Failure of testicular development associated with a rearrangement of 9p24.1 proximal to the SNF2 gene

In 46,XY individuals, testes are determined by the activity of the SRY gene (sex-determining region Y), located on the short arm of the Ychromosome. The other genetic components of the cascade that leads to testis formation are unknown and may be located on the Xchromosome or on the autosomes. Evidence for the existence of several loci associated with failure of male sexual development is indicated by reports of 46,XY gonadal dysgenesis associated with structural abnormalities of the Xchromosome or of autosomes (chromosomes9, 10, 11 and 17). In this report, we describe the investigation of a child presenting with multiple congenital abnormalities, mental retardation and partial testicular failure. The patient had a homogeneous de novo 46,XY,inv dup(9)(pter→p24.1::p21.1 →p23.3::p24.1→qter) chromosome complement. No deletion was found by either cytogenetic or molecular analysis. The SRY gene and DSS region showed no abnormalities. Southern blotting dosage analysis with 9p probes and fluorescent in situ hybridisation data indicated that the distal breakpoint of the duplicated fragment was located at 9p24.1, proximal to the SNF2 gene. We therefore suggest that a gene involved in normal testicular development and/or maintenance is present at this position on chromosome 9. Received: 20 January 1997 / Accepted: 5 November 1997     

Structural Transitions and Energy Landscape for Cowpea Chlorotic Mottle Virus Capsid Mechanics from Nanomanipulation in?Vitro and in Silico

Physical properties of capsids of plant and animal viruses are important factors in capsid self-assembly, survival of viruses in the extracellular environment, and their cell infectivity. Combined AFM experiments and computational modeling on subsecond timescales of the indentation nanomechanics of Cowpea Chlorotic Mottle Virus capsid show that the capsid’s physical properties are dynamic and local characteristics of the structure, which change with the depth of indentation and depend on the magnitude and geometry of mechanical input. Under large deformations, the Cowpea Chlorotic Mottle Virus capsid transitions to the collapsed state without substantial local structural alterations. The enthalpy change in this deformation state ΔH_ind = 11.5–12.8 MJ/mol is mostly due to large-amplitude out-of-plane excitations, which contribute to the capsid bending; the entropy change TΔS_ind = 5.1–5.8 MJ/mol is due to coherent in-plane rearrangements of protein chains, which mediate the capsid stiffening. Direct coupling of these modes defines the extent of (ir)reversibility of capsid indentation dynamics correlated with its (in)elastic mechanical response to the compressive force. This emerging picture illuminates how unique physico-chemical properties of protein nanoshells help define their structure and morphology, and determine their viruses’ biological function.     

The Ciliopathy Protein CC2D2A Associates with NINL and Functions in RAB8-MICAL3-Regulated Vesicle Trafficking

Ciliopathies are a group of human disorders caused by dysfunction of primary cilia, ubiquitous microtubule-based organelles involved in transduction of extra-cellular signals to the cell. This function requires the concentration of receptors and channels in the ciliary membrane, which is achieved by complex trafficking mechanisms, in part controlled by the small GTPase RAB8, and by sorting at the transition zone located at the entrance of the ciliary compartment. Mutations in the transition zone gene CC2D2A cause the related Joubert and Meckel syndromes, two typical ciliopathies characterized by central nervous system malformations, and result in loss of ciliary localization of multiple proteins in various models. The precise mechanisms by which CC2D2A and other transition zone proteins control protein entrance into the cilium and how they are linked to vesicular trafficking of incoming cargo remain largely unknown. In this work, we identify the centrosomal protein NINL as a physical interaction partner of CC2D2A. NINL partially co-localizes with CC2D2A at the base of cilia and ninl knockdown in zebrafish leads to photoreceptor outer segment loss, mislocalization of opsins and vesicle accumulation, similar to cc2d2a-/- phenotypes. Moreover, partial ninl knockdown in cc2d2a-/- embryos enhances the retinal phenotype of the mutants, indicating a genetic interaction in vivo, for which an illustration is found in patients from a Joubert Syndrome cohort. Similar to zebrafish cc2d2a mutants, ninl morphants display altered Rab8a localization. Further exploration of the NINL-associated interactome identifies MICAL3, a protein known to interact with Rab8 and to play an important role in vesicle docking and fusion. Together, these data support a model where CC2D2A associates with NINL to provide a docking point for cilia-directed cargo vesicles, suggesting a mechanism by which transition zone proteins can control the protein content of the ciliary compartment.     

Mutations in CSPP1 Cause Primary Cilia Abnormalities and Joubert Syndrome with or without Jeune Asphyxiating Thoracic Dystrophy

Joubert syndrome (JBTS) is a recessive ciliopathy in which a subset of affected individuals also have the skeletal dysplasia Jeune asphyxiating thoracic dystrophy (JATD). Here, we have identified biallelic truncating CSPP1 (centrosome and spindle pole associated protein 1) mutations in 19 JBTS-affected individuals, four of whom also have features of JATD. CSPP1 mutations explain ∼5% of JBTS in our cohort, and despite truncating mutations in all affected individuals, the range of phenotypic severity is broad. Morpholino knockdown of cspp1 in zebrafish caused phenotypes reported in other zebrafish models of JBTS (curved body shape, pronephric cysts, and cerebellar abnormalities) and reduced ciliary localization of Arl13b, further supporting loss of CSPP1 function as a cause of JBTS. Fibroblasts from affected individuals with CSPP1 mutations showed reduced numbers of primary cilia and/or short primary cilia, as well as reduced axonemal localization of ciliary proteins ARL13B and adenylyl cyclase III. In summary, CSPP1 mutations are a major cause of the Joubert-Jeune phenotype in humans; however, the mechanism by which these mutations lead to both JBTS and JATD remains unknown.     

Allosteric communication in dihydrofolate reductase: signaling network and pathways for closed to occluded transition and back

Escherichia coli dihydrofolate reductase (DHFR) catalyzes the reduction of dihydrofolate to tetrahydrofolate. During the catalytic cycle, DHFR undergoes conformational transitions between the closed (CS) and occluded (OS) states that, respectively, describe whether the active site is closed or occluded by the Met20 loop. The CS→OS and the reverse transition may be viewed as allosteric transitions. Using a sequence-based approach, we identify a network of residues that represents the allostery wiring diagram. Many of the residues in the allostery wiring diagram, which are dispersed throughout the adenosine-binding domain as well as the loop domain, are not conserved. Several of the residues in the network have been previously shown by NMR experiments, mutational studies, and molecular dynamics simulations to be linked to equilibration conformational fluctuations of DHFR. To further probe the nature of events that occur during conformational fluctuations, we use a self-organized polymer model to monitor the kinetics of the CS→OS and the reverse transitions. During the CS→OS transition, coordinated changes in a number of residues in the loop domain enable the Met20 loop to slide along the α-helix in the adenosine-binding domain. Sliding is triggered by pulling of the Met20 loop by the βG-βH loop and the pushing action of the βG-βH loop. The residues that facilitate the Met20 loop motion are part of the network of residues that transmit allosteric signals during the CS→OS transition. Replacement of M16 and G121, whose C^α atoms are about 4.3 Å in the CS, by a disulfide cross-link impedes that CS→OS transition. The order of events in the OS→CS transition is not the reverse of the forward transition. The contact Glu18-Ser49 in the OS persists until the sliding of the Met20 loop is nearly complete. The ensemble of structures in the transition state in both the allosteric transitions is heterogeneous. The most probable transition-state structure resembles the OS (CS) in the CS→OS (OS→CS) transition, which is in accord with the Hammond postulate. Structures resembling the OS (CS) are present as minor (∼ 1-3%) components in equilibrated CS (OS) structures.     

Systemic inflammatory markers in patients with aortic sclerosis

The aim of the study was to evaluate several mediators of inflammation in patients with aortic sclerosis in relation to severity of cardiovascular disease. Serum level of cytokines, soluble intracellular adhesion molecule 1, matrix metalloproteinase (MMP) 2 and 9 and their tissue inhibitor TIMP-1, were measured by ELISA and MMPs activity by zymography in 51 aortic sclerosis patients. The increase in MMPs expression positively correlated with their gelatinase activity; also there was a positive correlation between MMP-9 and TIMP-1 serum levels. Moreover, IL-6 concentration positively correlated with both serum level and activity of MMP-9. The level of IL-6 and IL-1Ra were higher in patients with a great burden of atherosclerosis. Noteworthy, statistically significant higher levels of IL-6 were noticed for patients with coronary artery disease. There was a significant increase in IL-6 serum level as well as a significant decrease in IL-1Ra for patients with a history of myocardial infarction. A trend toward higher concentration of inflammatory mediators was noticed in relation to the increase in severity of the aortic valve disease. Our results support the hypothesis of an "inflammatory pattern" associated with AS pathology and suggest the persistence of a chronic inflammation in patients who experienced acute coronary events.     

Highly efficient constant-current electroporation increases in vivo plasmid expression

Electroporation has been demonstrated as an effective technique for enhancing the delivery of plasmids coding for DNA vaccines and therapeutic proteins into skeletal muscle. Nevertheless, constant-voltage techniques do not take into account the resistance of the tissue and result in tissue damage, inflammation, and loss of plasmid expression. In the present study, we have used a software-driven constant-current electroporator to deliver plasmids to mice and small and large pigs. The voltage, amperage, and resistance of the tissue during pulses were recorded and analyzed. Optimal conditions of electroporation were identified in both species, and found to be highly dependent on the individual tissue resistance. Six- to 10-week-old pigs had higher muscle resistance compared to 1- to 2-year-old pigs, but both values were four to five times lower than the resistance of the mouse muscle. In mice, optimum amperage, pulse length, and lag time between plasmid injection and electroporation were identified to be 0.1 Amps, 20 msec and 0 sec. The electroporation pulse pattern among the electrodes also affected plasmid expression. These results indicate that age- and tissue-specific resistance, pulse pattern, and other variables associated with the electroporation need to be optimized for each separate species to achieve maximum plasmid expression.     

C3a and C3b activation products of the third component of complement (C3) are critical for normal liver recovery after toxic injury

Although the complement system has been implicated in liver regeneration after toxic injury and partial hepatectomy, the mechanism or mechanisms through which it participates in these processes remains ill-defined. In this study, we demonstrate that complement activation products (C3a, C3b/iC3b) are generated in the serum of experimental mice after CCl(4) injection and that complement activation is required for normal liver regeneration. Decomplementation by cobra venom factor resulted in impaired entry of hepatocytes into S phase of the cell cycle. In addition, livers from C3-deficient (C3(-/-)) mice showed similarly impaired proliferation of hepatocytes, along with delayed kinetics of both hepatocyte hyperplasia and removal of injured liver parenchyma. Restoration of hepatocyte proliferative capabilities of C3(-/-) mice through C3a reconstitution, as well as the impaired regeneration of C3a receptor-deficient mice, demonstrated that C3a promotes liver cell proliferation via the C3a receptor. These findings, together with data showing two waves of complement activation, indicate that C3 activation is a pivotal mechanism for liver regeneration after CCl(4) injury, which fulfills multiple roles; C3a generated early after toxin injection is relevant during the priming of hepatocytes, whereas C3 activation at later times after CCl(4) treatment contributes to the clearance of injured tissue.     

Course of pregnancies in women with Cushing's disease treated by gamma-knife

Data concerning pregnancy in women with Cushing's disease treated by gamma-knife (GK) are scanty. We present and discuss the course and outcome of five pregnancies in two women with Cushing's disease (CD), the first of whom was treated only by GK, and the second one treated by surgery, GK and ketoconazole. In the first patient, pregnancy was uneventful and full-term. During gestation, plasma ACTH, serum cortisol and 24-h urinary free cortisol (UFC) levels were steady, and always in the normal range for healthy non-pregnant individuals. The newborn was healthy and normal-weight. In the second woman, two pregnancies, occurring 3 years after GK and few months after ketoconazole withdrawal, were interrupted by spontaneous abortion or placental disruption despite normal cortisol levels. This patient became again pregnant 3 years later and delivered vaginally a healthy full-term infant. Seven months after the delivery, the patient became pregnant again and at the 39th week of gestation delivered vaginally a healthy male. Hypoprolactinemia and/or central hypothyroidism occurred in both cases. In women with CD treated by GK, pregnancy can occur. However, pregnancy is at risk even when ACTH and cortisol levels are normalized by treatment. After GK, evaluation of pituitary function is mandatory due to the risk of hypopituitarism.