Identification of 16 novel mutations in the argininosuccinate synthetase gene and genotype-phenotype correlation in 38 classical citrullinemia patients

Classical citrullinemia (CTLN1), a rare autosomal recessive disorder, is caused by mutations of the argininosuccinate synthetase (ASS) gene, localized on chromosome 9q34.1. ASS functions as a rate-limiting enzyme in the urea cycle. Previously, we identified 32 mutations in the ASS gene of CTLN1 patients mainly in Japan and the United States, and to date 34 different mutations have been described in 50 families worldwide. In the present study, we report ASS mutations detected in 35 additional CTLN1 families from 11 countries. By analyzing the entire coding sequence and the intron-exon boundaries of the ASS gene using RT-PCR and/or genomic DNA-PCR, we have identified 16 novel mutations (two different 1-bp deletions, a 67-bp insertion, and 13 missense) and have detected 12 known mutations. Altogether, 50 different mutations (seven deletion, three splice site, one duplication, two nonsense, and 37 missense) in 85 CTLN1 families were identified. On the basis of primary sequence comparisons with the crystal structure of E. coli ASS protein, it may be concluded that any of the 37 missense mutations found at 30 different positions led to structural and functional impairments of the human ASS protein. It has been found that three mutations are particularly frequent: IVS6-2A>G in 23 families (Japan: 20 and Korea: three), G390R in 18 families (Turkey: six, U.S.: five, Spain: three, Israel: one, Austria: one, Canada: one, and Bolivia: one), and R304W in 10 families (Japan: nine and Turkey: one). Most mutations of the ASS gene are "private" and are distributed throughout the gene, except for exons 5 and 12-14. It seems that the clinical course of the patients with truncated mutations or the G390R mutation is early-onset/severe. The phenotype of the patients with certain missense mutations (G362V or W179R) is more late-onset/mild. Eight patients with R86H, A118T, R265H, or K310R mutations were adult/late-onset and four of them showed severe symptoms during pregnancy or postpartum. However, it is still difficult to prove the genotype-phenotype correlation, because many patients were compound heterozygotes (with two different mutations), lived in different environments at the time of diagnosis, and/or had several treatment regimes or various knowledge of the disease.     

ATP-sensitive K+ and Ca2+-activated K+ channels in lamprey ( Petromyzon marinus) red blood cell membrane

The patch-clamp technique was used to demonstrate the presence of ATP-sensitive K(+) channels and Ca(2+)-activated K(+) channels in lamprey ( Petromyzon marinus) red blood cell membrane. Whole-cell experiments indicated that the membrane current under isosmotic (285 mosmol l(-1)) conditions is carried by K(+). In the inside-out configuration an ATP-sensitive K(+) channel (70-80 pS inward, 35-40 pS outward) was present in 35% of patches. Application of ATP to the intracellular side reduced unitary current with half-maximal inhibition in the range 10-100 microM. A block was obtained with 100 microM lidocaine and inhibition was obtained with 0.5 mM barium acetate. A Ca(2+)-activated K(+) channel (25-30 pS inward, 10-15 pS outward) was present in 57% of patches. Inhibition was produced by 10 mM TEA and 500 nM apamin and sensitivity to Ba(2+) was lower than for ATP-sensitive channels. No spontaneous channel activity was recorded in the cell-attached configuration under isotonic conditions. With hypotonic saline 68% of patches showed spontaneous single-channel activity, and, of 75 active patches, 66 cell-attached patches showed channel activity corresponding to Ca(2+)-activated K(+) channels.     

Herpesvirus, cytomegalovirus, human sperm and assisted fertilization

BACKGROUND: The effect of viral particles on the motility of human sperm and the relationship between sperm and virus are of importance particularly in assisted fertilization. METHODS: We incubated ejaculated sperm with or without seminal fluid with either herpes simplex virus type 2 (HSV2) or human cytomegalovirus (HCMV). For each experiment, 5 x 10(5) sperm were incubated with a viral load of between 10(4) and 10(6) plaque-forming units. RESULTS: We detected no apparent variations in the percentage of motile forms when sperm were incubated with either HSV2 or HCMV. Using a computer-aided semen analysis system, a slight difference was reported in the percentage of motile forms when seminal fluid-free sperm were incubated with HSV2 (57.18 versus 64.43 in the control). Although the mean amplitude of lateral head displacement and the curvilinear velocity were significantly higher in infected sperm, the difference in straight line velocity was not statistically significantly different. Few viral particles (HSV2 or HCMV) adhered to the sperm membrane in the presence of seminal fluid. However, more particles stuck when in the absence of seminal fluid, particularly with HSV2 (8% of sperm sections for HSV2; 4% for HCMV). CONCLUSIONS: The relationship between sperm and viruses depends on the type of virus present as well as the presence or absence of seminal fluid. Motility is not a good enough criterion on which to prove the presence of viral elements, either in the medium or on the sperm.     

Mouse Sperm Rosette: Assembling During Epididymal Transit,in vitro Disassemble,and Oligosaccharide Participation in the Linkage Material

In many mammals, sperm associations had been observed, but not in the mouse. In this work, mouse sperm rosettes are morphologically described inside the epididymis and during its dissolution in a culture medium. Also characterized are the saccharides present in the linking material. Sperm association and other epididymal actions are supported by sperm during epididymal transit and are verified at the caudal region, suggesting a relation between epididymal transit and sperm maturation. In drops of epididymal content obtained from distal (cauda), but not from proximal (caput and corpus) regions; dissolved in culture medium, rosettes appear to be 10 to 15 motile sperm joined by their heads. After 3 min, sperm progressively detach, disassembling the rosette. These structures are studied by several techniques, including optic, electronic (scanning electron microscopy and transmission electron microscopy), and video microscopy. At the ultrastructural level, a dense network of electron‐dense material was observed between sperm heads, joining them. Based on previous works in rat, several lectins were used to characterize the type of saccharides present in this linking material. To avoid the contact between sperm and epididymal fluid from distal region—that probably exerts an influence on sperm association—a ligature was placed between caput and corpus. This epididymal content isolated from caput did not display any rosettes after 28 days. Anat Rec, 2007. © 2007 Wiley‐Liss, Inc.     

Susceptibility of corneal allografts and xenografts to antibody-mediated rejection

The effects of passive transfer of antisera containing cytotoxic antibodies to allo- and xenoantigens on survival of corneal allografts and xenografts were evaluated in experimental models. Corneas from allogeneic B10 or xenogeneic rat Lewis donors were grafted orthotopically into BALB/c mice. Recipient mice were treated with donor-specific antisera administered at the period of grafting or at 2 weeks after transplantation. Rejection was determined by the severity of corneal opacity using a standard scoring system. Treatment of graft recipients with donor-specific antisera accelerated the onset of graft rejection and significantly shortened survival times of both corneal allografts and xenografts. Corneal xenografts, which had been accepted after treatment with anti-CD4 monoclonal antibody, were acutely rejected by the passive transfer of antiserum against xenoantigens. The results suggest that corneal grafts are vulnerable to antibody-dependent immunity and that cytotoxic antibodies against graft donor antigens can mediate rejection of both corneal allografts and xenografts.     

Decreased prefrontal 5-HT2A receptor binding in subjects at enhanced risk for schizophrenia

The brain serotonin-2A receptor (5-HT_2AR) has been implicated in both the pathology of schizophrenia and the therapeutic action of atypical antipsychotics. However, little is known about the 5-HT_2AR status before the onset of schizophrenia and before the exposure to antipsychotics. We used [¹⁸F] altanserin and positron emission tomography (PET) in a pilot study of 6 individuals suspected to be at elevated risk for schizophrenia and seven age-matched controls to test the hypothesis that regional 5-HT_2AR binding is altered in the prodromal stages of schizophrenia. Distribution volume ratios (DVRs) as a proxy for 5-HT_2AR availability were significantly reduced in prefrontal cortex regions of at-risk subjects, implicating early abnormalities of serotonergic neurotransmission that antecede the onset of schizophrenia.     

Functional aspects of serotonin transmission in the basal ganglia: a review and an in vivo approach using the push-pull cannula technique

Peripheral deafferentation of the rodent olfactory bulb results in loss of dopamine content, tyrosine hydroxylase activity and immunocytochemical staining for tyrosine hydroxylase in juxtaglomerular dopamine neurons. Reinnervation of the bulb by afferent neurons results in the return of all parameters to control levels suggesting that the dopamine neurons did not degenerate but that the expression of tyrosine hydroxylase enzyme was transneuronally regulated in a static population of juxtaglomerular cells. To evaluate this possibility, we determined the activity and immunocytochemical localization of the second enzyme in the dopamine biosynthetic pathway, DOPA decar?ylase. At a time when tyrosine hydroxylase activity was reduced to 25% of control values, DOPA decar?ylase activity in the lesioned bulb was maintained at about 65% of that in the unlesioned bulb. Immunocytochemical staining with antibodies to both enzymes, performed sequentially in the same sections, demonstrated that in the unlesioned bulb tyrosine hydroxylase and DOPA decar?ylase are co-localized in the same population of juxtaglomerular neurons. Similar results were obtained in adjacent sections each stained with one of the two antibodies. In contrast, in the deafferented bulb, about three times as many neurons were stained with DOPA decar?ylase as with tyrosine hydroxylase antibodies. The DOPA decar?ylase activity measurements and immunocytochemistry argue for the continued presence, in the lesioned olfactory bulb, of a population of tyrosine hydroxylase deficient dopamine neurons.The data suggest that olfactory receptor cell innervation transneuronally regulates the expression of tyrosine hydroxylase by mechanisms separate from those controlling the levels of DOPA decar?ylase.     

Disturbances of cell-mediated immunity in ornithosis

27 cases of ornithosis were observed during an epidemia in 1980 in Kielce and subsequently followed with respect to immunological characteristics of peripheral blood lymphocytes. Blastic transformation of these cells was tested after stimulation in vitro with three different mitogens. Identification of peripheral blood T and B lymphocytes was done using rosette tests (E,EA,EAC) and the occurrence of surface immunoglobulins was determined by the immunofluorescent method with polyvalent anti-immunoglobulin serum. The counts of T and B lymphocytes in the peripheral blood were normal throughout the whole period of the observation, but from the 3rd week on a significant impairment of 3H-thymidine incorporation into the cells stimulated with Con A was observed, and from the 10th week on, this impairment appeared also in cells stimulated with PHA and PWM. These observations revealed considerable disturbances in cell-mediated reactivity in patients with ornithosis and seem to be connected with chronic infection with Chlamydia psittaci.     

A morphometric comparison of the nasopharyngeal airway of laboratory animals and humans

Solid silicone rubber casts of the nasopharyngeal and laryngeal regions of a human cadaver (child, 3 years old) and a laboratory primate (baboon, 10 years old) were made, and cross-sectional areas were measured in detail. Cross-sectional areas of other species reported in the published literature were used for comparison. In the child's nose cast, the frontal nasal duct (frontonasal duct), which enters the anterior part of the middle meatus, and the sphenoidal recess were almost absent. The ethmoidal turbinates (superior and middle concha) and the maxillary turbinates (inferior concha) were present but were not fully developed. In the baboon nose, the different turbinates were well defined and smooth but of a less complex nature than the child's nose. Of the species compared, the baboon's upper airways had the greatest similarity to the human child's. The present study shows that for the species investigated and for those from the literature, the cross-sectional area increases from the external nares to the maxilloturbinate region (inferior concha). There is a relatively sudden drop in cross-sectional area about halfway through the nose. The present study suggests a functional relationship between nasal structure and cross-sectional area across species.