7116 was observed in the 0.05 mg/kg dose group with 27.5%ID/g at 144 h after tracer injection. MicroPET imaging revealed specific tumor uptake of ⁸⁹Zr-RG7116 in FaDu and H441 models with an increase in tumor uptake over time. Biodistribution data was consistent with the microPET findings in FaDu, H441, QG56 and Calu-1 xenografts, which correlated with HER3 expression levels. In conclusion, ⁸⁹Zr-RG7116 specifically accumulates in HER3 expressing tumors. PET imaging with this tracer provides real-time non-invasive information about RG7116 distribution, tumor targeting and tumor HER3 expression levels.     

Hypothesis for a serine proteinase-like domain at the COOH terminus of Slowpoke calcium-activated potassium channels

Bovine pancreatic trypsin inhibitor (BPTI) is a 58-residue protein with three disulfide bonds that belongs to the Kunitz family of serine proteinase inhibitors. BPTI is an extremely potent inhibitor of trypsin, but it also specifically binds to various active and inactive serine proteinase homologs with KD values that range over eight orders of magnitude. We previously described an interaction of BPTI at an intracellular site that results in the production of discrete subconductance events in large conductance Ca2+ activated K+ channels (Moss, G.W.J., and E. Moczydlowski. 1996, J. Gen. Physiol, 107:47-68). In this paper, we summarize a variety of accumulated evidence which suggests that BPTI binds to a site on the KCa channel protein that structurally resembles a serine proteinase. One line of evidence includes the finding that the complex of BPTI and trypsin, in which the inhibitory loop of BPTI is masked by interaction with trypsin, is completely ineffective in the production of substate events in the KCa channel. To further investigate this notion, we performed a sequence analysis of the alpha-subunit of cloned slowpoke KCa channels from Drosophila and mammals. This analysis suggests that a region of approximately 250 residues near the COOH terminus of the KCa channel is homologous to members of the serine proteinase family, but is catalytically inactive because of various substitutions of key catalytic residues. The sequence analysis also predicts the location of a Ca(2+)-binding loop that is found in many serine proteinase enzymes. We hypothesize that this COOH-terminal domain of the slowpoke KCa channel adopts the characteristic double-barrel fold of serine proteinases, is involved in Ca(2+)-activation of the channel, and may also bind other intracellular components that regulate KCa channel activity.