Kinetics of human sperm acrosomal exocytosis.

The acrosome reaction is a unique event in the lifespan of sperm characterized by the exocytosis of the acrosomal content and the release of hybrid vesicles formed by patches of the outer acrosomal membrane and the plasma membrane. This unique regulated exocytosis is mediated by essentially the same membrane fusion machinery present in neuroendocrine cells. However, whereas secretion in neuroendocrine cells occurs in less than a second, the acrosome reaction is normally assessed after several minutes of incubation with inducers. In this report, we measured the kinetics of human sperm exocytosis triggered by two stimuli (calcium ionophore and progesterone) by using electron microscopy and three different approaches based on the incorporation of fluorescent Pisum sativum agglutinin into the acrosome upon opening of fusion pores connecting the extracellular medium with the acrosomal lumen. The results with the different methods are consistent with a slow kinetics (t½ = 14 min). We also manipulated the system to measure different steps of the process. We observed that cytosolic calcium increased with a relatively fast kinetics (t½ = 0.1 min). In contrast, the swelling of the acrosomal granule that precedes exocytosis was a slow process (t½ = 13 min). When swelling was completed, the fusion pore opening was fast (t½ = 0.2 min). The results indicate that acrosomal swelling is the slowest step and it determines the kinetics of the acrosome reaction. After the swelling is completed, the efflux of calcium from intracellular stores triggers fusion pores opening and the release of hybrid vesicles in seconds.

A novel myelin protein zero (V136G) homozygous mutation causing late onset demyelinating polyneuropathy with brain white matter lesions.

Although less common than peripheral myelin protein 22 (PMP22) duplication, there are mutations in myelin protein zero (MPZ) responsible for Charcot-Marie-Tooth disease (CMT) with a number of different clinical profiles. We report here a novel MPZ homozygous mutation, with a peculiar pattern characterized by a late-onset demyelinating profile. In addition, the patient presented brain white matter lesions seemingly ascribable to the mutation.

Cholesterol content regulates acrosomal exocytosis by enhancing Rab3A plasma membrane association.

The acrosome is an exocytic granule that overlies the spermatozoan nucleus. In response to different stimuli, it undergoes calcium-regulated exocytosis. Freshly ejaculated mammalian sperm are not immediately capable of undergoing acrosome reaction. The acquisition of this ability is called capacitation and involves a series of still not well-characterized changes in the sperm physiology. Plasma membrane cholesterol removal is one of the sperm modifications that are associated with capacitation. However, how sterols affect acrosomal exocytosis is unknown. Here, we show that short incubations with cyclodextrin, a cholesterol removal agent, just before stimulation promote acrosomal exocytosis. Moreover, the effect was also observed in permeabilized cells stimulated with calcium, indicating that cholesterol plays a direct role in the calcium-dependent exocytosis associated with acrosome reaction. Using a photo-inhibitable calcium chelator, we show that cholesterol affects an early event of the exocytic cascade rather than the lipid bilayers mixing. Functional data indicate that one target for the cholesterol effect is Rab3A. The sterol content does not affect the Rab3A activation-deactivation cycle but regulates its membrane anchoring. Western blot analysis and immunoelectron microscopy confirmed that cholesterol efflux facilitates Rab3A association to sperm plasma membrane. Our data indicate that the cholesterol efflux occurring during capacitation optimizes the conditions for the productive assembly of the fusion machinery required for acrosome reaction.

alpha-SNAP and NSF are required in a priming step during the human sperm acrosome reaction.

The acrosome is a membrane-limited granule that overlies the nucleus of the mature spermatozoon. In response to physiological or pharmacological stimuli it undergoes a special type of Ca2+-dependent exocytosis termed the acrosome reaction (AR), which is an absolute prerequisite for fertilization. Aided by a streptolysin-O permeabilization protocol developed in our laboratory, we have previously demonstrated requirements for Rab3A, N-ethylmaleimide-sensitive factor (NSF), several soluble NSF-attachment protein receptor (SNARE) proteins, and synaptotagmin VI in the human sperm AR. Here, we show that alpha-soluble NSF-attachment protein (alpha-SNAP), a protein essential for most fusion events through its interaction with NSF and the SNARE complex, exhibits a direct role in the AR. First, the presence of alpha-SNAP is demonstrated by the Western blot of human sperm protein extracts. Immunostaining experiments reveal an acrosomal localization for this protein. Second, the Ca2+ and Rab3A-triggered ARs are inhibited by anti-alpha-SNAP antibodies. Third, bacterially expressed alpha-SNAP abolishes exocytosis in a fashion that depends on its interaction with NSF. Fourth, we show a requirement for alpha-SNAP/NSF in a prefusion step early in the exocytotic pathway, after the tethering of the acrosome to the plasma membrane and before the efflux of intra-acrosomal Ca2+. These results suggest a key role for alpha-SNAP/NSF in the AR, and strengthen our understanding of the molecular players involved in the vesicle-to-plasma membrane fusion taking place during exocytosis.

Requirement of protein tyrosine kinase and phosphatase activities for human sperm exocytosis.

The acrosome is a membrane-limited granule that overlies the nucleus of the mature spermatozoon. In response to physiological or pharmacological stimuli, sperm undergo calcium-dependent exocytosis termed the acrosome reaction, which is an absolute prerequisite for fertilization. Protein tyrosine phosphorylation and dephosphorylation are a mechanisms by which multiple cellular events are regulated. Here we report that calcium induces tyrosine phosphorylation in streptolysin O (SLO)-permeabilized human sperm. As expected, pretreatment with tyrphostin A47-a tyrosine kinase inhibitor-abolishes the calcium effect. Interestingly, the calcium-induced increase in tyrosine phosphorylation has a functional correlate in sperm exocytosis. Masking of phosphotyrosyl groups with a specific antibody or inhibition of tyrosine kinases with genistein, tyrphostin A47, and tyrphostin A51 prevent the acrosome reaction. By reversibly sequestering intra-acrosomal calcium with a photo-inhibitable chelator, we show a requirement for protein tyrosine phosphorylation late in the exocytotic pathway, after the efflux of intra-acrosomal calcium. Both mouse and human sperm contain highly active tyrosine phosphatases. Importantly, this activity declines when sperm are incubated under capacitating conditions. Inhibition of tyrosine phosphatases with pervanadate, bis(N,N-dimethylhydroxoamido)hydroxovanadate, ethyl-3,4-dephostatin, and phenylarsine oxide prevents the acrosome reaction. Our results show that both tyrosine kinases and phosphatases play a central role in sperm exocytosis.

[Intraoperative monitoring of the facial nerve: anesthesia and neurophysiology considerations]. / Monitorización intraoperatoria del nervio facial: consideraciones anestésicas y neurofisiológicas.

Intraoperative monitoring of the motor component of the 7th cranial pair or any other nerve should be routine during any surgical procedure involving risk of neural damage, whether or not the skull is opened. This paper discusses the main indications for monitoring facial and acoustic nerves during ear and neurosurgery involving the pontocerebellar angle (for acoustic neuroma or in surgery on the 8th cranial pair). Intraoperative electromyographic monitoring of the facial nerve should be used routinely in acoustic neuroma surgery to reduce the degree of postoperative neurological impairment and avoid possible malpractice suits. Although such monitoring requires that the muscle remain unblocked, intraoperative neuromuscular relaxants can be used if doses are administered along with appropriate monitoring of the level of peripheral neuromuscular block.

Rab3A and calmodulin regulate acrosomal exocytosis by mechanisms that do not require a direct interaction.

The interaction between Rab3A and calmodulin is necessary for the inhibitory effect of Rab3A in neuroendocrine cells. Contrastingly, Rab3A triggers the exocytosis known as acrosome reaction in permeabilized spermatozoa. Here we show that a Rab3A mutant that cannot bind calmodulin was fully capable of triggering acrosomal exocytosis. Additionally, calmodulin by itself abrogated the exocytosis triggered by Rab3A. The effect was observed with both the wild type protein and the calmodulin binding deficient mutant. Our results indicate that the inhibitory and stimulatory effects of Rab3A in different exocytic processes are mediated by different effectors.

Event-related evoked potential P300 in frontotemporal dementia.

There are no studies on event-related cognitive potentials in frontotemporal dementia (FTD). In order to evaluate the aptitude and usefulness of the event-related P300 potential in this disease, we prospectively examined 60 cases: 11 patients with FTD diagnosed according to the Lund and Manchester criteria and Neary consensus criteria, 33 patients with a probable Alzheimer's disease diagnosis following NINCDS-ADRDA criteria, and 16 normal controls. P300 latency, amplitude and reaction time were recorded using an auditory oddball paradigm. In this sample, P300 potential could be reliably performed by 10/11 FTD patients, notwithstanding their language or executive function deficiencies. The FTD group P300 mean latency was midway between the normal controls and the Alzheimer's disease group (ANOVA F(2, 74199) = 16.5; p = 0.00003). The latency range of the FTD patients were within normal values (average plus 1.96 standard deviation of the values of the control group), except for one case with a latency of 448 ms. Post hoc Newman-Keuls analysis showed that the P300 latencies of the control and FTD groups did not differ significantly (p = 0.15) and that the Alzheimer's disease group had a delayed P300 latency that differed significantly from that of the FTD (p = 0.002) and control group (p = 0.0002). However, there was overlapping in P300 latency values of the three groups. Despite these differences in latencies, the reaction time was significantly increased in the FTD and the Alzheimer's disease groups. These findings indicate that the P300 potential is less affected in patients with FTD than those with Alzheimer's disease. This fact could aid in FTD diagnosis, differential diagnosis with Alzheimer's disease and possibly its clinical management.

Synaptotagmin VI participates in the acrosome reaction of human spermatozoa.

Acrosomal exocytosis is a calcium-dependent secretion event causing the release of the acrosomal contents and the loss of the membranes surrounding the acrosome. The synaptotagmins are a family of calcium-binding proteins that participate in the exocytosis of synaptic vesicles. The ubiquitous synaptotagmin VI isoform was found in human sperm cells by Western blot analysis. Immunocytochemistry at the optical and electron microscopy levels localized the protein to the outer acrosomal membrane. Calcium-triggered acrosomal exocytosis in permeabilized sperm cells was abrogated by a specific anti-synaptotagmin VI antibody, indicating that the protein is required for the process. Moreover, a recombinant fusion protein between glutathione S-transferase and the two calcium and phospholipid binding domains of synaptotagmin VI completely inhibited calcium-triggered exocytosis. Interestingly, phorbol ester-dependent in vitro phosphorylation of this recombinant protein abolished its inhibitory effect. We previously showed that, in permeabilized spermatozoa, addition of active Rab3A triggers acrosomal exocytosis at very low calcium concentration. Rab3A-promoted exocytosis was inhibited by the cytosolic domain of synaptotagmin VI and by the anti-synaptotagmin VI antibody, indicating that synaptotagmin is also necessary for Rab-mediated acrosomal content release. In conclusion, the results strongly indicate that synaptotagmin VI is a key component of the secretory machinery involved in acrosomal exocytosis.

Clinical-molecular study of a family with essential tremor, late onset seizures and periodic paralysis.

We report the clinical features of, and the molecular study performed on, a Spanish family with essential tremor (ET), late onset epilepsy and autosomal dominant hypokalemic periodic paralysis (hypoPP). The presence of hypoPP in this kindred suggested an ion channel as a candidate gene for ET. Our study identified an Arg528His CACNL1A3 mutation in patients with hypoPP, and excluded this mutation as the cause of tremor or epilepsy in this kindred.

Calcium-triggered acrosomal exocytosis in human spermatozoa requires the coordinated activation of Rab3A and N-ethylmaleimide-sensitive factor.

The acrosome reaction of spermatozoa is a complex, calcium-dependent, regulated exocytosis. Fusion at multiple sites between the outer acrosomal membrane and the cell membrane causes the release of the acrosomal contents and the loss of the membranes surrounding the acrosome. However, very little is known about the molecules that mediate and regulate this unique fusion process. Here, we show that N-ethylmaleimide-sensitive factor (NSF), a protein essential for most fusion events, is present in the acrosome of several mammalian spermatozoa. Moreover, we demonstrate that calcium-dependent exocytosis of permeabilized sperm requires active NSF. Previously, we have shown that the addition of the active (GTP-bound) form of the small GTPase Rab3A triggers exocytosis in permeabilized spermatozoa. In the present report we show that Rab3A is necessary for calcium-dependent exocytosis. The activation of Rab3A protects NSF from N-ethylmaleimide inhibition and precludes the exchange of the endogenous protein with recombinant dominant negative mutants of NSF. Furthermore, Rab3A activation of acrosomal exocytosis requires active NSF. Our results suggest that, upon calcium stimulation, Rab3A switches to its active GTP-bound form, triggering the formation of a protein complex in which NSF is protected. This process is suggested to be an essential part of the molecular mechanism of membrane fusion leading to the release of the acrosomal contents.

Suspected sexual abuse: an unusual presentation form of congenital myotonic dystrophy.

UNLABELLED: In children, anal abnormalities due to various causes may be confused with sexual abuse. We present the case of a child in whom the suspicion of abuse due to anal dilatation led to the previously unknown diagnosis of myotonic dystrophy. Myopathic involvement of the perianal musculature is a known feature of congenital myotonic dystrophy that usually appears in late childhood or adolescence. CONCLUSION: We stress the importance of considering an underlying myopathic condition in the differential diagnosis of anal laxity. Further studies, such as anal ultrasonography, may help when the diagnosis of abuse is not clear.

[Automatic methods of measurement of motor potential units]. / Métodos automáticos de cuantificación de potenciales de unidad motora.

INTRODUCTION: The study of motor unit potentials (MUP) may be carried out using qualitative or quantitative methods. The considerable usefulness of quantitative methods in the diagnosis of neuromuscular disorders has led to development of new automatic techniques which permit greater speed and accuracy of clinical investigation. In this review we consider traditional electromyographic techniques and describe some automatic methods. DEVELOPMENT: Qualitative techniques are simpler but give limited information since they only permit recording of very marked alterations. The introduction of trigger and averaging permit quantitative analysis and thus the evaluation of less obvious changes. Although they do not identify all the potentials of an electromyographic signal, the new automatic techniques using partial decomposition avoid the loss of time occurring with earlier techniques, give reliable objective measurements of the MUP and also the possibility of measuring new parameters, such as the area or frequency of discharge. CONCLUSIONS: New methods of evaluation of the MUP have significant advantages over traditional methods, namely increased speed and objectivity. However, this makes it necessary to redefine normal values since different methods are used for obtaining and processing the signals, otherwise the results may not be strictly comparable.

Transthyretin Tyr77 familial amyloid polyneuropathy: a clinicopathological study of a large kindred.

More than 40 point mutations (producing different clinical manifestations) have been described in diverse points of the plasma protein transthyretin (TTR). The Met30 is considered the most common mutation, the Tyr77 mutation being the second most prevalent. However, data from patients with this late mutation are scarce, and usually come from isolated case reports or tables. The Tyr77 mutation is not as well characterized as the Met30 mutation, especially with respect to such aspects as prognosis or possible treatment by liver transplantation. We therefore present the clinical and pathological features of an extensive family with the Tyr77 TTR mutation, comprising 12 affected individuals over four generations. Six living individuals were followed over a 10-year period. Retrospective data were obtained with regard to the deceased family members. We found that an initial and sometimes prolonged carpal tunnel syndrome, beginning between the 6th and 7th decades, characterizes the Tyr77 mutation. In most cases this evolved to generalized peripheral nerve involvement, restrictive cardiomyopathy, and intestinal malabsortion. Although survival is usually high, there are progressive cases that should be candidates for liver transplant, before severe impairment has developed.

[The association of the Eaton-Lambert myasthenic syndrome and subacute cerebellar degeneration of nonparaneoplastic origin]. / Asociación de síndrome miasteniforme de Eaton-Lambert y degeneración cerebelosa subaguda de origen no paraneoplásico.

A 52-year-old male with Eaton-Lambert syndrome associated to seven years subacute cerebellar degeneration of non paraneoplastic origin is presented. Immunosuppressive treatment with azathioprine allowed the neuromuscular symptoms to be controlled although no appreciable effects were observed in the cerebellar symptoms which remained stable.

[Demyelinating polyradiculitis in neuro borreliosis]. / Polirradiculitis desmielinizante en la neuroborreliosis.

Demyelinating neuropathy is considered a rare complication of Lyme borreliosis. We report a case of meningoradiculitis due to Borrelia burgdorferi in which the neurophysiological analysis showed evidence of restricted demyelinating involvement of the nerve roots and plexus, with no peripheral involvement or signs of distal axonal lesions. Lyme disease, therefore, can in fact be associated with demyelinating polyradiculitis with no peripheral nerve damage.