Immuno-electron microscopic evidence for two different types of partial somatic repair of the mutant Brattleboro vasopressin gene.

In homozygous Brattleboro rats a frame-shift mutation in the vasopressin gene prevents secretion of vasopressin by magnocellular neurosecretory neurons and thus causes diabetes insipidus. Whereas most "vasopressin" neurons in Brattleboro homozygotes apparently lack vasopressin and its associated neurophysin and glycopeptide, some isolated cells overcome the mutation and "revert" to producing readily detectable amounts of vasopressin. We describe here two morphologically and immunocytochemically distinct subsets of such "revertant" cells. One subset contain, in their rough endoplasmic reticulum cisterns, electron-dense aggregates immunoreactive for vasopressin, for parts of oxytocin-neurophysin, and for CP14 (a peptide with a sequence deduced from the mutated precursor), but not for vasopressin-associated glycopeptide ("glycopeptide") or vasopressin-neurophysin. In Brattleboro heterozygotes, which have one mutant and one normal copy of the vasopressin gene, morphologically similar revertant cells exist; the aggregates in the rough endoplasmic reticulum of these cells do not immuno-label for CP14, but the cells do produce 160-nm neurosecretory granules immunoreactive for vasopressin, vasopressin-neurophysin and glycopeptide. In Brattleboro homozygotes, the second, more abundant subset of neurons which recover vasopressin immunoreactivity also express vasopressin-associated glycopeptide and CP14 but not oxytocin-neurophysin; both glycopeptide and CP14 are restricted to the rough endoplasmic reticulum but do not form aggregates. We conclude that two different somatic repairs of the Brattleboro mutation can occur. We propose that, in aggregate-containing neurons, exons B and C have been exchanged between the vasopressin and oxytocin genes; glycopeptide-immunoreactive neurons have either undergone mismatch repair or exchanged exon B.     

Vesicular targeting and the control of ion secretion in epithelial cells: implications for cystic fibrosis.

Non-polarized HT-29 colonic epithelial cells fail to respond to cyclic AMP-generating agonists with increases in plasma membrane anion conduction. Radio-isotopic efflux and patch-clamp experiments revealed that both undifferentiated and differentiated HT-29 colonocytes possess volume- and Ca(2+)-activated Cl- channels. However, only within the apical plasma membranes of the latter were cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channels found. CFTR was expressed equally well in both non-polarized and polarized colonocytes. Lack of CFTR-dependent anion conduction was shown to be the result of CFTR retention within a peripheral intracellular compartment. We demonstrate that upon polarization, CFTR moves to the apical plasma membrane via a Brefeldin A (BFA)-sensitive intracellular trafficking pathway.     

The potentiating effect of rheumatoid arthritis serum in the immediate phase of nephrotoxic nephritis

Nephrotoxic nephritis induced in rats was employed as an experimental model to investigate the possible effects of rheumatoid factor on in vivo antigen–antibody reactions. Rats injected simultaneously with rheumatoid arthritis serum and rabbit nephrotoxic globulin showed a three-fold increase in immediate proteinuria compared with rats injected with nephrotoxic globulin alone. This potentiating effect of rheumatoid arthritis serum was evident even when the serum was injected 48 hr after the nephrotoxic globulin and was also apparent to a lesser extent in rats decomplemented by a prior injection of aggregated human IgG. Normal human serum had no effect on the proteinuria produced by a standard dose of nephrotoxic globulin while rheumatoid arthritis serum injected with normal rabbit globulin did not increase urinary protein excretion above baseline levels. In rats injected with rheumatoid arthritis serum and nephrotoxic globulin, human IgM (presumably rheumatoid factor) was detected by immunofluorescence on the glomerular basement membrane along with the nephrotoxic globulin and rat complement and persisted at this site for as long as 42 days after the initial injections. Rheumatoid factor activity was also recovered by elution from glomeruli isolated from rat kidneys 24 hr after the injection of rheumatoid arthritis serum and nephrotoxic globulin.     

Attentional characteristics of infants and toddlers with Williams syndrome during triadic interactions

Two studies were conducted to consider the looking behavior of infants and toddlers with Williams syndrome (WS).In Study 1,the looking behavior of a 10-month-old girl with WS during play sessions with her mother and with a stranger was compared to that of 2 groups of infants who were developing normally (ND),1 matched for chronological age and the other for developmental age. The infant with WS spent more than twice as much time looking at her mother as the infants in either contrast group did. She also spent twice as much time looking at the stranger.In addition, during 78%of this time, her gaze at the stranger was coded as extremely intense. Looks of this intensity were virtually never made by the ND infants. In Study 2,the looking behavior of 31 individuals with WS ages 8 to 43 months during a genetics evaluation was compared to that of 319 control children in the same age range (242 with developmental delay due to causes other than WS).Twenty-three of the 25 participants with WS aged 33 months or younger demonstrated extended and intense looking at the geneticist. In contrast, none of the control participants looked extensively or intently at the geneticist. Findings are discussed in the context of previous research on arousal and focused attention during normal development and on temperament and personality of older children and adults with WS. It is argued that the unusual looking patterns evidenced by infants and toddlers with WS presage the unusual temperament and personality of older individuals with WS, and the possibility of a genetic basis for these behaviors is addressed.     

Delineation of the male phenotype in carniofrontonasal syndrome

We are reporting on a 4-generation family in which 6 individuals had frontonasal dysplasia with variable extracranial abnormalities. All affected persons had hypertelorism, bifid or broad nose, and highly arched palate. Associated abnormalities included cleft lip and palate (1/6), webbed neck (2/6), Sprengel anomaly (2/6), pseudoarthrosis of the clavicle (2/6), pectus excavatum (3/6), narrow, sloping shoulders (3/6), diaphragmatic hernia (2/6), broad first toe (4/6), brachydactyly (3/6), fifth finger clinodactyly (5/6), longitudinal grooves of nails (5/6), shawl scrotum (2/3 males), first degree hypospadias (1/3), and mild mental retardation (1/6). Only one affected female had brachycephaly and right coronal synostosis. Four other affected relatives had varying degrees of facial asymmetry, but normal skull contour. No male to male transmission is observed, and both daughters of an affected male were affected. Based on the phenotype of the 3 affected females, craniofrontonasal syndrome (CFNS) is the likely diagnosis. However, there are 3 affected males in this kindred, and 2 of the 3 had significant anomalies. Affected males also had genital abnormalities and pectus deformity of the chest, not previously reported in this condition. Two of the 3 males have posterolateral diaphragmatic hernia. This family expands the phenotype of affected males.     

Differential effects of interleukin-4 in peptide induced autoimmunity

BALB/c mice immunized with multimeric DWEYSVWLSN develop IgG1 anti-DNA antibodies and glomerular immunoglobulin deposits, leading us to investigate the role of IL-4 in this model of antigen induced lupus. Splenocytes from DWEYSVWLSN immunized mice secreted IL-4 but not gamma-interferon. Following peptide immunization, IgG1 anti-peptide and anti-DNA antibodies were significantly higher in IL-4 wild type mice, while IgM and IgG3 anti-DNA levels were significantly higher in IL-4 knockout mice. Titers of IgG anti-laminin and anti-histone, but not anti-Sm/RNP and anti-cardiolipin antibodies, were significantly higher in the IL-4 wild type group. Glomerular immunoglobulin deposition was substantially decreased in IL-4 knockout mice. We conclude that while IL-4 does not materially affect the generation of some autoantibody responses associated with peptide induced autoimmunity, IL-4 deficiency inhibits kidney immunoglobulin deposition. The effect of IL-4 on humoral autoimmunity in lupus is complex, and is dependent on genetic background, the antigenic trigger and stage of disease.     

Immunoradiometric assay for cytomegalovirus-specific IgG antibodies: assay development and evaluation in blood transfusion practice

An immunoradiometric assay (radio-immunosorbent test; RIST) for the detection of IgG antibodies to human herpesvirus 4 [human cytomegalovirus (CMV)] has been developed. The technique utilizes CMV antigen passively adsorbed to a polyvinyl microtitration plate and a radiolabelled murine monoclonal anti-human IgG antibody to detect binding of human antibody to the 'solid phase' reagent. The assay was optimized, and its specificity confirmed by testing paired acute and convalescent sera from patients with acute CMV or other human herpesvirus infections. To determine the assay's sensitivity 1433 blood donor sera were examined. The RIST was more sensitive than a standard complement fixation (CFT), in that 53% of these sera were positive by RIST and 48% positive by CFT. There were 1303 concordant results, 88 sera positive only by RIST and 19 sera were only positive by CFT. These discrepant results remained after an attempt to exclude false positive reactivity; their significance is discussed. Use of a monoclonal anti-human IgG antibody in the RIST reduced non-specific binding to the control uninfected cell antigen such that blood donor sera could be tested in the assay using only a CMV antigen without generating an unacceptable false positive rate.     

Bacterial superantigen-treated intestinal epithelial cells upregulate heat shock proteins 25 and 72 and are resistant to oxidant cytotoxicity

While the pathological events evoked by infection are commonly described, effective host responses to bacteria and their products should primarily be protective. Heat shock protein (Hsp) expression is upregulated by many stimuli and serves to maintain intracellular protein integrity. The ability of the prototypic superantigen, Staphylococcus aureus enterotoxin B (SEB) to induce Hsps was investigated with BALB/c mice and by in vitro addition to the murine small intestinal epithelial cell line MSIE. SEB-treated (5 or 100 microg intraperitoneally) mice revealed increased Hsp25 and Hsp72, but not Hsc73, in jejunal lymphocytes and epithelial cells. A similar Hsp response to SEB occurred in MSIE cells and was preceded by activation of the ERK1/2 and p38 mitogen-activated protein kinases but not the SAPK/JNK pathway; pharmacological inhibition of ERK1/2, but not p38, significantly reduced SEB-induced Hsps. Moreover, SEB-treated MSIE cells were protected against oxidant-induced cytotoxicity (measured by 51Cr release) and F-actin depolymerization. Thus, SEB exposure results in a rapid induction of the Hsp25 and Hsp72 in intestinal epithelial cells, both directly and through lymphocyte activation, and we suggest that this event is important in protecting the gut from damage by Staphylococcus infection or in the reparatory process and may be a generalized response to lumen-derived bacterial toxins.     

A novel dystrophin isoform is required for normal retinal electrophysiology

Dystrophin is present in the outer plexiform layer of the retinaand is required for normal retinal function as measured by electroretinography.We describe the identification of a novel isoform of dystrophln(Dp260) present in the mouse retina. The unIque 5' terminusof the mRNA originates from a newly identified exon and is splicedin frame to exon 30 of the Duchenne muscular dystrophy (DMD)gene. The retinal isoform of dystrophln has 13 novel amino acidsas its N-terminus followed by most of the dystrophin rod domainand the cysteine-rich C-terminal domains. Analysis of mousetissues indicated this isoform of dystrophin Is expressed inretina, brain and cardiac tissue. Comparison of retinal electrophysiologyin mdx and mdx^cv3 mouse suggests that Dp260 is required fornormal retinal function.