Gas phase characterization of the noncovalent quaternary structure of Cholera toxin and the Cholera toxin B subunit pentamer

Cholera toxin (CTx) is an AB5 cytotonic protein that has medical relevance in cholera and as a novel mucosal adjuvant. Here, we report an analysis of the noncovalent homopentameric complex of CTx B chain (CTx B5) using electrospray ionization triple quadrupole mass spectrometry and tandem mass spectrometry and the analysis of the noncovalent hexameric holotoxin usingelectrospray ionization time-of-flight mass spectrometry over a range of pH values that correlate with those encountered by this toxin after cellular uptake. We show that noncovalent interactions within the toxin assemblies were maintained under both acidic and neutral conditions in the gas phase. However, unlike the related Escherichia coli Shiga-like toxin B5 pentamer (SLTx B), the CTx B5 pentamer was stable at low pH, indicating that additional interactions must be present within the latter. Structural comparison of the CTx B monomer interface reveals an additional α-helix that is absent in the SLTx B monomer. In silico energy calculations support interactions between this helix and the adjacent monomer. These data provide insight into the apparent stabilization of CTx B relative to SLTx B

The spectral transmission of ocular media suggests ultraviolet sensitivity is widespread among mammals

Although ultraviolet (UV) sensitivity is widespread among animals it is considered rare in mammals, being restricted to the few species that have a visual pigment maximally sensitive (λmax) below 400 nm. However, even animals without such a pigment will be UV-sensitive if they have ocular media that transmit these wavelengths, as all visual pigments absorb significant amounts of UV if the energy level is sufficient. Although it is known that lenses of diurnal sciurid rodents, tree shrews and primates prevent UV from reaching the retina, the degree of UV transmission by ocular media of most other mammals without a visual pigment with λmax in the UV is unknown. We examined lenses of 38 mammalian species from 25 families in nine orders and observed large diversity in the degree of short-wavelength transmission. All species whose lenses removed short wavelengths had retinae specialized for high spatial resolution and relatively high cone numbers, suggesting that UV removal is primarily linked to increased acuity. Other mammals, however, such as hedgehogs, dogs, cats, ferrets and okapis had lenses transmitting significant amounts of UVA (315–400 nm), suggesting that they will be UV-sensitive even without a specific UV visual pigment

A rare de novo nonsense mutation in OTX2 causes early onset retinal dystrophy and pituitary dysfunction

PurposeTo describe the clinical findings of a patient with an early onset retinal dystrophy and a novel mutation in OTX2, and to compare these findings with previously reported cases.MethodsUsing direct sequencing, we screened 142 patients, who had either Leber congenital amaurosis (LCA) or early onset retinal dystrophy (EORD), for mutations in OTX2. All patients received a detailed ophthalmic examination including electroretinography and retinal imaging.ResultsOnly one mutation in OTX2 was identified. A novel heterozygous p.S138X stop mutation was identified in a seven-year-old male who had an infantile onset retinal dystrophy. The mutation was not present in either parent or in 181 blood donor samples. There was a history of failure to thrive in infancy, poor feeding, and growth hormone deficiency. Poor vision and nyctalopia was present from the first year. Funduscopy revealed a hyperpigmented peripapillary ring with a fine granular pigmentation of the RPE throughout the fundus. The scotopic bright flash ERG a-wave was subnormal and the waveform electronegative, in keeping with dysfunction both at the level of the photoreceptor and post-phototransduction. Visual function has been stable to date.ConclusionsMutations in OTX2 have been reported in association with major developmental malformations of the eye, with retinal dystrophies such as LCA, and with pituitary dysfunction and seizure activity in some cases. This case adds further support for a role of OTX2 both in retinal development and pituitary function, and highlights a novel retinal dystrophy phenotype seen in association with mutations in OTX2

Medical Sequencing of Candidate Genes for Nonsyndromic Cleft Lip and Palate

Nonsyndromic or isolated cleft lip with or without cleft palate (CL/P) occurs in wide geographic distribution with an average birth prevalence of 1/700. We used direct sequencing as an approach to study candidate genes for CL/P. We report here the results of sequencing on 20 candidate genes for clefts in 184 cases with CL/P selected with an emphasis on severity and positive family history. Genes were selected based on expression patterns, animal models, and/or role in known human clefting syndromes. For seven genes with identified coding mutations that are potentially etiologic, we performed linkage disequilibrium studies as well in 501 family triads (affected child/mother/father). The recently reported MSX1 P147Q mutation was also studied in an additional 1,098 cleft cases. Selected missense mutations were screened in 1,064 controls from unrelated individuals on the Centre d'Étude du Polymorphisme Humain (CEPH) diversity cell line panel. Our aggregate data suggest that point mutations in these candidate genes are likely to contribute to 6% of isolated clefts, particularly those with more severe phenotypes (bilateral cleft of the lip with cleft palate). Additional cases, possibly due to microdeletions or isodisomy, were also detected and may contribute to clefts as well. Sequence analysis alone suggests that point mutations in FOXE1, GLI2, JAG2, LHX8, MSX1, MSX2, SATB2, SKI, SPRY2, and TBX10 may be rare causes of isolated cleft lip with or without cleft palate, and the linkage disequilibrium data support a larger, as yet unspecified, role for variants in or near MSX2, JAG2, and SKI. This study also illustrates the need to test large numbers of controls to distinguish rare polymorphic variants and prioritize functional studies for rare point mutations