Functional consequences of ROMK mutants linked to antenatal Bartter's syndrome and implications for treatment

The antenatal variant of Bartter's syndrome is an autosomal recessive kidney disease characterized by polyhydramnios, premature delivery, hypokalemic alkalosis and hypercalciuria. It is genetically heterogeneous, having been linked recently to mutations in an ATP- sensitive, renal outer medullary K+channel, ROMK, and earlier to mutations in the Na-K-2Cl co-transporter, NKCC2. We characterized four of the mutations reported in three heterozygous ROMK variants of antenatal Bartter's and found that each expressed a distinct phenotype in Sf9 cells. One mutation expressed normal function and appears to be an allelic polymorphism. The other three mutations produced channels with significantly reduced K+fluxes. However, the mechanisms in each case were different and reflected abnormalities in phosphorylation, proteolytic processing or protein trafficking. The different mechanisms may be important in the design of appropriate therapy for patients with this disease.      

A model of corrective gene transfer in X-linked ichthyosis

Single gene recessive genetic skin disorders offer attractive prototypes for the development of therapeutic cutaneous gene delivery. We have utilized X-linked ichthyosis (XLI), characterized by loss of function of the steroid sulfatase arylsulfatase C (STS), to develop a model of corrective gene delivery to human skin in vivo. A new retroviral expression vector was produced and utilized to effect STS gene transfer to primary keratinocytes from XLI patients. Transduction was associated with restoration of full-length STS protein expression as well as steroid sulfatase enzymatic activity in proportion to the number of proviral integrations in XLI cells. Transduced and uncorrected XLI keratinocytes, along with normal controls, were then grafted onto immunodeficient mice to regenerate full thickness human epidermis. Unmodified XLI keratinocytes regenerated a hyperkeratotic epidermis lacking STS expression with defective skin barrier function, effectively recapitulating the human disease in vivo. Transduced XLI keratinocytes from the same patients, however, regenerated epidermis histologically indistinguishable from that formed by keratinocytes from patients with normal skin. Transduced XLI epidermis demonstrated STS expression in vivo by immunostaining as well as a normalization of histologic appearance at 5 weeks post-grafting. In addition, transduced XLI epidermis demonstrated a return of barrier function parameters to normal. These findings demonstrate corrective gene delivery in human XLI patient skin tissue at both molecular and functional levels and provide a model of human cutaneous gene therapy.      

Mutational analysis of the SOX9 gene in campomelic dysplasia and autosomal sex reversal: lack of genotype/phenotype correlations

It has previously been shown that, in the heterozygous state, mutations in the SOX9 gene cause campomelic dysplasia (CD) and the often associated autosomal XY sex reversal. In 12 CD patients, 10 novel mutations and one recurrent mutation were characterized in one SOX9 allele each, and in one case, no mutation was found. Four missense mutations are all located within the high mobility group (HMG) domain. They either reduce or abolish the DNA-binding ability of the mutant SOX9 proteins. Among the five nonsense and three frameshift mutations identified, two leave the C-terminal transactivation (TA) domain encompassing residues 402-509 of SOX9 partly or almost completely intact. When tested in cell transfection experiments, the recurrent nonsense mutation Y440X, found in two patients who survived for four and more than 9 years, respectively, exhibits some residual transactivation ability. In contrast, a frameshift mutation extending the protein by 70 residues at codon 507, found in a patient who died shortly after birth, showed no transactivation. This is apparently due to instability of the mutant SOX9 protein as demonstrated by Western blotting. Amino acid substitutions and nonsense mutations are found in patients with and without XY sex reversal, indicating that sex reversal in CD is subject to variable penetrance. Finally, none of 18 female patients with XY gonadal dysgenesis (Swyer syndrome) showed an altered SOX9 banding pattern in SSCP assays, providing evidence that SOX9 mutations do not usually result in XY sex reversal without skeletal malformations.      

Polyphenols and Human Health: The Role of Bioavailability

Polyphenols are a group of phytochemicals with potential health-promoting effects. They are classified as flavonoid (flavonols, flavanols, flavones, flavanones, isoflavones, and anthocyanins) and non-flavonoid molecules (phenolic acids, hydroxycinnamic acids, lignans, stilbenes, and tannins). Although an increasing number of trials have shown a correlation among polyphenol consumption and a reduction in risk factors for chronic diseases, discrepancies in explaining their positive effects have been found in terms of the bioavailability. In fact, polyphenols show a low bioavailability due to several factors: interaction with the food matrix, the metabolic processes mediated by the liver (phase I and II metabolism), intestine and microbiota. On the other hand, the biological activities of phenol compounds may be mediated by their metabolites, which are produced in vivo, and recent studies have confirmed that these molecules may have antioxidant and anti-phlogistic properties. This review discusses the studies performed in vivo, which consider the polyphenol bioavailability and their different food sources. Factors influencing the biological effects of the main classes of polyphenols are also considered.     

Changes in allograft bone irradiated at different temperatures

Secondary sterilisation of allograft bone by gamma irradiation is common, but the conditions under which it is performed vary between tissue banks. Some do so at room temperature, others while the bone is frozen. Bone is made brittle by irradiation because of the destruction of collagen alpha chains, probably mediated by free radicals generated from water molecules. Freezing reduces the mobility of water molecules and may therefore decrease the production of free radicals. We found that bone irradiated at -78 degrees C was less brittle and had less collagen damage than when irradiated at room temperature. These findings may have implications for bone-banking.     

Expression and immunogenicity of a liver stage malaria epitope presented as a foreign peptide on the surface of RNA-free MS2 bacteriophage capsids.

We have designed a novel vaccine strategy which enables display of short peptides expressed from chimeras of the gene encoding the coat protein of the RNA bacteriophage MS2 and inserted foreign DNA. MS2 coat protein has a beta-hairpin loop at the N-terminus which forms the most radially distinct feature of the mature capsid. The coat protein gene was modified to enable insertion of DNA at the central part of the beta-hairpin loop. Upon expression of the recombinant gene in E. coli, the MS2 coat protein subunits self-assemble into capsids, each comprising 180 copies of the monomer. This system was used to produce chimeras containing a putatively protective epitope, T1, from the immunodominant liver stage antigen-1 (LSA-1) of the malaria parasite Plasmodium falciparum. The immunogenicity of the native MS2 capsid and the recombinant construct was investigated in BALB/c (H-2(d)) mice. The native protein appeared to elicit both humoral and cellular immune responses, observed as a predominance of type 2 cytokines but with a mixed profile of immunoglobulin isotypes. In contrast, the LSA-1 chimera stimulated a type 1-polarised response, with significant upregulation of interferon-gamma, a finding which corroborates naturally acquired resistance to liver stage malaria. These results validate RNA phage capsid display of immunogenic determinants as a basis for the development of novel peptide vaccines and indicate that further evaluation of MS2 coat protein as a vector for malaria epitopes is merited.     

Serum and sputum alpha 2 macroglobulin in patients with chronic obstructive airways disease.

Serum alpha 2 macroglobulin concentrations were measured in patients with chronic obstructive airways disease and an age-matched group of control subjects. The mean serum level of alpha 2 macroglobulin was significantly lower in bronchitic subjects with acute chest infections than the mean value of the controls. No significant differences were observed between serum alpha 2 macroglobulin values in controls, subjects with "emphysema", and bronchitic patients who did not have chest infections. Sputum alpha 2 macroglobulin concentrations were compared in sputum samples from bronchitic patients with and without acute chest infections. The protein was detected (greater than 0.2 mg/l) in 94% of sputum samples from infected subjects but only 60% of non-infected sputum samples. Concentrations of alpha 2 macroglobulin in infected samples were significantly higher than the non-infected samples. Sputum/serum concentration ratios of alpha 2 macroglobulin were also significantly higher in infected samples but this difference was eliminated by "correcting" the values with the albumin sputum/serum ratios of the same samples. The results suggest that alpha 2 macroglobulin concentrations are higher in bronchial secretions during chest infection because of increased transudation from the blood rather than the presence of significant local secretion.     

The influence of reported paternal attitudes on the decision to breast-feed

Objective: To identify factors that influence a woman's decision to breast-feed.
Methodology: Five hundred and fifty-six women were recruited from the maternity wards of two Perth hospitals. Data were collected from a self-administered questionnaire completed by participants prior to discharge. Logistic regression analysis was used to determine factors influencing the initiation of breast-feeding.
Results: At discharge from hospital 83.8% of women were breast-feeding, including 6% who were giving complementary formula feeds. After controlling for potentially confounding demographic and biomedical factors, the father's reported preference for breast-feeding was found to be the most important factor influencing a woman's decision to breast-feed (OR 10.18).
Conclusion: Fathers participate in and influence the choice of infant feeding method and should be included in breast-feeding discussions.