Bacterioruberin extracts from a genetically modified hyperpigmented Haloferax volcanii strain: antioxidant activity and bioactive properties on sperm cells.

AIMS: To examine the antioxidant activity of Bacterioruberin (Bctr)-rich extracts isolated from a hyperpigmented, genetically modified Haloferax volcanii strain (HVLON3) and to investigate the effect on cold-sensitive ram sperm cells. METHODS AND RESULTS: The strain HVLON3 produces higher Bctr amounts than most haloarchaea (220 ± 13 mg g-1 DW). HVLON3-Bctr extract has higher antioxidant activity than ß-carotene (threefold) as evaluated using 2,2 diphenyl-1-picrylhydrazyl combined with Electron Paramagnetic Resonance analysis (EC50 4·5 × 10-5  mol l-1 vs 13·9 × 10-5  mol l-1 respectively). Different concentrations of HVLON3-Bctr extracts were assayed on ram sperm after freezing/thawing and physiologically relevant parameters were examined. Extracts containing 7 and 20 µmol l-1 Bctr significantly improved cell viability (P < 0·0001), total and progressive motility (P < 0·0001) and sperm velocities (P = 0·0172 for curvilinear velocity VCL, P = 0·0268 for average path velocity VAP and P = 0·0181 for straight line velocity VSL) and did not affect other parameters evaluated. CONCLUSIONS: HVLON3 is an excellent source of natural microbial C50 carotenoids with applicability in Biotechnology, Biomedical and Veterinary fields. HVLON3 Bctr extract improves the quality of cryopreserved ram sperm cells and could be applied to increase insemination yields. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides an insight on the bioactive properties of a bioproduct derived from haloarchaea (carotenoids) which are so far underexploited.

Objective evaluation of ram and buck sperm motility by using a novel sperm tracker software.

This work offers researchers the first version of an open-source sperm tracker software (Sperm Motility Tracker, V1.0) containing a novel suit of algorithms to analyze sperm motility using ram and buck sperm as models. The computer-assisted semen analysis is used in several publications with increasing trend worldwide in the last years, showing the importance of objective methodologies to evaluate semen quality. However, commercial systems are costly and versatility is constrained. In the proposed method, segmentation is applied and the tracking stage is performed by using individual Kalman filters and a simplified occlusion handling method. The tracking performance in terms of precision (number of true tracks), the percentage of fragmented paths and percentage of correctly detected particles were manually validated by three experts and compared with the performance of a commercial motility analyzer (Microptic's SCA). The precision obtained with our sperm motility tracker was higher than the one obtained with a commercial software at the current acquisition frame rate of 25 fps (P < 0.0001), concomitantly with a similar percentage of fragmentized tracks (P = 0.0709) at sperm concentrations ranging 25-37 × 106 cells/mL. Moreover, our tracker was able to detect trajectories that were unseen by SCA. Kinetic values obtained by using both methods were contrasted. The higher values found were explained based on the better performance of our sperm tracker to report speed parameters for very fast motile sperm. To standardize results, acquisition conditions are suggested. This open-source sperm tracker software has a good plasticity allowing researchers to upgrade according requirements and to apply the tool for sperm from a variety of species.

Heterologous recombinant protein with decapacitating activity prevents and reverts cryodamage in ram sperm: An emerging biotechnological tool for cryobiology.

During the last decades fundamental and applied aspects of mammalian ram sperm cryopreservation have been increasingly explored by scientists and biotechnologists. Many works report modifications in the composition of the freezing extenders and explore the beneficial and detrimental effects of seminal plasma or seminal plasma components in cryopreservation. Seminal plasma is known to contain stabilizing proteins, thereby this is a good start point to study the maintenance of membrane stability based on the basic knowledge of sperm physiology. However, seminal plasma composition is variable among rams and also the introduction of exogenous seminal plasma or its fractions to commercial semen can be associated with the transmission of viral diseases. Our work shows that a mouse protein, called SPINK3 (Serine Protease Inhibitor Kazal type 3) with decapacitating activity interacts with heterologous ram sperm when it is produced as a recombinant molecule. By immunocytochemistry assays we demonstrate that this protein (naturally expressed by mouse seminal vesicle under androgenic control) binds to the apical portion of both fresh and frozen ram sperm, the same localization described in mouse homologous sperm. Furthermore, it significantly improves sperm progressive motility compared to non-treated samples when it is added to freezing extenders and to dilution media after thawing. On the contrary, addition of SPINK3 does not modify sperm viability. The percentage of sperm with intact acrosome after ionophore induction was also significantly higher in sperm frozen in the presence of SPINK3 compared to control samples and the addition of SPINK3 after thawing significantly reduced both induced and non induced acrosomal loss, indicating that heterologous SPINK3 might act as a calcium inhibitor transport as described in mouse. Based on our results SPINK3 may find a place as a desirable biotechnological tool to achieve a higher proportion of competent sperm to fertilize.

Reorganization of Azospirillum brasilense cell membrane is mediated by lipid composition adjustment to maintain optimal fluidity during water deficit.

AIMS: We study the Azospirillum brasilense tolerance to water deficit and the dynamics of adaptive process at the level of the membrane. METHODS AND RESULTS: Azospirillum brasilense was exposed to polyethylene glycol (PEG) growth and PEG shock. Tolerance, phospholipids and fatty acid (FA) composition and membrane fluidity were determined. Azospirillum brasilense was able to grow in the presence of PEG; however, its viability was reduced. Cells grown with PEG showed membrane fluidity similar to those grown without, the lipid composition was modified, increasing phosphatidylcholine and decreasing phosphatidylethanolamine amounts. The unsaturation FAs degree was reduced. The dynamics of the adaptive response revealed a decrease in fluidity 20 min after the addition of PEG, indicating that the PEG has a fluidizing effect on the hydrophobic region of the cell membrane. Fluidity returned to initial values after 60 min of PEG exposure. CONCLUSION: Azospirillum brasilense is able to perceive osmotic changes by changing the membrane fluidity. This effect is offset by changes in the composition of membrane phospholipid and FA, contributing to the homeostasis of membrane fluidity under water deficit. SIGNIFICANCE AND IMPACT OF THE STUDY: This knowledge can be used to develop new Azospirillum brasilense formulations showing an adapted membrane to water deficit.

Effect of seminal plasma on post-thaw quality and functionality of corriedale ram sperm obtained by electroejaculation and artificial vagina.

We have already shown that seminal collection method affects seminal plasma composition and sperm quality in Corriedale rams. In this study, we evaluated the effect of seminal plasma collected by electroejaculation or artificial vagina on sperm resistance to cryodamage. Seminal plasma of five rams of the Corriedale breed collected by artificial vagina or electroejaculation was added before freezing to sperm cells collected by the two methods, and post-thaw quality parameters were evaluated. We found that seminal plasma has no effect on sperm resistance to cryodamage. However, we observed significantly higher percentages of sperm with intact and functional plasma membrane, intact acrosome and greater fertilizing potential after thawing in samples obtained by electroejaculation. This study demonstrates that sperm collected by electroejaculation are more resistant to damage caused by cryopreservation than those collected by artificial vagina.

Combined epidermal growth factor and hyaluronic acid supplementation of in vitro maturation medium and its impact on bovine oocyte proteome and competence.

The conditions for in vitro oocyte maturation impact on cytoplasmic and nuclear processes in the oocyte. These events are differentially influenced by the nature of the maturation inducer and the presence of intact cumulus in cumulus-oocyte complexes. Epidermal growth factor is the main growth factor promoting oocyte maturation. Also, hyaluronic acid (HA) produced by cumulus cells is known to be responsible for the correct structural and functional organization of the cumulus during oocyte maturation. Therefore, we evaluated the developmental competence of bovine oocytes matured in vitro in a maturation medium supplemented with both EGF and HA, compared to FSH and fetal bovine serum (FBS). In addition, the impact of IVM conditions on the proteomic profile of metaphase II bovine oocytes was analyzed by two-dimensional electrophoresis. Cumulus-oocyte complexes were matured in two media: (1) 10 ng/mL EGF, 15 µg/mL HA, and 100-µM cysteamine and (2) 0.01 UI/mL rh-FSH and 10% FBS. The percentages of first polar body and embryo production and the kinetics of embryo development and oocyte proteomic profiles were analyzed. Oocytes matured in the presence of EGF-HA showed an increase (6%, P < 0.05) in the percentage of polar body extrusion. The blastocyst rate was 3% (P < 0.05) higher in the FSH-FBS group, but no differences were found in the rate of expanded blastocyst neither in total embryo production between IVM conditions. Cleavage rate of oocytes matured with FSH-FBS was 5% higher (P < 0.05) with respect to EGF-HA-matured oocytes when evaluated 30 hours after fertilization. However, at Day 7, those inseminated oocytes that underwent division at a correct timing showed that although there are still early blastocysts in the FSH-FBS condition, EGF-HA embryos have developed completely into blastocysts. Still, the production rate of those embryos that achieved expansion was similar between both maturation conditions. On the other hand, noncleaved presumptive zygotes at Day 7 developed into the different stages with similar rates (∼4%) independently of the medium condition. Modifications of IVM medium composition markedly affected protein profile of bovine oocytes in a differential manner. The proteomic approach revealed the presence of 68 spots in both treatments, 41 exclusively found in the FSH-FBS group and 64 exclusive for the EGF-HA group. Taken together, these results indicate that combined EGF-HA supplementation of in vitro maturation medium could be used to improve oocyte meiotic competence and ensure a better timing to develop into the blastocyst stage.

Electroejaculation increases low molecular weight proteins in seminal plasma modifying sperm quality in Corriedale rams.

This study was conducted to evaluate the effect of seminal collection method (artificial vagina or electroejaculation) on the protein composition of seminal plasma and sperm quality parameters in Corriedale rams. To address this question, we assessed the effect of seminal collection method on motility, plasma membrane integrity and functionality, mitochondrial functionality and the decondensation state of nuclear chromatin in sperm cells. Volume, pH, osmolarity, protein concentration, total protein content and protein profile using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and 2-D polyacrylamide electrophoresis of seminal plasma collected with artificial vagina and electroejaculation were also analysed. The main findings from this study were that ejaculates obtained with electroejaculation had (i) a higher number of spermatozoa with intact plasma membrane and functional mitochondria and (ii) a higher proportion of seminal plasma, total protein content and relative abundance of low molecular weight proteins than ejaculates obtained with artificial vagina. Five of these proteins were identified by mass spectrometry: binder of sperm 5 precursor; RSVP14; RSVP22; epididymal secretory protein E1 and clusterin. One protein spot with molecular weight of approximately 31 kDa and isoelectric point of 4.8 was only found in the seminal plasma from electroejaculation.

An alternative easy method for antibody purification and analysis of protein-protein interaction using GST fusion proteins immobilized onto glutathione-agarose.

Immobilization of small proteins designed to perform protein-protein assays can be a difficult task. Often, the modification of reactive residues necessary for the interaction between the immobilized protein and the matrix compromises the interaction between the protein and its target. In these cases, glutathione-S-transferase (GST) is a valuable tag providing a long arm that makes the bait protein accessible to the mobile flow phase of the chromatography. In the present report, we used a GST fusion version of the 8-kDa protein serine protease inhibitor Kazal-type 3 (SPINK3) as the bait to purify anti-SPINK3 antibodies from a rabbit crude serum. The protocol for immobilization of GST-SPINK3 to glutathione-agarose beads was modified from previously reported protocols by using an alternative bifunctional cross-linker (dithiobis(succinimidyl propionate)) in a very simple procedure and by using simple buffers under physiological conditions. We concluded that the immobilized protein remained bound to the column after elution with low pH, allowing the reuse of the column for alternative uses, such as screening for other protein-protein interactions using SPINK3 as the bait.

SPINK3 modulates mouse sperm physiology through the reduction of nitric oxide level independently of its trypsin inhibitory activity.

Serine protease inhibitor Kazal-type (SPINK3)/P12/PSTI-II is a small secretory protein from mouse seminal vesicle which contains a KAZAL domain and shows calcium (Ca(2+))-transport inhibitory (caltrin) activity. This molecule was obtained as a recombinant protein and its effect on capacitated sperm cells was examined. SPINK3 inhibited trypsin activity in vitro while the fusion protein GST-SPINK3 had no effect on this enzyme activity. The inactive GST-SPINK3 significantly reduced the percentage of spermatozoa positively stained for nitric oxide (NO) with the specific probe DAF-FM DA and NO concentration measured by Griess method in capacitated mouse sperm; the same effect was observed when sperm were capacitated under low Ca(2+) concentration, using either intracellular (BAPTA-AM) or extracellular Ca(2+) (EDTA) chelators. The percentage of sperm showing spontaneous and progesterone-induced acrosomal reaction was significantly lower in the presence of GST-SPINK3 compared to untreated capacitated spermatozoa. Interestingly, this decrease was overcome by the exogenous addition of the NO donors, sodium nitroprusside (SNP), and S-nitrosoglutathione (GSNO). Phosphorylation of sperm proteins in tyrosine residues was partially affected by GST-SPINK3, however, only GSNO was able to reverse this effect. Sperm progressive motility was not significantly diminished by GST-SPINK3 or BAPTA-AM but enhanced by the addition of SNP. This is the first report that demonstrates that SPINK3 modulates sperm physiology through a downstream reduction of endogenous NO concentration and independently of SPINK3 trypsin inhibitory activity.

Conserved ram seminal plasma proteins bind to the sperm membrane and repair cryopreservation damage.

Whole seminal plasma (SP) enhances the function and fertility of frozen/thawed ram sperm. The objective of the current study was to investigate whether SP proteins capable of binding to molecules from the sperm plasma membrane were conserved among ram breeds, and whether these proteins were sufficient to overcome cryopreservation-induced reductions in sperm quality. Whole ram SP, obtained from rams of various breeds, improved progressive motility of frozen/thawed sperm at all times evaluated (P < 0.05); however, it did not improve total motility (15 min, P = 0.480; 30 min, P = 0.764; and 45 min, P = 0.795). To identify SP proteins responsible for this effect, a new method was developed to retain SP proteins that bound specifically to the sperm membrane by immobilization of sperm membrane proteins. These proteins specifically bound to the sperm surface, especially the acrosomal region. Lactotransferrin, epididymal secretory protein E1, Synaptosomal-associated protein 29, and RSVP-20 were identified (mass spectrometry) in this fraction. The retained SP proteins fraction repaired ultrastructural damage of frozen/thawed sperm and, with the addition of fructose, significantly improved motility of frozen/thawed sperm. We concluded that SP proteins that bound to the sperm membrane were conserved among ram breeds, and that when added to frozen/thawed semen (along with an energy source), they repaired ram sperm damage and enhanced sperm motility.

Clinicopathological features and outcome for dogs with mast cell tumors and bone marrow involvement.

BACKGROUND: Mast cell tumors (MCTs) with bone marrow (BM) involvement are poorly documented in dogs and are associated with a poor prognosis. Successful treatment strategies have not been described. HYPOTHESIS: Clinicopathologic findings of affected dogs are not specific. Administration of lomustine or imatinib is beneficial. ANIMALS: Fourteen dogs with MCT and BM involvement. METHODS: Clinical and laboratory evaluations were performed in each dog on admission and during follow-up. All dogs received prednisone. Additionally, 8 dogs received lomustine and 3 dogs received imatinib. Imatinib was administered if tumor-associated tyrosine kinase KIT was aberrant. RESULTS: On admission, 11 dogs had a single cutaneous nodule and 3 dogs had multiple nodules. Involvement of regional lymph nodes, liver, or spleen was observed in each dog. BM infiltration with mast cells (MCs) was observed in all dogs. On CBC, nonregenerative anemia, leukopenia, or thrombocytopenia was common. Four dogs had circulating MCs. Increased alkaline phosphatase or alanine transferase activity was observed in 12 and 10 dogs, respectively. Treatment with lomustine induced partial remission in 1 of 8 dogs. Median survival time was 43 days (range, 14-57). Dogs on imatinib experienced complete remission. Two dogs survived for 117 and 159 days, and the third was alive after 75 days. Dogs treated symptomatically did not improve and were euthanized after 1, 14, and 32 days. CONCLUSIONS AND CLINICAL IMPORTANCE: A combination of clinical and laboratory evaluation helps in identifying dogs with MCT and BM infiltration. Administration of lomustine is not helpful in affected dogs. The beneficial effect of imatinib warrants further investigation.

Seasonal variations in the composition of ram seminal plasma and its effect on frozen-thawed ram sperm.

It has been proposed that seminal plasma (SP) in the extender or in post-thaw media can prevent and revert cold-shock damage in cryopreserved ram sperm; however, this was dependent on season. We evaluated sperm parameters from Frisian ram semen incubated for various intervals with SP from all seasons and stored at -18 or -196 degrees C. At both temperatures, SP from autumn or winter increased (P<0.05) sperm motility, whereas no SP, or SP from spring or summer, had no effect. However, neither viability nor membrane or acrosomal status were modified by SP. Thirteen SP proteins were bound to the sperm surface (16.1, 16.7, 17.4, 23.3, 25.2, 27.5, 35.0, 40.0, 49.0, 53.5, 55.5, 61.0, and 86.0kDa). The SP proteins that bound to sperm were affected by season, but not by conservation temperature. Sperm incubated with SP from autumn had increased concentrations of five proteins; two were identified (with specific antibodies) as RSVP14 and RSVP20. In conclusion, SP from autumn and winter improved sperm motility of frozen-thawed ram sperm, and storage of ram SP at -18 or -196 degrees C did not affect protein composition. The SP proteins that bound to the sperm surface may be responsible for sperm membrane stabilization and should be further investigated.

Integrated morphophysiological assessment of two methods for sperm selection in bovine embryo production in vitro.

Extensive work was done regarding the ability of Swim up and Percoll gradient to select functional sperm for in vitro embryo production (IVP) systems. The aim of this work was to compare Swim up and Percoll as methods of sperm selection by ultrastructural, biochemical and functional studies. Frozen-thawed semen from two bulls (Experiments 1 and 2, respectively) were treated using Swim up or Percoll discontinuous gradients. Motility, sperm membrane ultrastructure, sperm proteins, in vitro embryo production (insemination doses, cleavage, embryo yield and quality) and embryo sex ratio were scored and compared. Electron transmission microscopy of outer sperm membranes showed higher (P<0.05) percentage of sperm with lost acrosomes in Percoll treated samples compared to Swim up. A differential protein pattern was also detected. When in vitro embryo production was performed, Percoll gradient produced higher (P<0.05) number of fertilizing doses (7.6 versus 5.9, Bull 1; 13.5 versus 7.8, Bull 2) and higher sperm motility (90% versus 76.6%, Bull 1; 81.7% versus 68.3%, Bull 2) than Swim up. The percentage of cleavage (Day 3) was similar in both treatment groups, whereas embryo production rate (Day 7) was higher (39.4% versus 30.2%, Bull 1; 38% versus 32.4%, Bull 2; P<0.05) when Percoll gradient was used. The percentage of hatched embryos (Day 11) and sex ratio did not differ. Total cell counting and embryo differential staining (inner cell mass and trophoblast cells) of Day 7 embryos showed that Percoll treated sperm produced better quality embryos compared to Swim up. We concluded that Percoll had a better performance selecting sperm and an enhanced capacity for embryo production when compared with the Swim up procedure; this could be attributed to a better acrosome exocytosis, associated to the absence of certain membrane proteins.

A view to regressive autism through home movies. Is early development really normal?

OBJECTIVE: To describe early development of children with regressive autism. METHOD: Home movies from the first 18 months of life of three groups of children with early onset autism (EOA), regressive autism (RA) and typical development (TD) were rated through the Grid for Attention in Infants in three age ranges. Different ANOVA and post hoc-tests were conducted on frequencies of behaviours. RESULTS: Differently from TD, for both RA and EOA non-social attention is higher than social attention across ages. While EOA is characterized by an early deficit in social attention, in RA social attention increases until the first birthday when its decrease proceeds at the same rate as the increase of non-social attention. CONCLUSION: We hypothesize that the intense interest towards objects can be the first sign of an atypical development also in RA; this distinguishing feature lead us to believe that regression, based on the assumption of a previous normal development, is only apparent.

Serine protease activity, bovine sperm protease, 66 kDa (BSp66), is present in hamster sperm and is involved in sperm-zona interaction.

Bovine sperm protease, 66 kDa (BSp66) is a serine protease previously characterized in bovine spermatozoa. Like other proteases, it may be present in sperm from other mammalian species different from bovine, playing a role in the fertilization process. In this study, we looked for BSp66 in hamster spermatozoa using heterologous antibodies against bovine BSp66. An immunoreactive protein was detected by Western blotting in mature and immature sperm. The detected protein had two isoforms similar to the ones reported in bovine sperm. Furthermore, indirect immune detection by fluorescence and electron microscopy assays, showed BSp66 signal at the acrosomal region similar to bovine sperm. As it was determined in bovine sperm, the acrosomal reaction displays the antigen within the acrosomal content. When live hamster sperm was incubated with polyclonal antibody against bovine BSp66 a decrease in the number of sperm bound to zona pellucida in homologous IVF and an impairment of head-head agglutination, were observed. These results suggest that a protease homologous to bovine BSp66 is present in golden hamster spermatozoa, with a conserved molecular mass and cellular location. Moreover, hamster BSp66 is probably involved in zona pellucida recognition.

Course of autism signs in the first year of life.

Autism spectrum disorders (ASD) are thought to be present right from birth, even if a minority of children displays a normal course during infancy followed by a regression during the second year of life. However, established criteria are not yet available to differentiate these different courses of ASD, and data coming from different sources have not yet been organized into a clear definition. The aim of this study was to elucidate the time of onset, as well as type, frequency and stability of symptoms during the first year of life in ASD. The behavioral summarized evaluation scale, applied to 40 home movies of children later diagnosed as having ASD, showed that most of the subjects (87.5%) display symptoms within the first year of life, when only a small group (12.5%) is completely symptom free. A group of more rated symptoms was found, constituting a typical pattern characterized by being withdrawn, and displaying poor social initiative, hypoactivity, and lack of emotional modulation. The importance of these findings is discussed in relation to early diagnosis and treatment.

BSp66 protease is widespread in the acrosomal region of sperm from several mammalian species.

Fertilization in mammals comprises a sequence of events leading to the fusion of sperm and oocyte membranes. Although proteases are known to be involved in this process, their role in fertilization is controversial. There is extensive work on the characterization of proteolytic systems, including serine proteases, which demonstrates that acrosomal proteases can be distinguished among the sperm of different mammalian species on the basis of the gelatin-hydrolyzing activity on SDS-PAGE by the quantity and variety of the enzymes. In this report, we investigated the occurrence and activity of the serine protease BSp66, previously characterized in bovine spermatozoa, in various mammalian sperm. A protein with a molecular mass of 66 kDa cross-reacted with heterologous antibodies against bovine BSp66 when sperm extracts of several mammalian species were analyzed by Western blot. In agreement, proteolytic activity corresponding to the molecular mass of BSp66 was detected by gelatin zymography in all the species analyzed. This protein was located on the acrosomal region of sperm cells by immunofluorescence methods. We concluded that BSp66 is widespread in mammalian sperm, with a conserved location in the acrosomal region.

The communicative use of pointing in autism: developmental profile and factors related to change.

PURPOSE: To describe different longitudinal profiles in communicative abilities and symptoms severity in early autism. METHODS: Five children with autism, aged from 3;3 to 4;10 years at baseline, were tested for production and comprehension of imperative and declarative pointing at about 4-month intervals. Concurrently with these sessions, children were evaluated in terms of cognitive and communicative abilities, and symptoms severity. RESULTS: Four subjects showed a mild to severe retardation in communicative and linguistic abilities. For production, all children exhibited the imperative pointing and only one the declarative pointing. For comprehension, two subjects showed the same profile as in production ('only imperative' and 'first imperative-later declarative', respectively). One child did not show any clear comprehension of the pointing gestures produced by the experimenter, and one child was able to understand both pointing in the same session. Childhood autism rating scale (CARS) global scores tended to decrease across sessions for all subjects and different individual profiles were identified. DISCUSSION: Declarative or experience-sharing pointing emerged later in one child only; it remained absent in four children as production, and in two children as comprehension. A preliminary conclusion based on CARS rating, is that autism involves a symptomatology that may decrease across time even if children differ in the decreasing profile relative to specific scores.

Autism and cerebellum. An unusual finding with MRI.

In recent years neuroimaging techniques have shown in young autistic children morphological disorders of the brainstem and cerebellum. After a review of the literature the authors present a three-year-old child, with a pervasive developmental disorder (autistic type) whose MRI shows an unusual finding in the right cerebellar hemisphere. The achieved levels in many developmental areas (linguistic, cognitive, relational, communicative) have been referred too. This developmental description represents additional information to the research about possible correlations between autistic symptoms and neuroanatomic abnormalities. The focus is about the role of neocerebellum in complex processes such as joint attention, shift attention, cognitive and linguistic functions which are probably involved in pathogenesis of autistic disorder. The presence of such inabilities in a case with cerebellar hemisphere abnormalities, without vermis compromission, could suggest that in autism exist deficits in more complex connections between neocerebellum and others regions of the brain.

Early behavioral development in autistic children: the first 2 years of life through home movies.

OBJECTIVE: The main aim of the research is to study the early behavioral development in autistic children through home movies. METHODS: fifteen home movies, regarding the first 2 years of life of autistic children are compared with the home movies of 15 normal children. The films of the two groups were mixed and rated by blind ratings with the Grid for the Assessment of Normal Behavior in Infants and Toddlers. The grid is composed of 17 items grouped into three developmental areas: social competence, intersubjectivity and symbolic activity. For every area, we have identified specific children's behaviors. RESULTS: Significant differences between the two groups are found both in the range of age 0-6 for intersubjectivity, and in the ranges of age 6-12 and 18-24 for symbolic activity. CONCLUSIONS: The authors pose some hypotheses about an early-appearing impairment of intersubjectivity, the ability to represent other's state of mind, in subjects with autistic disorder.