Absence of the tight junctional protein AF-6 disrupts epithelial cell-cell junctions and cell polarity during mouse development.

BACKGROUND: The establishment, maintenance and rearrangement of junctions between epithelial cells are extremely important in many developmental, physiological and pathological processes. AF-6 is a putative Ras effector; it is also a component of tight and adherens junctions, and has been shown to bind both Ras and the tight-junction protein ZO-1. In the mouse, AF-6 is encoded by the Af6 gene. As cell-cell junctions are important in morphogenesis, we generated a null mutation in the murine Af6 locus to test the hypothesis that lack of AF-6 function would cause epithelial abnormalities. RESULTS: Although cell-cell junctions are thought to be important in early embryogenesis, homozygous mutant embryos were morphologically indistinguishable from wild-type embryos through 6.5 days post coitum (dpc) and were able to establish all three germ layers. The earliest morphological abnormalities were observed in the embryonic ectoderm of mutant embryos at 7.5 dpc. The length of the most apical cell-cell junctions was reduced, and basolateral surfaces of those cells were separated by multiple gaps. Cells of the embryonic ectoderm were less polarized as assessed by histological criteria and lateral localization of an apical marker. Mutant embryos died by 10 dpc, probably as a result of placental failure. CONCLUSIONS: AF-6 is a critical regulator of cell-cell junctions during mouse development. The loss of neuroepithelial polarity in mutants is consistent with a loss of efficacy of the cell-cell junctions that have a critical role in establishing apical/basolateral asymmetry.

pH-dependent and ligand induced conformational changes of eucaryotic protein synthesis initiation factor eIF-(iso)4F: a circular dichroism study.

The structural features of wheat germ protein synthesis initiation factor eIF-(iso)4F, which has a cap binding protein as one of its two subunits, are unknown. In this study, circular dichroism (CD) spectra and secondary structure prediction were obtained for eIF-(iso)4F and its two subunits, p28 and p86. The alpha-helix content of eIF-(iso)4F changed from 42% at pH 6.3 to 15% at pH 7.6, the optimum pH for cap binding. The beta-sheet content increased from 14% (pH 6.3) to 38% at pH 7.6. The CD spectra of the two subunits, p28 and p86 were also measured and analyzed. The separated subunits both had a higher alpha-helix content at pH 7.6 than the native protein, giving values of 60% and 34% alpha-helix for p28 and p86, respectively. Binding of the dinucleotide cap analog to p28 reduced the alpha-helix content to approximately 8% with an increase in the beta sheet content from 10% to 37%. The conformational changes in eIF-(iso)4F upon binding with mRNA are dependent on cap or oligonucleotide structure. A conformation consisting of approximately the same alpha-helix and beta-sheet content can be induced by ligands even at non-optimal pH values. This large conformational transition suggests eIF-(iso)4F binds nucleic acids by interaction of a beta-sheet motif and that this conformational transition may have a regulatory role.

A fluorescence study of the interaction of protein synthesis initiation factors 4A, 4E, and 4F with mRNA and oligonucleotide analogs.

The initial interaction of mRNA with the protein synthesis machinery presumably involves recognition of the 5'-cap (m7GpppN), although it is not clear at the present time whether this recognition is by eIF-4E or eIF-4F. This process has been studied by direct fluorescence titration experiments. The equilibrium constants for the formation of the binary protein: m7GpppG, protein:mRNA, and protein:protein complexes as well as the ternary mRNA:eIF-4E:eIF-4A complexes were measured. These studies show, for the first time, direct evidence for an eIF-4A:eIF-4E interaction. In contrast to earlier studies, we show that the affinity of eIF-4E and eIF-4F for globin mRNA is similar. Furthermore, the relative affinities of mRNA analogs (capped oligonucleotides) for these initiation factors indicate that the cap is the predominant feature recognized for binding, but other features also contribute to the eIF-4E:mRNA interaction.

Development of educational image databases and e-books for medical physics training.

Medical physics education and training requires the use of extensive imaging material and specific explanations. These requirements provide an excellent background for application of e-Learning. The EU projects Consortia EMERALD and EMIT developed five volumes of such materials, now used in 65 countries. EMERALD developed e-Learning materials in three areas of medical physics (X-ray diagnostic radiology, nuclear medicine and radiotherapy). EMIT developed e-Learning materials in two further areas: ultrasound and magnetic resonance imaging. This paper describes the development of these e-Learning materials (consisting of e-books and educational image databases). The e-books include tasks helping studying of various equipment and methods. The text of these PDF e-books is hyperlinked with respective images. The e-books are used through the readers' own Internet browser. Each Image Database (IDB) includes a browser, which displays hundreds of images of equipment, block diagrams and graphs, image quality examples, artefacts, etc. Both the e-books and IDB are engraved on five separate CD-ROMs. Demo of these materials can be taken from www.emerald2.net.

Lower limb venous pressure in diabetic neuropathy.

OBJECTIVE: Distended turgid veins have been noted in the feet of patients with diabetic neuropathy. This study assessed the supine venous pressure and its correlation with diabetic neuropathy and arteriovenous (AV) shunting in the feet of type I and type II diabetic subjects. RESEARCH DESIGN AND METHODS: Seventeen patients with chronic Charcot joints, 11 patients with a history of neuropathic foot ulceration, 14 diabetic control subjects, and 11 normal control subjects were studied. Neuropathy was assessed by vibration and thermal thresholds and standard cardiovascular autonomic neuropathy tests. Supine venous pressure was measured by insertion of a 21-gauge needle connected to a strain gauge manometer into the veins on the dorsum of the foot. Venous PO2 was used as a measure of AV shunting. RESULTS: Venous pressure was raised in the group with chronic Charcot joints (18.9 +/- 4.2 mmHg, P < 0.001 vs. the diabetic control group [11.3 +/- 2.6 mmHg]), the normal control group (12.3 +/- 2.3 mmHg, mean +/- SD), and the diabetic group with a history of neuropathic foot ulceration (16.0 +/- 3.7 mmHg, P = 0.008 vs. diabetic control subjects and P = 0.04 vs. normal control subjects), with a maximum of 28 mmHg. Venous pressure was correlated with heart rate variation to deep breathing, r = -0.61 (P = 0.001), vibration threshold, r = 0.56 (P = 0.001), Valsalva ratio, r = -0.64 (P -= 0.001), warm threshold, r = 0.69 (P = 0.001), and venous PO2, r = 0.43 (P = 0.02). There was no correlation with skin temperature, duration of diabetes, age, HbA1c, random blood glucose, ankle/brachial Doppler index, height, or serum creatinine. CONCLUSIONS: Venous pressure is much higher in the neuropathic diabetic limb and is correlated with the severity of neuropathy and with AV shunting.

Identification of the cap binding domain of human recombinant eukaryotic protein synthesis initiation factor 4E using a photoaffinity analogue.

Binding of eIF-4E to the 5' m7G cap structure of eukaryotic mRNA signals the initiation of protein synthesis. In order to investigate the molecular basis for this recognition, photoaffinity labeling with [gamma-32P]8-N3GTP was used in binding site studies of human recombinant cap binding protein eIF-4E. Competitive inhibition of this cap analogue by m7GTP and capped mRNA indicated probe specificity for interaction at the protein binding site. Saturation of the binding site with [gamma-32P]8-N3GTP further demonstrated the selectivity of photoinsertion. Aluminum (III)-chelate chromatography and reverse-phase HPLC were used to isolate the binding site peptide resulting from digestion of photolabeled eIF-4E with modified trypsin. Amino acid sequencing identified the binding domain as the region containing the sequence Trp 113-Arg 122.Lys 119 was not identified in sequencing analysis nor was it cleaved by trypsin. These results indicate that Lys 119 is the residue directly modified by photoinsertion of [gamma-32P]8-N3GTP. A detailed understanding of eIF-4E.m7G mRNA cap interactions may lead the way to regulating this essential protein-RNA interaction for specific mRNA in vivo.

Transplant renal artery stenosis in 77 patients--does it have an immunological cause?

Transplant renal artery stenosis (TRAS) is a common complication after transplantation and is an important cause of graft dysfunction. Damage from graft rejection, trauma, and atherosclerosis have been implicated as possible causes. We reviewed all 917 patients transplanted in our unit since 1978 to study the prevalence, clinical features, and possible causes of TRAS. Seventy-seven patients with TRAS were identified. The detected incidence was 2.4% before the introduction of color doppler ultrasonography (CDU) and rose to 12.4% after CDU was introduced in 1985, giving an overall incidence of 8.4% during a mean follow-up period of 6.9 years. The TRAS group was compared with a control group of 77 transplanted patients matched for age, year of transplant, sex, and number of previous grafts. Mean ages for the study and control groups were 43.6 +/- 15 and 44.8 +/- 13.7 yr. A total of 25% of cases of TRAS were diagnosed within the first 8 wk of transplantation and in 60% within the first 30 wk (median = 23 wk). All patients were treated with angioplasty, 28 patients had recurrence of TRAS requiring multiple angioplasties (maximum 5) and 1 went on to have surgery. Angioplasty resulted in a significant fall in plasma creatinine. Patient and graft survival were significantly worse in the TRAS group: 69% vs. 83% (P < 0.05) and 56% vs. 74% (P < 0.05) (TRAS vs. Control), respectively. There was a significantly higher incidence of rejection, especially cellular rejection in the TRAS group, 0.67 vs. 0.35 episodes per patient (P < 0.01) (TRAS vs. Control). Recurrence but not occurrence of TRAS was associated with the use of cyclosporine.

A six year prospective study of fibrinogen and other risk factors associated with mortality in stable claudicants.

A total of 333 patients with stable intermittent claudication at recruitment were followed up for 6 years to determine risk factors associated with subsequent mortality. Cardiovascular diseases were the underlying cause of death in 78% of the 114 patients who died. The strongest independent predictor of death during the follow-up period was the plasma fibrinogen level, an increase of 1 milligram being associated with a nearly two-fold increase in the probability of death within the next 6 years. Age, low ankle/brachial pressure index and a past history of myocardial infarction also increased the probability of death during the study period. The plasma fibrinogen level is a valuable index of those patients with stable intermittent claudication at high risk of early mortality. The results also provide further evidence for the involvement of fibrinogen in the pathogenesis of arterial disease.

Menstrual dysfunction following use of oral contraceptives.

PIP: An evaluation of 13 patients with menstrual dysfunction following discontinuation of oral contraceptives is presented. All had normal ovulatory cycles of 26-34 days before beginning oral contraceptive medication. The duration of therapy was 3-42 months with 14 months the mean. Relationship between occurrence of menstrual dysfunction and duration of therapy or type of agent administered was not noted. 9 were classified as amenorrheic (6 months or more of secondary amenorrhea) and their condition lasted from 6 to 36 months. Cervical mucus from 6 demonstrated a 3 to 4+ fern phenomena, indicating significant endogenous estrogen production. Gynecologic endocrine status was evaluated by basal body temperature, endometrial biopsy, and cervical mucus arborization. Total gonadotropins were measured in those demonstrating poor or absent fern phenomenon. Adrenal and thyroid functions were measured by 17-ketosteroid and 17-hydroxysteroid excretion and PBI respectively. Patients were screened for pituitary tumors by X-ray and visual examinations. Clomiphene (Clomid) was administered to 6 patients in 5 day courses of 50 or 100 mg per day. Of these 4 responded with ovulatory cycles. One became pregnant. 2 patients not responding to several such courses, were given human menopausal gonadotropin (HMG Pregova) in doses of 140 I.U. a day until cervical mucus demonstrated 4+ fern phenomenon. Human chorionic gonadotropin (HCG) was then given in a single dose of 5000 I.U. One responded to this treatment and became pregnant. 3 were not treated because they did not desire conception or ovulation induction. The 4 patients who were amenorrheic for less than 6 months were considered oligomenorrheic. 2 responded to clomiphene but the pattern of prolonged follicular phase has recurred in both. Their menstrual dysfunction is thought to be due to delayed recovery from suppression of some hypothalamic-pituitary mechanisms involved in normal gonadotropin regulation. Gonadtropin therapy is the only effective method of ovulation induction in those patients who fail to respond to clomiphene.^ieng

Neoplasia in the rat induced by N-hydroxyphenacetin, a metabolite of phenacetin.

N-hydroxyphenacetin, a phenacetin metabolite, was fed to rats as a 0.05-0.5% dietary supplement. After 9 months, tumours of the liver were found in 36 of 64 animals. One animal also developed a renal tumour. No tumours were found in control animals. The findings implicate phenacetin as a carcinogen and suggest that N-hydroxyphenacetin may be the metabolite responsible.

Rapid micro-isolation of mammalian oxymyoglobin for biophysical studies.

A rapid, single-step procedure is described for the isolation of mammalian oxymyoglobin for physical studies. The procedure employs a high-capacity, reusable, Hg-resin through which the myoglobin is filtered. The mammalian myoglobins for which sequences have been reported lack reactive cysteine. The column binds hemoglobin and other sulfhydryl-containing proteins, and retards other proteins. The procedure requires less than twenty minutes for the isolation of oxymyoglobin from homogenized muscle tissue. Virtually no metmyoglobin is formed. The yields are 2-3 times greater than by other methods. The procedure can easily be scaled down for isolation of myoglobin from a few milligrams of muscle. Identical rate constants for oxygen association, dissociation, and CO association were measured by laser photolysis for beef heart myoglobin prepared by this procedure and that of Yamazaki et al. (Yamazaki, I., Yokota, K. and Shikama, K. (1964) J. Biol. Chem. 239, 4151). Gel electrophoresis indicates that the preparation is at least 95% pure.

Distraction-flexion injury in a neurologically intact pediatric patient treated nonoperatively: case study.

STUDY DESIGN: A case study was conducted. OBJECTIVE: To detail the successful nonoperative management of a distraction-flexion Salter-Harris Type 1 midcervical spine injury in a young child. SUMMARY OF BACKGROUND DATA: Documented cases of neurologically intact pediatric patients with unstable flexion-distraction injuries of the midcervical spine are rare. METHODS: A case report and literature review are presented. RESULTS: A rare distraction-flexion injury was successfully treated nonoperatively. CONCLUSIONS: Although distraction-flexion cervical spine injuries are common in adults and often occur with concomitant neurologic sequelae, they also can occur in the pediatric patient. Whereas these injuries require surgery in adults, they can be managed nonoperatively in the pediatric population.

The effect of caffeine on the accommodative response/accommodative stimulus function and on the response AC/A ratio.

Using a phoro-accommodometer, accommodation and convergence responses were measured for 17 experimental subjects before and after they ingested 200 mg caffeine. For 16 control subjects accommodation and convergence responses were determined at two test sessions separated by 45 min. For the experimental group, the accommodative response/accommodative stimulus (Ar/As) slope increased 0.05 D/D after ingestion of caffeine, and the response AC/A ratio decreased 3.05 delta/D. The y-intercept of the convergence response/accommodative response increased by 3.7 delta in the experimental group. These changes were statistically significant. For the control group, the differences in Ar/As slope, response AC/A ratio, and convergence response/accommodative response y-intercept between first and second test sessions were not statistically significant.

Leaching of accelerator-produced radionuclides.

Leaching of radionuclides produced in soil and rock by high energy proton-induced radiation was studied for the SSC site. Comparison was made with predictions of a Monte Carlo code CASIM and previous results for the Fermilab site. The principal long-lived radionuclides were 3H and 22Na in agreement with Fermilab results. A few other radionuclides were present at lower concentrations in a subset of the samples. For example, 134Cs was detected in a few SSC water samples. Leaching from SSC chalk was dependent on previous weathering and on leaching time. The more soil-like marl and shale were leached more rapidly. Results of this study, in conjunction with the SSC groundwater model, show that adequate groundwater protection would have been maintained for an accidental loss of the entire proton beam at a point in the SSC Collider tunnel. Early warning techniques developed are directly applicable to soil activation monitoring at other facilities.

Groundwater activation at the Superconducting Super Collider: a new design model.

A groundwater activation model was developed for use in designing the accelerators at the Superconducting Super Collider Laboratory. This model is based on the concept of a 4-m-thick "activation zone" surrounding the accelerator enclosure, which contains over 99% of the soil activation caused by beam losses. Empirical shielding formulae based on computer simulations indicate that the soil activation in the activation zone decreases exponentially with distance from the tunnel enclosure. From this assumption, the average activation in the activation zone is derived. It is shown that the average activity concentration in the activation zone is equal to the activity concentration 1 m from the accelerator enclosure. The activation concentration in the water averaged over the volume of the activation zone is compared to the drinking water standards. The goal of this model is to meet the drinking water regulatory standards by averaging the activation in the activation zone. Groundwater activation concentrations have been calculated for the Super Collider utilizing experimental measurements of production cross sections and leachability factors. Comparison is made to the groundwater activation criterion for both routine and accidental beam losses.

Reliability of rotary prism fusional vergence ranges.

BACKGROUND: The measurement of fusional vergence ranges is an important clinical test in the assessment of binocular vision status. Fusional vergence ranges are typically measured by recording a patient's reports of blur, break, and recovery to base-in (BI) and base-out (BO) prism. Published reliability data on fusional vergence ranges are very limited. METHODS: Eight subjects underwent four testing sessions, at which repeated measurements of fusional vergence ranges were taken. Near ranges were tested at the first session only Distance ranges were tested at all four sessions. Intra-examiner standard deviations were calculated for each fusional vergence test result (BI and BO; blur, break, and recovery) for each session. Intra-examiner standard deviations were averaged. These values were used to determine 95% limits of agreement. RESULTS: The 95% limits of agreement were between 2 delta and 2.5 delta for the distance BI break and recovery and for the near BI recovery; between 3 and 4 delta for near BI break and near BO break; between 4 and 5 delta for distance BO blur and recovery and for near BI blur; and between 5 and 5.5 delta for distance BO break and near BO blur and recovery.