Extensive in vitro cross-reactivity to seed storage proteins is present among walnut (Juglans) cultivars and species.

BACKGROUND: Tree nuts, including English walnuts (Juglans regia), are sources of food allergens often associated with life-threatening allergic reactions. It is unknown if seed storage proteins from other Juglans species have IgE epitopes similar to those of the important English walnut allergens, Jug r 1 (2S albumin) and Jug r 2 (vicilin-like). OBJECTIVE: To screen for potential germplasm sources of hypoallergenic seed storage proteins of relevance in walnut food allergy. We sought to identify English walnut cultivars (cvs) or other Juglans species that showed decreased IgE binding to major seed storage proteins or an inability to cross-react with Jug r 1 or Jug r 2. METHODS: We determined if IgE in sera of patients who have had life-threatening systemic reactions to English walnut bound protein extracts from all tested walnut cvs (57 cvs total) or species (six) by Western immunoblot. Further, we used immunoblot inhibition to determine the in vitro cross-reactivity of Jug r 1 and Jug r 2, native and recombinant, with several walnut species. RESULTS: All walnut cvs and species contain allergenic proteins. Furthermore, as shown by in vitro immunoblot inhibition, the major walnut allergens in the species tested cross-reacted with those in J. regia cv. Chandler and J. nigra cv. Thomas extracts. CONCLUSIONS: Based on our findings, it is unlikely that a composite hypoallergenic walnut could be bred from available germplasm. In addition, patients with severe allergy to English walnut are likely to be clinically allergic to all commercial English walnut cvs and other closely related Juglans species.

The West Point Study: 40 years of follow-up.

Completion of cardiovascular evaluations of 387 members marked the end of 40 years of follow-up in the West Point Study. Coronary artery disease (CAD) caused 4 cases of sudden death, 14 cases of myocardial infarction (MI), 13 cases of angina, and 17 cases of silent CAD. Using risk factors (serum cholesterol, estimated HDL-cholesterol, systolic blood pressure, and smoking status) measured before age 28, we derived a multivariate regression formula for predicting which members of the study, had they been pilots, would have been grounded for CAD before age 55. This derivation used data from only those subjects with CAD or with no evidence of CAD. We then used the formula to compute a risk-related score for each member of the study. In the tertile group with the highest risk-related scores, 17% manifested CAD by age 55 and the first event occurred at age 39. In the tertile group of lowest scores, 2% experienced CAD by age 55 and the first event occurred at age 51. We conclude that it is possible to select pilot candidates with the lowest risk for CAD.

Transformation of pecan and regeneration of transgenic plants.

A gene transfer system developed for walnut (Juglans regia L.) was successfully applied to pecan (Carya illinoensis [Wang] K. Koch). Repetitively embryogenic somatic embryos derived from open-pollinated seed of 'Elliott', 'Wichita', and 'Schley' were co-cultivated with Agrobacterium strain EHA 101/pCGN 7001, which contains marker genes for beta-glucuronidase activity and resistance to kanamycin. Several modifications of the standard walnut transformation techniques were tested, including a lower concentration of kanamycin and a modified induction medium, but these treatments had no measurable effect on efficiency of transformation. Nineteen of the 764 viable inoculated embryos produced transgenic subclones; 13 of these were from the line 'Elliott'6, 3 from 'Schley'5/3, and 3 from 'Wichita'9. Transgenic embryos of 'Wichita'9 germinated most readily and three subclones were successfully micropropagated. Three transgenic plants of one of these subclones were obtained by grafting the tissue cultured shoots to seedling pecan rootstock in the greenhouse. Gene insertion, initially detected by GUS activity, was confirmed by detection of integrated T-DNA sequences using Southern analysis.

Improved efficiency of the walnut somatic embryo gene transfer system.

AnAgrobacterium-mediated gene transfer system which relies on repetitive embryogenesis to regenerate transgenic walnut plants has been made more efficient by using a more virulent strain ofAgrobacterium and vectors containing genes for both kanamycin resistance and beta-glucuronidase (GUS) activity to facilitate early screening and selection. Two plasmids (pCGN7001 and pCGN7314) introduced individually into the disarmedAgrobacterium host strain EHA101 were used as inoculum. Embryos maintained on medium containing 100 mg/l kanamycin after co-cultivation produced more transformed secondary embryos than embryos maintained on kanamycin-free medium. Of the 186 GUS-positive secondary embryo lines identified, 70% were regenerated from 3 out of 16 primary embryos inoculated with EHA101/pCGN7314 and grown on kanamycin- containing medium, 28% from 4 out of 17 primary embryos inoculated with EHA101/ pCGN7001 and grown on kanamycin medium, and 2% from one out of 13 primary embryos inoculated with EHA101/pCGN7001 but not exposed to kanamycin. Because kanamycin inhibits but does not completely block new embryo formation in controls, identification of transformants formerly required repetitive selection on kanamycin for several months. Introduction of the GUS marker gene allowed positive identification of transformant secondary embryos as early as 5-6 weeks after inoculation. DNA analysis of a representative subset of lines (n=13) derived from secondary embryos confirmed transformation and provided evidence for multiple insertion events in single inoculated primary embryos.

Origin of somatic embryos from repetitively embryogenic cultures of walnut (Juglans regia L.): Implications forAgrobacterium-mediated transformation.

Early stages of somatic embryo development from embryogenic cultures ofJuglans regia (Persian or English walnut) are described. Histological examination reveals that secondary somatic embryos arise from cotyledons and hypocotyls of primary embryos cultured in the dark. The embryos originate by transverse to oblique divisions of surface cells. Single-cell origin of the secondary embryos confirms the potential of the repetitive embryogenesis system forAgrobacterium-mediated transformation and regeneration of non-chimeric, transgenic walnut plants.

Regeneration by somatic embryogenesis of triploid plants from endosperm of walnut, Juglans regia L. cv Manregian.

Plants were regenerated by somatic embryogenesis from endosperm tissue of open-pollinated seeds of Juglans regia L. cv Manregian. These plants were obtained by growing endosperm tissue on media similar to those used for plant regeneration from walnut cotyledons (Tulecke and McGranahan 1985). The plants appear morphologically uniform and have a triploid chromosome number of 3n=48. Nine plants have been grown to a young sapling stage in soil. This embryogenic line from endosperm has been maintained in culture for two years by the process of repetitive somatic embryogenesis.

The effects of intraventricular gradients on left ventricular ejection dynamics.

The generation of abnormal gradients between the apical cavity and the subaortic valvular region of the left ventricle in patients with hypertrophic cardiomyopathy (HCM) has traditionally been equated to a dynamic obstruction to left ventricular outflow. To examine this concept in more detail, left ventricular ejection dynamics were studied during cardiac catheterization in 30 patients with HCM and 29 patients with no evidence of cardiovascular disease. Using multisensor catheterization techniques, ascending aortic flow velocity and micromanometer left ventricular and aortic pressures were simultaneously recorded during rest (n = 47). Dynamic left ventricular emptying was also analyzed with frame-by-frame angiography (n = 46). The temporal distribution of left ventricular outflow was independently derived from both flow velocity and angiographic techniques. The HCM patients were subdivided into three groups: I, intraventricular gradients at rest (n = 9); II, intraventricular gradients only with provocation (n = 12); III, no intraventricular gradients despite provocation (n = 9). Expressed as a precentage of the available systolic ejection period (%SEP), the time required for ejection of the total stroke volume was (mean +/- 1 S.D.): Group I, 69 +/- 17% (flow), 64 +/- 6% (angio); Group II, 63 +/- 14% (flow), 65 +/- 6% (angio); Group III, 61 +/- 16% (flow), 62 +/- 4% (angio); control group, 90 +/- 5% (flow) 86 +/- 10% (angio). No significant difference was observed between any of the three HCM subgroups, but, compared with the control group, ejection was completed much earlier in systole independent of the presence or absence of intraventricular gradients. The presence of coexisting mitral regurgitation in 12 of the HCM patients did not alter these results. This study demonstrates that 'outflow obstruction', as traditionally defined by the presence of an abnormal intraventricular pressure gradient and systolic anterior motion of the mitral valve, does not impede left ventricular outflow in HCM. In a pure fluid dynamic sense, we believe that outflow obstruction does not exist in this disease entity.

Dynamics of left ventricular ejection in obstructive and nonobstructive hypertrophic cardiomyopathy.

The purpose of this study was to examine the dynamics of left ventricular ejection in patients with obstructive and nonobstructive hypertrophic cardiomyopathy (HCM). 30 patients with HCM and 29 patients with no evidence of cardiovascular disease were studied during cardiac catheterization. Using a single multisensor catheter, electromagnetically derived ascending aortic flow velocity and high fidelity left ventricular and aortic pressures were recorded during rest (n = 47) and provocative maneuvers (n = 23). Dynamic ventricular emptying during rest was also analyzed with frame-by-frame angiography (n = 46). Left ventricular outflow was independently derived from both flow velocity and angiographic techniques. The HCM patients were subdivided into three groups: (I) intraventricular gradients at rest (n = 9), (II) intraventricular gradients only with provocation (n = 12), and (III) no intraventricular gradients despite provocation (n = 9). During rest, the percentage of the total systolic ejection period during which forward aortic flow existed was as follows (mean +/- 1 SD): group I, 69 +/- 17% (flow), 64 +/- 6% (angio); group II, 63 +/- 14% (flow), 65 +/- 6% (angio); group III, 61 +/- 16% (flow), 62 +/- 4% (angio); control group, 90 +/- 5% (flow), 86 +/- 9% (angio). No significant difference was observed between any of the HCM subgroups, but compared with the control group, ejection was completed much earlier in systole independent of the presence or absence of intraventricular gradients. These results suggest that "outflow obstruction," as traditionally defined by the presence of an abnormal intraventricular pressure gradient and systolic anterior motion of the mitral valve, does not impede left ventricular outflow in HCM.

Detection of coronary artery disease in asymptomatic aircrew members with thallium-201 scintigraphy.

Thallium-201 exercise myocardial perfusion scintigraphy was accomplished in 130 aircrew members prior to their undergoing coronary angiography. Most were undergoing cardiac catheterization for an abnormal exercise response to treadmill testing. Of these, 22 men had arteriographic evidence of obstructive coronary disease of at least 50% narrowing in a single vessel. All had abnormal myocardial scintigrams. There were 12 other aviators who had minimal degrees of coronary artery disease with lesions less than 50% as the maximum degree of obstruction. Of these, 8 had abnormal thallium scans showing a perfusion defect in the area of the myocardium, presumably supplied by the diseased coronary artery. Of the 96 men with normal angiograms, only 4 had abnormal myocardial scintigraphy. An abnormal myocardial scintigram was often associated with significant obstructive disease. A normal scan accurately ruled out the presence of high-grade obstructive lesions and missed only four cases of minimal coronary disease. The application of gated thallium myocardial perfusion scans in the practice of aerospace cardiology has important significant applications for followup of therapeutic modalities as well as screening for evidence of myocardial ischemia in apparently healthy aircrew members.

Traumatic right coronary artery-right ventricular fistula with retained intramyocardial bullet.

A case of traumatic right coronary artery-right ventricular fistula secondary to a gunshot wound is presented. In addition, the bullet was retained within the interventricular septum. The diagnostic approach, surgical findings and operative procedure of this and other reported cases are discussed. Several key points are emphasized. First, extended follow-up is necessary after trauma to the heart since fistulas may develop years after the initial injury. Second, surgery is generally indicated for fistulas although some data are presented suggesting that small to moderate fistulas may be treated medically. Third, if surgery is undertaken, very careful operative technique must be utilized to locate and close the fistula. Surgical treatment of choice may be coronary arterial ligation with a distal bypass graft if necessary. Postoperative evaluation is mandatory because fistulas may recur. Indications for removal of a foreign body within the myocardium are also discussed.