Molecular interaction of connexin 30.3 and connexin 31 suggests a dominant-negative mechanism associated with erythrokeratodermia variabilis

Connexins are homologous four-transmembrane-domain proteins and major components of gap junctions. We recently identified mutations in either GJB3 or GJB4 genes, encoding respectively connexin 31 (Cx31) or 30.3 (Cx30.3), as causally involved in erythrokeratodermia variabilis (EKV), a mostly autosomal dominant disorder of keratinization. Despite slight differences, phenotypes of EKV Mendes Da Costa (Cx31) and EKV Cram-Mevorah (Cx30.3) show major clinical overlap and both Cx30.3 and Cx31 are expressed in the upper epidermal layers. These similarities suggested to us that Cx30.3 and Cx31 may interact at a molecular level. Indeed, expression of wild-type Cx30.3 in HeLa cell resulted only in minor amounts of protein addressed to the plasma membrane. Mutant Cx30.3 was hardly detectable and disturbed intercellular coupling. In sharp contrast, co-expression of both wild-type proteins led to a gigantic increase of stabilized heteromeric gap junctions. Furthermore, co-expressed wild-type Cx30.3 and Cx31 coprecipitate, which demonstrates a physical interaction. Inhibitor experiments revealed that this interaction begins in the endoplasmic reticulum. These results not only provide new insights into epidermal connexin synthesis and polymerization, but also allow a novel molecular explanation for the similarity of EKV phenotypes.     

Reporting of conflicts of interest in guidelines of preventive and therapeutic interventions

Background

Guidelines published in major medical journals are very influential in determining clinical practice. It would be essential to evaluate whether conflicts of interests are disclosed in these publications. We evaluated the reporting of conflicts of interest and the factors that may affect such disclosure in a sample of 191 guidelines on therapeutic and/or preventive measures published in 6 major clinical journals (Annals of Internal Medicine, BMJ, JAMA, Lancet, New England Journal of Medicine, Pediatrics) in 1979, 1984, 1989, 1994 and 1999.

Results

Only 7 guidelines (3.7%) mentioned conflicts of interest and all were published in 1999 (17.5% (7/40) of guidelines published in 1999 alone). Reporting of conflicts of interest differed significantly by journal (p=0.026), availability of disclosure policy by the journal (p=0.043), source of funding (p < 0.001) and number of authors (p=0.004). In the entire database of 191 guidelines, a mere 18 authors disclosed a total of 24 potential conflicts of interest and most pertained to minor issues.

Conclusions

Despite some recent improvement, reporting of conflicts of interest in clinical guidelines published in influential journals is largely neglected.

     

The mouse Mid1 gene: implications for the pathogenesis of Opitz syndrome and the evolution of the mammalian pseudoautosomal region

We have recently reported isolation of the gene responsible for X- linked Opitz G/BBB syndrome, a defect of midline development. MID1 is located on the distal short arm of the human X chromosome (Xp22. 3) and encodes a novel member of the B box family of zinc finger proteins. We have now cloned the murine homolog of MID1 and performed preliminary expression studies during development. Mid1 expression in undifferentiated cells in the central nervous, gastrointestinal and urogenital systems suggests that abnormal cell proliferation may underlie the defect in midline development characteristic of Opitz syndrome. We have also found that Mid1 is located within the mouse pseudoautosomal region (PAR) in Mus musculus , while it seems to be X- specific in Mus spretus. Therefore, Mid1 is likely to be a recent acquisition of the M. musculus PAR. Genetic and FISH analyses also demonstrated a high frequency of unequal crossovers in the murine PAR, creating spontaneous deletion/duplication events involving Mid1. These data provide evidence for the first time that genetic instability of the PAR may affect functionally important genes. In addition, we show that MID1 is the first example of a gene subject to X-inactivation in man while escaping it in mouse. These data contribute to a better understanding of the molecular content and evolution of the rodent PAR.      

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.      

Determination of the parent of origin in nine cases of prenatally detected chromosome aberrations found after intracytoplasmic sperm injection

Prenatal cytogenetic analysis of 71 fetuses conceived by intracytoplasmic sperm injection (ICSI) resulted in the detection of nine (12.7%) chromosome aberrations including two cases of 47,XXY, four cases involving a 45,X cell line and three autosomal trisomies. Molecular analysis of the parental origin of the deleted or supernumerary chromosome was performed by using polymorphic microsatellite markers. Six cases involving a sex chromosome abnormality were found to be of paternal origin while the two trisomic cases that could be analysed were of maternal origin. Two cases involved the same infertile couple who had two consecutive ICSI pregnancies terminated because of a chromosome abnormality. The replaced embryos in both cases originated from a single batch of ICSI fertilized oocytes of which part was used to initiate the first pregnancy and part was cryopreserved and used to initiate the second pregnancy.      

Multiple endometrial stromal nodules with sparse cysts and glands in the lung--a nodular variation of endometriosis that may mimic metastases of sarcoma

We report an unusual case of a nodular variation of pulmonary endometriosis. To our knowledge, there is no previous report on a morphological investigation of this entity. The etiology of this rare condition is still a matter of discussion. The well-circumscribed nodular mass is composed of cells identical to, or closely resembling, those of endometrial stroma containing sparse cysts and glands. Immunohistochemically, the cells showed an extensive co-expression of cytokeratin AE1/AE3 and vimentin and were highly positive for progesterone receptor (PRICA) and estrogen receptor (ERICA). Cells lining the cysts and glands as a monolayer were reactive for Ber-Ep4, cytokeratin Pan and cytokeratin AE1/AE3 and negative to all other markers used including PRICA and ERICA. The differential diagnosis of this entity included fibrous tumor of the pleura and metastatic low-grade-endometrial-stromal-sarcoma. The morphological findings are correlated with immunohistochemical studies and results of cell image analysis. This study details the clinicopathological features of the nodular variation of pulmonary endometriosis.     

Experimental investigation of a fast Monte Carlo photon beam dose calculation algorithm

An experimental verification of the recently developed XVMC code, a fast Monte Carlo algorithm to calculate dose distributions of photon beams in treatment planning, is presented. The treatment head is modelled by a point source with energy distribution (primary photons) and an additional head scatter contribution. Utility software is presented, allowing the determination of the parameters for this model using a single measured depth dose curve in water. The simple beam model is considered to be a starting point for more complex models being planned for future versions of the code. This paper is mainly focused on the influence of the different techniques on variance reduction and material property determination for dose distributions. It is demonstrated that XVMC and the simple beam model reproduce measured (by a diamond detector) relative dose distributions with an accuracy of better than +/-2% in various homogeneous and inhomogeneous phantoms. Furthermore, relative dose distributions in solid state phantoms have been measured by film. Also for these cases, measured and calculated dose distributions agree within experimental uncertainty. The short calculation time (depending on voxel resolution, statistical accuracy, field size and energy, a span of 1 min to 1 h using a present-day personal computer) and an interface to a commercial planning system will allow the implementation of the code for routine treatment planning of clinical electron and photon beams.     

A longitudinal study of maternal serum inhibin-A, inhibin-B, activin-A, activin-AB, pro-alphaC and follistatin during pregnancy

Maternal serum concentrations of inhibin-A, inhibin-B, activin-A, activin-AB, pro-alphaC-related inhibin forms, total follistatin, steroids and gonadotrophins were measured longitudinally in six normal singleton pregnancies. Maternal venous blood was collected randomly during a spontaneous follicular phase prior to donor insemination, at 5, 7, 9, 11, 16, 20, 24, 28, 32 and 36 weeks after the first missed menses and in the early puerperium. Steroid and gonadotrophin profiles conformed to previous reports. While at week 5 of gestation inhibin-A, activin-A and follistatin concentrations were similar to those at the follicular phase, all three increased progressively (P < 0.001) to maximal concentrations in week 36: approximately 48-fold (3740 +/- 1349 ng inhibin-A/ml), approximately 22-fold (6109 +/- 1443 ng activin-A/ml) and approximately 10-fold (3563 +/- 418 ng follistatin/ml) higher. Pro- alphaC concentrations reached a maximum in weeks 5 (approximately 5- fold, P < 0.001) and 36 (1027 +/- 174 pg/ml, P < 0.01). Inhibin-B (71 +/- 23 pg/ml prior to pregnancy) was undetectable (<12 pg/ml) between week 5-16 of gestation but increased slightly in the third trimester (26 +/- 7 pg/ml in week 36). Activin-AB was undetectable throughout pregnancy. Post-partum concentrations of inhibin-A (41 +/- 12 ng/ml), inhibin-B (<12 pg/ml), activin-A (950 +/- 149 pg/ml), pro-alphaC (128 +/- 22 pg/ml) and follistatin (990 +/- 79 ng/ml) were substantially lower than at week 36 of gestation. The activin-A:follistatin ratio increased from 0.5 in week 5 to 1.8 in week 36, suggesting that more free activin-A is available in the maternal circulation during late pregnancy.      

Treatment of keratoconjunctivitis sicca with topical androgen

Objective: Androgens have been reported to influence lipid production of sebaceous glands and even many ocular tissues. The effect of topical androgen therapy on a 54-year-old patient with keratoconjunctivitis sicca (KCS) and decreased lipid phase of the tear film is reported. Methods: For assessment of the lipid phase of the tear film, break up time (BUT) and lipid layer thickness (LLT) were monitored during 6 months before treatment as well as 3 months while using a daily topical androgen therapy. Results: During the topical androgen therapy the pathological lipid phase of the tear film was completely restored indicated by the normalisation of the values of BUT and LLT. Conclusion: These findings are consistent with animal experiments indicating that topical administered androgen can restore the decreased lipid phase of the tear film. This may open up new therapeutic strategies for KCS.     

Endothelial nitric oxide synthase gene polymorphism in women with idiopathic recurrent miscarriage

BACKGROUND: Lack of endothelium-derived nitric oxide is associated with vasospasm and vascular infarction. We investigated the relationship between idiopathic recurrent miscarriage and a polymorphism of the gene encoding endothelial nitric oxide synthase (NOS3). METHOD: In a prospective case-control study, 105 women with idiopathic recurrent miscarriage and 91 healthy controls were investigated. We used the polymerase chain reaction to identify the different alleles of a 27 base pair tandem repeat polymorphism in intron 4 of the NOS3 gene. RESULTS: The wild type B allele was identified on 329 out of 392 chromosomes (frequency 0.84). The polymorphic A allele was present on 63 chromosomes (frequency 0.16). The genotype frequencies were as follows: 68% (B/B), 31% (A/B) and.5% (A/A). The distribution of genotype frequencies was significantly different between the study and control groups for allele A/B heterozygotes (NOS3(A/B)) (36.7 versus 23.8%, P = 0.03, OR 1.6, 95% CI 1.1--3.8). Only one individual was homozygous for the A allele (NOS3(A/A)). She was in the study group. Between women with primary and secondary recurrent miscarriages, no statistically significant difference between the distribution of NOS3(A/B) genotypes (28 versus 34%) was observed. CONCLUSIONS: These data support a role for the NOS3 gene as a genetic determinant of the risk of idiopathic recurrent miscarriage.