p.R254Q mutation in the aquaporin‐2 water channel causing dominant nephrogenic diabetes insipidus is due to a lack of arginine vasopressin‐induced phosphorylation

Vasopressin regulates human water homeostasis by re‐distributing homotetrameric aquaporin‐2 (AQP2) water channels from intracellular vesicles to the apical membrane of renal principal cells, a process in which phosphorylation of AQP2 at S256 by cAMP‐dependent protein kinase A (PKA) is thought to be essential. Dominant nephrogenic diabetes insipidus (NDI), a disease in which the kidney is unable to concentrate urine in response to vasopressin, is caused by AQP2 gene mutations. Here, we investigated a reported patient case of dominant NDI caused by a novel p.R254Q mutation. Expressed in oocytes, AQP2‐p.R254Q appeared to be a functional water channel, but was impaired in its transport to the cell surface to the same degree as AQP2‐p.S256A, which mimics non‐phosphorylated AQP2. In polarized MDCK cells, AQP2‐p.R254Q was retained and was distributed similarly to that of unstimulated wt‐AQP2 or AQP2‐p.S256A. Upon co‐expression, AQP2‐p.R254Q interacted with, and retained wt‐AQP2 in intracellular vesicles. In contrast to wild‐type AQP2, forskolin did not increase AQP2‐p.R254Q phosphorylation at S256 or its translocation to the apical membrane. Mimicking constitutive phosphorylation in AQP2‐p.R254Q with the p.S256D mutation, however, rescued its apical membrane expression. These date indicate that a lack of S256 phosphorylation is the sole cause of dominant NDI here, and thereby, p.R254Q is a loss of function instead of a gain of function mutation in dominant NDI. © 2009 Wiley‐Liss, Inc.     

Elastin point mutations cause an obstructive vascular disease, supravalvular aortic stenosis

Supravalvular aortic stenosis (SVAS) is an inherited obstructive vascular disease that affects the aorta, carotid, coronary and pulmonary arteries. Previous molecular genetic data have led to the hypothesis that SVAS results from mutations in the elastin gene, ELN. In these studies, the disease phenotype was linked to gross DNA rearrangements (35 and 85 kb deletions and a translocation) in three SVAS families. However, gross rearrangements of ELN have not been identified in most cases of autosomal dominant SVAS. To define the spectrum of ELN mutations responsible for this disorder, we refined the genomic structure of human ELN and used this information in mutational analyses. ELN point mutations co-segregate with the disease in four familial cases and are associated with SVAS in three sporadic cases. Two of the mutations are nonsense, one is a single base pair deletion and four are splice site mutations. In one sporadic case, the mutation arose de novo. These data demonstrate that point mutations of ELN cause autosomal dominant SVAS.      

CD40 expression by gingival fibroblasts: correlation of phenotype with function

CD40, a member of the tumor necrosis factor-alpha receptor family, is constitutively expressed by cells of hematopoietic and non- hematopoietic origin, including fibroblasts. Signaling through this receptor molecule regulates inflammatory cytokine secretion by many cell types. Based on the recently described cytokine secretory heterogeneity of fibroblast cell subsets, we hypothesized that secretion of inflammatory cytokines by gingival fibroblast cultures may be dictated by the existence of differential proportions of cytokine- secreting subpopulations which express high levels of CD40. After examining a large number of gingival fibroblast (GF) cultures we find that the frequency of IL-6- and IL-8-secreting cells mirrors the frequency of cells expressing high levels of CD40 in these cultures. In addition, we demonstrate a direct functional relationship between CD40 expression and IL-6 or IL-8 secretion by showing that ligation of this molecule on GF, and CD40+ fibroblast subsets in particular, up- regulates secretion of these cytokines in vitro.      

Isolation and characterization of human and rabbit sperm tail fibrous sheath

Using mechanical and chemical dissection methods, fibrous sheath was isolated both from normal ejaculated human spermatozoa and from rabbit cauda epididymal spermatozoa. The same techniques did not produce a pure preparation of fibrous sheath from ejaculated rabbit spermatozoa, suggesting that further cross-linking and stabilization of sperm structures occurs in response to components of the seminal plasma. The isolation procedures were monitored by phase contrast microscopy and the purity of the fibrous sheath was verified by electron microscopy. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) of isolated human fibrous sheath revealed at least 14 protein bands of which the most intensely stained were of molecular weight 84, 72, 66.2, 57, 32 and 28.5 kDa. The rabbit fibrous sheath revealed at least 10 protein bands, of which the most intensely stained were 35.2, 32.7 and 28.5 kDa. The amino acid composition of the purified fibrous sheath from human and rabbit spermatozoa was similar, being high in aspartic acid and/or asparagine and glutamic acid and/or glutamine, serine, alanine, leucine, lysine and glycine, but low in histidine, tyrosine and isoleucine. This composition is similar to that reported for the rat and suggests that mammalian sperm tail fibrous sheaths are composed of similar types of proteins, although there are apparent differences in protein components between species.      

The CTLA-4 gene region of chromosome 2q33 is linked to, and associated with, type 1 diabetes. Belgian Diabetes Registry

Susceptibility to autoimmune insulin-dependent (type 1) diabetes mellitus is determined by a combination of environmental and genetic factors, which include variation in MHC genes on chromosome 6p21 (IDDM1) and the insulin gene on chromosome 11p15 (IDDM2). However, linkage to IDDM1 and IDDM2 cannot explain the clustering of type 1 diabetes in families, and a role for other genes is inferred. In the present report we describe linkage and association of type 1 diabetes to the CTLA-4 gene (cytotoxic T lymphocyte associated-4) on chromosome 2q33 (designated IDDM12). CTLA-4 is a strong candidate gene for T cell- mediated autoimmune disease because it encodes a T cell receptor that mediates T cell apoptosis and is a vital negative regulator of T cell activation. In addition, we provide supporting evidence that CTLA-4 is associated with susceptibility to Graves' disease, another organ- specific autoimmune disease.      

The efficacy of prophylactic intravenous antibiotics in elective foot and ankle surgery

A retrospective chart review of 555 patients who received elective foot and ankle surgeries between 1995 and 2001 at 1 outpatient podiatric hospital clinic was performed to evaluate the efficacy of preoperative intravenous antibiotic use. Only those patients who were having elective foot or ankle surgery for the first time, were being followed up at the hospital's outpatient clinic, and had a nontraumatic cause for their surgery were included in this study. A wound was considered infected when purulent material from the wound sites was noted and an organism(s) was cultured. A wound complication was defined as a superficial dehiscence, edema, erythema, or stitch abscess. Three hundred six (55.1%) patients received a preoperative antibiotic and 249 (44.9%) patients did not. Of the 306 patients who received a preoperative antibiotic, 9 (1.6%) acquired a postoperative wound infection, whereas 8 (1.4%) of the 249 patients who did not receive preoperative antibiotics acquired a postoperative infection. A logistic regression model and chi square tests of association were used to determine if preoperative antibiotic use, age, gender, type of surgical procedure, operative time, tourniquet use, past medical history, and internal fixation were predictive of or associated with postoperative wound infection or complication. None of the study factors was predictive of postoperative wound infection or complication (P >.01). Preoperative antibiotic use was associated with surgical category and internal fixation use (P <.001) but not postoperative wound infection or complication (P >.01). The results suggest that prophylactic intravenous antibiotic use in routine elective foot and ankle surgery is not warranted.     

Chondromalacia patellae: assessment with MR imaging

Magnetic resonance (MR) images of the posterior patellar hyaline articular cartilage were obtained in 23 subjects to determine if MR imaging could accurately demonstrate the patellar cartilage. Arthroscopy was used as the standard of reference. Three subjects were asymptomatic volunteers. In the remaining 20 who had patellofemoral pain, arthroscopy was performed before MR imaging in seven and afterward in 12; one did not undergo arthroscopy. MR imaging showed focal areas of swelling of the patellar cartilage, focal hypointensity, surface irregularity, areas of thinning, and areas of cartilage loss with exposure of subchondral bone. The surgical findings agreed with those from MR images in all seven patients who underwent arthroscopy before MR imaging and in ten of the 12 who underwent surgery afterward. MR imaging is an accurate means of examining the posterior patellar cartilage and should be considered as an alternative to diagnostic arthroscopy when chondromalacia patellae is suspected.     

Acute experimental cerebral ischemia: MR enhancement using Gd-DTPA

The effects of a paramagnetic contrast agent, gadolinium-DTPA (Gd-DTPA), on magnetic resonance (MR) imaging of acute cerebral ischemia was investigated in a feline model of middle cerebral artery occlusion. Imaging was performed both before and after administration of an intravenous dose of 0.2 mmol/kg of Gd-DTPA. The animals were then sacrificed for pathologic correlation. No changes in intensity or relaxation times were noted before or after Gd-DTPA administration in two animals with 2 hours of occlusion. Infarcts were noted before and after contrast enhancement in all six cats with ischemia of greater than 16-hours duration. Gd-DTPA caused significant increase in intensity of infarct but not in that of normal cerebral tissue. Rapid enhancement was visible in infarcts of 16-24 hours, but such enhancement was slower in infarcts of 72-168 hours, presumably owing to slowed inflow caused by increased vasogenic edema in the latter group. Contrast enhancement of acute cerebral ischemic lesions with Gd-DTPA offers no improvement in sensitivity of MR imaging, although the conspicuity of the lesion may be improved. Additionally, contrast media may provide potential temporal and pathophysiological data for better characterization of cerebral ischemia.     

Intracranial circulation: pulse-sequence considerations in three- dimensional (volume) MR angiography

The technique and feasibility of magnetic resonance (MR) angiography of intracranial vessels were studied in 35 healthy volunteers. Variations in image orientation, repetition time (TR), and flip angle were evaluated to determine their effects on flow-related enhancement. Gradient modifications--including echo time (TE), motion compensation, bandwidth, and field of view--were also studied in an effort to reduce motion-induced phase shifts. Results indicated that a FISP (fast imaging with steady precession) sequence with a TR of 50 msec, TE of 15 msec, velocity compensation in the read and section-select directions, acceleration compensation in the read direction, anisotropic volume, and a 1.25-mm partition thickness produced three-dimensional angiographic MR images that were accurate and reproducible in the depiction of the major intracranial vessels. Difficulties with field of view, persistent signal void secondary to higher-order motion, and spatial resolution remain major problems requiring additional study.