Vertebrate mesendoderm induction and patterning.

Many of the key molecular events underlying the induction and patterning of the vertebrate mesoderm and endoderm have recently been elucidated. T-box transcription factors and TGF-beta and Wnt signaling pathways play crucial roles in the initial induction of the mesendoderm and the subdivision of the posterior mesoderm into rostral and caudal domains.

The pink pulseless hand: a review of the literature regarding management of vascular complications of supracondylar humeral fractures in children.

Supracondylar fractures of the humerus are the commonest upper limb fractures in children, accounting for up to 70% of all paediatric elbow fractures [Wilson MJ, Hunter JB. Supracondylar fractures of the humerus in children--wire removal in the outpatient setting. Injury Extra 2006 Aug;37(8):313-315] and are often complicated by neurovascular injury. Much confusion surrounds the management of the child with a "pink pulseless hand" post-fracture reduction and several treatment options have been proposed including observation, immediate exploration and angiography. The literature contains a number of case series with variable follow-up. Both angiography and colour duplex ultrasound provide little benefit in the management of these patients. A child with a pink pulseless hand post-fracture reduction can be managed expectantly unless additional signs of vascular compromise develop, in which case exploration should be undertaken.

LKM-1 autoantibodies recognize a short linear sequence in P450IID6, a cytochrome P-450 monooxygenase.

LKM-1 autoantibodies, which are associated with autoimmune chronic active hepatitis, recognize P450IID6, a cytochrome P-450 monooxygenase. The reactivities of 26 LKM-1 antisera were tested with a panel of deletion mutants of P450IID6 expressed in Escherichia coli. 22 sera recognize a 33-amino acid segment of P450IID6, and 11 of these recognize a shorter segment, DPAQPPRD. PAQPPR is also found in IE175 of herpes simplex virus type 1 (HSV-1). Antibodies for HSV-1 proteins were detected by ELISA in 17 of 20 LKM-1 sera tested. An immobilized, synthetic peptide, DPAQPPRDC, was used to purify LKM-1 antibodies. Affinity purified LKM-1 autoantibodies react on immunoblots with a protein in BHK cells after infection with HSV-1. 11 of 24 LKM-1 sera, including 3 that recognize DPAQPPRD, also exhibit antibodies to the hepatitis C virus (HCV) protein, C100-3. Affinity purified LKM-1 antibodies did not recognize C100-3. However, partial sequence identity was evident between portions of the immunopositive 33-amino acid segment of P450IID6 and other portions of the putative HCV polyprotein. Immune cross-recognition of P450IID6 and HCV or HSV-1 proteins may contribute to the occurrence of LKM-1 autoantibodies.

Major antigen of liver kidney microsomal autoantibodies in idiopathic autoimmune hepatitis is cytochrome P450db1.

Type 1, liver kidney microsomal autoantibodies (LKM-1) are associated with a subgroup of idiopathic autoimmune type, chronic active hepatitis (CAH). The antigenic specificity of LKM-1 autoantibodies from 13 patients was investigated by immunoblot analysis of human liver microsomal proteins. Polypeptides of 50, 55, and 64 kD were detected with these antisera. A high titer LKM-1 serum was selected to screen a human liver lambda gt11 cDNA expression library, resulting in the isolation of several complementary (c)DNA clones. Autoantibodies affinity purified from proteins expressed by two of the immunopositive cDNA clones, HLD8.2 and HLD13.2, specifically react with a 50-kD protein of human liver microsomes and display immunofluorescence staining of the proximal renal tubular epithelia characteristic of LKM-1 sera. Determination of the sequence of HLD8.2 revealed that it encodes a recently described cytochrome P450db1. A bacterial fusion protein constructed from HLD8.2 proved to be a specific and sensitive diagnostic reagent. All sera from patients with LKM-1 positive liver disease react with this fusion protein. No reaction was seen, however, for sera from patients with other types of autoimmune liver diseases, viral hepatitis, systemic immunological disorders, or healthy controls.

Characterization of a cDNA encoding a human kidney, cytochrome P-450 4A fatty acid omega-hydroxylase and the cognate enzyme expressed in Escherichia coli.

A cDNA encoding a cytochrome P-450 4A (CYP4AII) was cloned from a human kidney cDNA library. Northern blot analysis and RNase protection assays indicate that related mRNAs occur in kidney and liver with the highest abundance found in kidney. The enzyme was expressed from its cDNA in Escherichia coli. A solubilized preparation of the enzyme reconstituted with cytochrome P-450 reductase catalyzed the omega-hydroxylation of lauric acid, palmitic acid, and arachidonic acid with turnover numbers of 9.8, 2.2 and 0.55 min-1, respectively. Little or no activity was detected toward prostaglandins A1 and E1.

A pleiomorphic GH pituitary adenoma from a Carney complex patient displays universal allelic loss at the protein kinase A regulatory subunit 1A (PRKARIA) locus.

Carney complex (CNC) is a familial multiple endocrine neoplasia syndrome associated with GH-producing pituitary tumours and transmitted as an autosomal dominant trait. Mutations of the PRKAR1A gene are responsible for approximately half the known CNC cases but have never found in sporadic pituitary tumours. Pituitary tissue was obtained from an acromegalic CNC patient heterozygote for a common (PRKARIA)i-inactivating mutation. Both immunohistochemistry and electron microscopy showed a highly pleiomorphic pituitary adenoma. The cell culture population appeared morphologically heterogeneous and remained so after more than 30 passages. The mixture was comprised of cells strongly immunostained for GH, spindle-shaped myofibroblast-like cells, and cuboid cells with large axonal projections (negative for GH). The population appeared to have both epithelial and mesenchymal cells. Both at baseline and at passage 30, cytogenetic analysis indicated the presence of normal 46, XY diploid karyotype, whereas losses of the PRKARIA(i) locus were demonstrated in more than 98% of the cells by fluorescent in situ hybridisation, supporting this gene's involvement in pituitary tumorigenesis. Allelic loss may have occurred in a single precursor cell type that differentiated and clonally expanded into several phenotypes. Epithelial-to-mesenchymal transition may also occur in CNC-associated pleiomorphic pituitary adenomas.

A transgenic mouse bearing an antisense construct of regulatory subunit type 1A of protein kinase A develops endocrine and other tumours: comparison with Carney complex and other PRKAR1A induced lesions.

BACKGROUND: Inactivation of the human type Ialpha regulatory subunit (RIalpha) of cyclic AMP dependent protein kinase (PKA) (PRKAR1A) leads to altered kinase activity, primary pigmented nodular adrenocortical disease (PPNAD), and sporadic adrenal and other tumours. METHODS AND RESULTS: A transgenic mouse carrying an antisense transgene for Prkar1a exon 2 (X2AS) under the control of a tetracycline responsive promoter (the Tg(Prkar1a*x2as)1Stra, Tg(tTAhCMV)3Uh or tTA/X2AS line) developed thyroid follicular hyperplasia and adenomas, adrenocortical hyperplasia and other features reminiscent of PPNAD, including late onset weight gain, visceral adiposity, and non-dexamethasone suppressible hypercorticosteronaemia, with histiocytic, epithelial hyperplasias, lymphomas, and other mesenchymal tumours. These lesions were associated with allelic losses of the mouse chromosome 11 Prkar1a locus, an increase in total type II PKA activity, and higher RIIbeta protein levels; the latter biochemical and protein changes were also documented in Carney complex tumours associated with PRKAR1A inactivating mutations and chromosome 17 PRKAR1A locus changes. CONCLUSION: We conclude that the tTA/X2AS mouse line with a downregulated Prkar1a gene replicates several of the findings in Carney complex patients and their affected tissues, supporting the role of RIalpha as a candidate tumour suppressor gene.

Aspirin therapy is associated with less compact fibrin networks and enhanced fibrinolysis in patients with abdominal aortic aneurysm.

OBJECTIVE: Thrombotic changes in fibrin networks contribute to increased cardiovascular risk in patients with abdominal aortic aneurysm (AAA). Given that aspirin modulates the fibrin network, we aimed to determine if aspirin therapy is associated with changes in ex-vivo fibrin clot characteristics in AAA patients and also conducted an exploratory analysis of 5-year mortality in these individuals. METHODS: We recruited 145 male patients, divided into controls (aortic diameter < 3 cm, n = 49), AAA not taking aspirin (AAA-Asp, n = 50) and AAA on 75 mg day(-1) aspirin (AAA+Asp, n = 46), matched for aneurysm size. Characteristics of clots made from plasma and plasma-purified fibrinogen were investigated using turbidimetric analysis, permeation studies, and confocal and electron microscopy. Plasma fibrinogen, D-dimer and inflammatory marker levels were also measured. RESULTS: Maximum absorbance (MA) of plasma clots from controls was lower than that of AAA patients not on aspirin (AAA-Asp) at 0.30 ± 0.01 and 0.38 ± 0.02 au, respectively (P = 0.002), whereas aspirin-treated subjects had MA similar to controls (0.31 ± 0.02 P = 0.9). Plasma clot lysis time displayed an identical pattern at 482 ± 15, 597 ± 24 and 517 ± 27 s for control, AAA-Asp and AAA+Asp (P = 0.001 and P = 0.8). The lysis time of clots made from purified fibrinogen of AAA-Asp was longer than that of AAA+Asp patients (756 ± 47 and 592 ± 52 s, respectively; P = 0.041). Permeation studies and confocal and electron microscopy showed increased clot density in AAA-Asp compared with the AAA+Asp group. Mortality in AAA-Asp and AAA+Asp was similar, despite increased cardiovascular risk in the latter group, and both exhibited higher mortality than controls. CONCLUSION: Aspirin improves fibrin clot characteristics in patients with AAA, which may have important clinical implications.

Protein kinase A and its role in human neoplasia: the Carney complex paradigm.

The type 1 alpha regulatory subunit (R1alpha) of cAMP-dependent protein kinase A (PKA) (PRKAR1A) is an important regulator of the serine-threonine kinase activity catalyzed by the PKA holoenzyme. Carney complex (CNC) describes the association 'of spotty skin pigmentation, myxomas, and endocrine overactivity'; CNC is in essence the latest form of multiple endocrine neoplasia to be described and affects the pituitary, thyroid, adrenal and gonadal glands. Primary pigmented nodular adrenocortical disease (PPNAD), a micronodular form of bilateral adrenal hyperplasia that causes a unique, inherited form of Cushing syndrome, is also the most common endocrine manifestation of CNC. CNC and PPNAD are genetically heterogeneous but one of the responsible genes is PRKAR1A, at least for those families that map to 17q22-24 (the chromosomal region that harbors PRKAR1A). CNC and/or PPNAD are the first human diseases to be caused by mutations in one of the subunits of the PKA holoenzyme. Despite the extensive literature on R1alpha and PKA, little is known about their potential involvement in cell cycle regulation, growth and/or proliferation. The presence of inactivating germline mutations and the loss of its wild-type allele in CNC lesions indicated that PRKAR1A could function as a tumor-suppressor gene in these tissues. However, there are conflicting data in the literature about PRKAR1A's role in human neoplasms, cancer cell lines and animal models. In this report, we review briefly the genetics of CNC and focus on the involvement of PRKAR1A in human tumorigenesis in an effort to reconcile the often diametrically opposite reports on R1alpha.

Early re-presentations and the potential role of catheter-directed thrombolysis in patients diagnosed with a lower limb deep vein thrombosis: a single-centre experience.

INTRODUCTION: Catheter-directed thrombolysis (CDT) for iliofemoral deep vein thrombosis (DVT) restores venous patency, reduces the risk of the post-thrombotic syndrome and may reduce longer term treatment costs. This study assessed the potential role of CDT in patients with DVT with regard to representation following the index event. METHODS: A retrospective review of all patients with a positive lower limb DVT scan. Potential suitability of each patient to undergo CDT was based on well-recognized inclusion/exclusion criteria. RESULTS: In total, 1689 patients underwent a DVT-specific lower limb venous duplex. A total of 269 were found to have a DVT. Fifty-three of these patients met the inclusion criteria for CDT (only 2 underwent CDT). Fifty-nine of the 269 patients with an index DVT re-presented to our institution with a venous thromboembolism-related clinical event. These patients were significantly younger than those who did not reattend. A higher proportion of patients who represented were deemed suitable for CDT for the index DVT compared with those who did not represent (17/59 versus 36/210; P = 0.04). CONCLUSION: This pragmatic study highlights the fact that significant number of patients return to secondary care with actual/perceived complications following initial diagnosis and treatment of a DVT which may have been amenable to CDT.

Compression of the dorsalis pedis artery: a novel cause of blue toe syndrome.

Blue toe syndrome (BTS) is an important vascular condition characterized by painful blue discoloration of one or more digits. It is frequently due to emboli and is important because of the risk of progressive ischemia and tissue loss. A 53-year-old male presented with recurrent episodes of painful blue discoloration and blistering of the skin of the right hallux. On examination, the patient was found to have a cool, blue-purple great toe; all peripheral pulses were present. The patient was investigated for coagulopathy and potential sources of emboli, but the only abnormality was significant stenosis of the dorsalis pedis artery due to extrinsic compression by the extensor hallucis brevis tendon. In the absence of any other embolic source or abnormality, we believe that this case presents a novel and potentially remediable cause of BTS and indicates the need for a careful search for an underlying lesion when common causes of BTS have been excluded.

Ovarian mass causing paradoxical MI and leg ischaemia.

Paradoxical embolus through a patent foramen ovale is a well-reported phenomenon. Clinical consequences include stroke, intestinal infarction, lower limb ischaemia, and even acute myocardial infarction (MI), via embolisation to the coronary arteries. We present a case of acute MI, cardiogenic shock, and cardiac arrest caused not by this mechanism, but by embolisation of thrombotic material to the aortic root with transient complete occlusion of the left main stem (LMS) coronary artery. During percutaneous coronary intervention to treat this occlusion the thrombus became lodged at the aortic bifurcation causing lower limb ischaemia. Despite successful treatment of this via bilateral groin exploration and thromboembolectomy the patient became increasingly acidotic and an abdominal and pelvic CT scan was performed. This revealed the source of the thrombus to be the patient's congested and compressed pelvic veins which were the result of a large, previously undiagnosed ovarian malignancy with metastatic spread. Although very unusual we feel this case highlights an important differential in the diagnosis of anterolateral MI and images similar to those presented here are previously unreported in the literature.

Aberrant medial sural artery causing popliteal vein entrapment syndrome.

Isolated popliteal venous entrapment is unusual and often caused by variation or aberrant origins of the gastrocnemius muscle, thickened perivenous fascia or an abnormal vascular bundle. We report a unique case of a fit and well 35-year-old man with popliteal venous entrapment after presenting to the vascular unit with symptomatic varicose veins. The cause of the entrapment was found to be an aberrant medial sural artery on operative exploration. The artery was ligated, releasing the entrapped vein. The patient made an uneventful recovery with resolution of symptoms of venous insufficiency without evidence of muscle ischaemia.

Incidence of contralateral occult inguinal hernia found at the time of laparoscopic trans-abdominal pre-peritoneal (TAPP) repair.

BACKGROUND: Trans-abdominal laparoscopic inguinal hernia repair allows rapid assessment and exploration of the contralateral groin and repair of an occult hernia. Although previous studies have shown that the totally extra-peritoneal (TEP) hernia repair can be used to assess the contralateral groin, there is little data pertaining to the trans-abdominal pre-peritoneal (TAPP) approach. The aim of this study was to document the incidence of occult contralateral hernia at the time of TAPP hernia repair. METHODS: Data were collected prospectively from all patients undergoing laparoscopic TAPP hernia repair in a District General Hospital over a three-year period. Two specialist laparoscopic/upper gastrointestinal surgeons undertook all of the operations and telephone follow-up was carried out by a dedicated laparoscopic specialist nurse. RESULTS: A total of 310 patients underwent hernia surgery. Four cases were excluded, leaving 306 patients in the study. The male:female ratio was 10.5:1, with a median age of 59 years. Two hundred and six (67%) patients were booked for a unilateral hernia repair; of these, a contralateral hernia was found and repaired in 45 (22%). In 76 cases where a bilateral repair was planned, 61 (80%) went on to have both groin defects repaired. In the remaining 20%, the clinical suspicion of bilateral hernia was revised at the time of surgery to unilateral only. Twenty (7%) patients were booked to undergo a unilateral repair with the possibility of a contralateral hernia--in this group, the suspected contralateral defect was confirmed in 6 (30%) cases. Four (1%) cases were booked as femoral repairs, one of which was found to be an inguinal hernia. The clinical diagnostic accuracy was 78%. CONCLUSION: Accurate incidence figures of an occult contralateral inguinal hernia will enhance the pre-operative information given to patients and may impact on resource allocation and planning theatre logistics. Finding and repairing an occult contralateral hernia at the time of TAPP has the distinct advantage that it saves the patient from further symptoms and from another operation with its associated potential morbidity.

Variations in hepatic progesterone 21-hydroxylase activity reflect differences in the microsomal concentration of rabbit cytochrome P-450 1.

A monoclonal antibody specific for cytochrome P-450 1 that extensively (greater than 95%) inhibits the hepatic 21-hydroxylation of progesterone was used in a two-site immunoradiometric assay to estimate the concentration of cytochrome P-450 1 in microsomes prepared from 24 individual, untreated New Zealand White rabbits. The progesterone 21-hydroxylase activities of these microsomes ranged from 0.2 to 5.8 nmol min-1 mg microsomal protein-1. Scatchard analysis revealed similar slopes and thus apparent affinities between the antibody and microsome samples that varied greater than 10-fold in 21-hydroxylase activity. The maximal extent of binding of the antibody to different microsomal preparations was greater for microsomes exhibiting high as compared to low 21-hydroxylase activity, suggesting that the level of binding reflects the microsomal content of P-450 1. Quantitation was based on the extent of binding of the 125I-labeled monoclonal antibody to P-450 1 sequestered from a sample by a heterologous monoclonal antibody adsorbed to the wells of a microtiter plate. These results indicate that the microsomal content of P-450 1 varies from less than 0.05 to 0.5 nmol/mg microsomal protein. The microsomal content of this antigen as determined in the two-site immunoradiometric assay was highly correlated (r = 0.97) with progesterone 21-hydroxylase activity. Linear regression analysis was used to estimate the turnover number for progesterone in situ, yielding a value of 11 nmol deoxycorticosterone formed min-1 nmol microsomal P-450 1(-1). This is similar to the value of 14 nmol deoxycorticosterone formed min-1 nmol-1 obtained for the reconstituted, purified P-450 1 used as a standard in the immunoquantitation assay.

Non-adherence in pediatric transplantation: a review of the existing literature.

The degree of medical adherence in pediatric solid-organ transplant recipients frequently correlates with the degree of psychological distress, family functioning, and the physiological side-effects of immunosuppressant medications. This article examines the current literature regarding each of these factors and proposes recommendations for increasing the medical compliance among childhood transplant recipients.