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Reconstruction of extensive palatal defects in growing patients aims to restore speech intelligibility and swallowing function while avoiding excessive scarring formation that may cause growth disturbances in the palate and midface region. Free flaps transfer healthy, well-vascularized tissue to the defect area, and their combination with pharyngeal flaps allow for restoration of the velopharyngeal function. We examined speech and swallowing after microsurgical palate reconstruction in a series of six pediatric patients.Radial forearm free flaps were used in all cases, in combination with a superiorly based pharyngeal flap in five cases. Mean age at surgery was 10.7 years. Etiologies included recurrent oronasal fistula due to failed primary cleft palate repair (n = 4), embryonal rhabdomyosarcoma of the maxilla (n = 1), and inflammatory fibrous hyperplasia (n = 1). Speech evaluations (with the Hirose standard and listener ratings) and swallowing assessments (based on videofluoroscopy swallowing studies and patient-reported swallowing and diet) were performed in average 44 months postoperatively.All flaps survived without major postoperative complications. Speech intelligibility was graded as "excellent" in four patients and "moderate" in two. Hypernasality and nasal obstruction were each judged as "none/minimal" in five cases and "moderate" in one case. All patients tolerated oral diet without significant nasal regurgitation. In five of six patients, the swallowing assessment showed good motion and velopharyngeal closure.Microsurgical reconstruction of extensive palatal defects using radial forearm free flap, with or without a superiorly based pharyngeal flap, is a reliable technique that can deliver substantial improvement of speech and swallowing in pediatric patients.

Most pain studies have focused on only two aspects of pain: the influence of pain on attentional processing and the modulation of pain perception by affective stimuli. However, the influence of tonic pain on the attentional processing of affective stimuli has not been studied. In this study, we investigated the effects of tonic pain on the attentional processing of affective stimuli, focusing on autonomic responses and their relationship with both EEG power and functional connectivity. Forty participants (20 men and 20 women) received tonically painful and nonpainful thermal stimulation while viewing blocks of pleasant, unpleasant, or neutral images. The galvanic skin conductance response (SCR), electrocardiographic activity, and electroencephalographic (EEG) activity in the delta and theta bands were recorded. Participants rated the unpleasantness of the pain at the end of each block. Typical affective SCR and heart rate (HR) patterns were found in the no-pain condition, but when the pain was delivered, these patterns disappeared. EEG power and functional connectivity results showed that tonic pain affected the delta band in the central region during pleasant and unpleasant image blocks. Our findings suggest that tonic pain captured attentional focus and reduced the cognitive resources available for processing affective stimuli, altering the emotional experience associated with pain.

Proteins with JAB1/MPN/MOV34 metalloenzyme (JAMM/MPN+) domains are widespread among all domains of life, yet poorly understood. Here we report the purification and characterization of an archaeal JAMM/MPN+ domain protein (HvJAMM1) from Haloferax volcanii that cleaves ubiquitin-like small archaeal modifier proteins (SAMP1/2) from protein conjugates. HvJAMM1 cleaved SAMP1/2 conjugates generated in H. volcanii as well as isopeptide- and linear-linked SAMP1-MoaE in purified form. Cleavage of linear linked SAMP1-MoaE was dependent on the presence of the SAMP domain and the C-terminal VSGG motif of this domain. While HvJAMM1 was inhibited by size exclusion chromatography and metal chelators, its activity could be restored by addition of excess ZnCl2 . HvJAMM1 residues (Glu31, His88, His90, Ser98 and Asp101) that were conserved with the JAMM/MPN+ active-site motif were required for enzyme activity. Together, these results provide the first example of a JAMM/MPN+ zinc metalloprotease that independently catalyses the cleavage of ubiquitin-like (isopeptide and linear) bonds from target proteins. In archaea, HvJAMM1 likely regulates sampylation and the pools of 'free' SAMP available for protein modification. HvJAMM1-type proteins are thought to release the SAMPs from proteins modified post-translationally as well as those synthesized as domain fusions.

Mammalian spermatozoa fertilize only after capacitation. The removal of decapacitation factors that inhibit the acrosome reaction (AR) is one of the events taking place during capacitation. In this report, human sperm were capacitated by 18-h incubation in Biggers, Whitten & Whittingham medium (BWW) medium and the proteins, on release, were analysed. After gel filtration by high-performance liquid chromatography a main peak with an approximate native molecular weight of 130 kDa was recognized by an antinormal seminal plasma antibody. This fraction was able to inhibit the follicular fluid as well as the progesterone-induced AR, when added to capacitated spermatozoa. Additionally, it reacted with an antibody directed against seminal plasma from vasectomized donors but not with an antibody against epididymal proteins. The AR inhibitory activity was heat-denatured, could be partially destroyed when treated with proteases, and bound to Concanavalin-A and wheat germ lectins. These results suggest that during in vitro capacitation, human spermatozoa release a glycoproteic decapacitation factor produced by accessory sex glands.

Problem: To determine the ability of IgGs isolated from follicular fluids (hFFIgGs) to induce the acrosome reaction (AR) in human spermatozoa and to inhibit sperm–zona pellucida (ZP) interaction. Method of study: Incubation of capacitated spermatozoa with hFFIgGs (n = 40) and assessment of their effect on the AR or hemizona (HZ) assay in a condition that allows sperm–ZP interaction, avoiding acrosomal exocytosis. Results: hFFIgGs from different women varied in their ability of inducing the AR. Those hFFIgGs with the highest AR-inducing capacity evoked the exocytotic response in most of the different sperm donors tested [high Induction Frequency (IF)]. Some of these antibodies were also able of inhibiting sperm binding to ZP [low HZ Index (HZI)]. A significant correlation was found between the IF and the HZI for each hFFIgG. Conclusions: Human follicular fluid contains antibodies capable of inducing the AR and inhibiting sperm–ZP binding, suggesting that they could be directed towards ZP receptors. hFFIgGs would constitute a tool for the identification of sperm entities involved in fertilization.

A part of sperm glycosidase activities was detected as detergent-insoluble after sequential extractions with Triton X-100. Sixty per cent of total beta-glucuronidase activity was found in the detergent-insoluble fraction. This portion of beta-glucuronidase was resistant to extractions in the presence of 1 M KCl, chaotropic agents, colchicine or cytochalasine B, being only partially solubilized by 3 M KCl or DNAse I treatment. Results demonstrate that beta-glucuronidase is tightly associated to the Triton X-100 resistant fraction.

β-N-acetylglucosaminidase (NAG) activity in human epididymal fluid was separated into two forms (I and II) after HPLC-hydrophobic interaction chromatography. Both forms exhibited maximal activity at a pH of around 4.5 and had a molecular weight of 125 kD when determined by Superose-HPLC. After incubation at 50°C, form I retained only 30% of its activity while form II retained 90% activity. When analysed by non-denaturing electrophoresis, form I displayed higher electrophoretic mobility than did form II. These features indicate that the I and II isoforms found in the human epididymis are the A and B forms present in other tissues. NAG activity was measured in the fluid obtained form the different epididymal regions of 13 different samples. An average four-fold increase in activity between the proximal caput and distal corpus was found. The contribution of each isoform to the total activity was studied. The proximal caput found to be rich in the A isoform (59%), whereas the B form was predominant in the distal corpus (65%). Human spermatozoa contain membrane-associated NAG activity with an isoform distribution similar to that found in cauda epididymal fluid (CEP, 80% B). Finally, enzyme activity in CEP was two-fold greater than in seminal plasma. Taken together these results suggest that NAG may become associated with human spermatozoa during epididymal transit.

The ability of human follicular fluid (hFF), retrieved from women undergoing IVF to induce the acrosome reaction (AR) in human sperm has been documented by several laboratories. However, the nature of the active factors in the hFF and the physiological meaning of the AR induction are highly controversial. We performed a three step purification scheme for hFF and all the fractions were screened for the AR-inducing activity. AR activity was associated with a protein fraction of M(r) > 180 kD that on further analysis under PAGE was found to be composed by subunits of apparent M(r) 50,000 and 29,000. The N-terminal sequences of these bands showed a 100% homology with the heavy and light chains of human IgG. A polyclonal antibody raised against the purified protein and anti-human IgG were both able to suppress the acrosome reaction-inducing activity of crude hFF. However, neither normal human serum nor a purified preparation of human IgG were able to mimic the AR-inducing activity of hFF. We concluded that the AR-inducing activity of hFF is, at least in part, due to the presence of antisperm antibodies.

Fertilization involves adhesive interactions between gametes similar to those mediated by fibronectin (FN) in other cellular systems. Fibronectin has been found on the equatorial segment of ejaculated human sperm. As sperm capacity to interact with the oocyte is acquired during epididymal transit, the possible participation of FN in human sperm maturation was studied. The presence of FN in both epididymal sperm and fluid was demonstrated by the detection of a major component of 220 kD in immunoblot studies using anti-FN antisera. The concentration of FN in soluble tissue extracts of epididymis was determined by enzyme-linked immunosorbent assay (ELISA). A gradual increase along the length of the organ, averaging 12-fold from proximal caput to distal corpus, was detected. Immunocytochemistry assays indicated that the number of spermatozoa with immmunoreactive FN over the equatorial segment increased from 18% in caput to 64% in distal corpus epididymis. Immunoprecipitation of medium from epididymal explants culture with anti-FN antiserum demonstrated the de novo synthesis of FN in vitro. The greater number of FN-positive sperm coincident with FN accumulation in distal regions of the epididymis supports the role of FN in sperm maturation. © 1992 Wiley-Liss, Inc.