Transmission of Fusarium oxysporum f. sp. fragariae Through Stolons in Strawberry Plants.

Fusarium wilt of strawberry, caused by the soilborne pathogen Fusarium oxysporum f. sp. fragariae, is a growing threat to the strawberry industry worldwide. Symptoms of the disease typically include stunting, wilting, crown discoloration, and eventual plant death. When Fusarium wilt was discovered in California, the disease was not known to occur anywhere else in North America. Long distance movement of the pathogen would most likely occur through transport of infected plants, which seems plausible if strawberry plants can sustain infections without showing symptoms of disease. The results of this study document that F. oxysporum f. sp. fragariae can move through stolons of infected mother plants and colonize first-generation daughter plants. The pathogen can also move through stolons from first to second-generation daughter plants. Daughter plants of both generations were always symptomless. The pathogen was recovered from both roots and petioles of infected daughter plants. Similar results were obtained for two cultivars known to be susceptible to Fusarium wilt, Albion and Monterey. Transmission through stolons from mother to daughter plants also occurred in the resistant cultivar, San Andreas, but less frequently than in Albion and Monterey.

The synthesis of sialylated oligosaccharides using a CMP-Neu5Ac synthetase/sialyltransferase fusion.

Large-scale enzymatic synthesis of oligosaccharides, which contain terminal N-acetyl-neuraminic acid residues requires large amounts of the sialyltransferase and the corresponding sugar-nucleotide synthetase, which is required for the synthesis of the sugar-nucleotide donor, CMP-Neu5Ac. Using genes cloned from Neisseria meningitidis, we constructed a fusion protein that has both CMP-Neu5Ac synthetase and alpha-2,3-sialyltransferase activities. The fusion protein was produced in high yields (over 1200 U/L, measured using an alpha-2,3-sialyltransferase assay) in Escherichia coli and functionally pure enzyme could be obtained using a simple protocol. In small-scale enzymatic syntheses, the fusion protein could sialylate various oligosaccharide acceptors (branched and linear) with N-acetyl-neuraminic acid as well as N-glycolyl- and N-propionyl-neuraminic acid in high conversion yield. The fusion protein was also used to produce alpha-2,3-sialyllactose at the 100 g scale using a sugar nucleotide cycle reaction, starting from lactose, sialic acid, phosphoenolpyruvate, and catalytic amounts of ATP and CMP.

Pulmonary/systemic flow ratio in children after cavopulmonary anastomosis.

OBJECTIVES: This study attempted to provide a formula for calculation of the pulmonary/systemic flow ratio in children after bidirectional cavopulmonary anastomosis. BACKGROUND: With the bidirectional cavopulmonary anastomosis, only the superior vena cava blood is oxygenated by the lungs. The inferior vena cava flow recirculates into the systemic circulation. The ratio of these flows will determine systemic arterial saturation. METHODS: According to the Fick principle, 1) Systemic cardiac output (liters/min) = Pulmonary venous flow + Inferior vena cava flow; 2) Systemic blood oxygen transport (ml/min) = Pulmonary venous blood oxygen transport + Inferior vena cava blood oxygen transport. By substituting the first equation into the second, Pulmonary/systemic flow ratio = (Systemic saturation - Inferior vena cava saturation)/(Pulmonary venous saturation - Inferior vena cava saturation). RESULTS: We applied the third formula to data obtained from 34 catheterizations in 29 patients after bidirectional cavopulmonary anastomosis. Mean [+/- SD] age at operation was 1.70 +/- 1.43 years, and mean age at catheterization was 2.95 +/- 1.65 years. The pulmonary/systemic flow ratio calculated for all 29 patients was 0.58 +/- 0.09. Of 17 patients with aortography, 10 had systemic to pulmonary collateral vessels. Patients with collateral vessels had a significantly higher pulmonary/systemic flow ratio (0.61 +/- 0.07 vs. 0.53 +/- 0.07, respectively, p < 0.02) and systemic saturation (88 +/- 4% vs. 82 +/- 4%, respectively, p < 0.002) than those without collateral vessels. The pulmonary/systemic flow ratio in those patients with no collateral vessels was similar to the previously reported echocardiographically derived superior vena cava/systemic flow ratio in normal children. CONCLUSIONS: The pulmonary/systemic flow ratio after bidirectional cavopulmonary anastomosis can be calculated. Pulmonary blood flow in these patients determines systemic saturation and accounts for the majority of venous return in young children.

Repair of anomalous origin of the left coronary artery in the infant and small child.

Anomalous origin of the left coronary artery from the pulmonary artery is associated with myocardial infarction, left ventricular dysfunction, mitral valve dysfunction and, occasionally, intracardiac congenital abnormalities. A technique that utilizes a flap of the anterior wall of the pulmonary artery to serve as a neocoronary artery to direct aortic flow from a created aortopulmonary window to the pulmonary artery orifice of the anomalous left coronary artery was used in five patients aged 2.5 months to 4.75 years. Two patients were less than 4 months of age at operation. There was one death 2 days after operation and one late death. The two youngest patients required mitral valve replacement. Two of the three surviving patients are well at follow-up at 7 to 44 months. One patient has been lost to follow-up study. One patient had postoperative catheterization which showed an intact repair. The pulmonary artery neocoronary procedure is applicable to infants and small patients with anomalous origin of the left coronary artery from the pulmonary artery.

Low molecular weight antigen arrays delete high affinity memory B cells without affecting specific T-cell help.

An ongoing, T-cell dependent, secondary antibody response to an epitope can be suppressed in vivo by low molecular weight, soluble polymers, bearing multiple copies of the same epitope. This study illustrates that such suppressive T-cell independent antigen arrays target the epitope-specific, high affinity, memory B cells for long-term functional elimination. Splenocytes from hyperimmune unsuppressed donors, when adoptively transferred into irradiated recipients will readily reconstitute a secondary anti-hapten response after antigenic challenge. No such response was observed with splenocytes transferred from hyperimmune donors suppressed with antigen arrays. The extent of suppression depended on antigen array dose and duration of exposure in the donor animals. The suppressive antigen array carryover from the donors into the recipients was negligible and insufficient to account for the observed suppression. B cells from hyperimmune mice producing high affinity anti-fluorescein antibodies, generated by multiple fluoresceinated ovalbumin (FL-OVA) injections, were helped efficiently by T cells from hyperimmune donors, which were either unsuppressed or suppressed with antigen arrays. Accordingly, help from T cells, specific for the carrier protein remains intact after such suppression. Neither lipopolysaccharide (LPS), nor additional transferred carrier-primed T cells could reverse the unresponsiveness of adoptively transferred splenocytes from suppressed animals. Flow cytometry showed that the number of hapten-specific B cells was markedly reduced after suppression. Collectively, these data show that the long term elimination of an ongoing T-cell dependent antibody response by suppressive exogenous antigen arrays is due to the functional deletion of high affinity, antigen-specific B cells, even in the presence of adequate T-cell help. The long-term nature of such functional deletion strongly suggests physical deletion of the antigen-specific B cell population.

Molecular basis for the lack of mimicry of Brucella polysaccharide antigens by Ab2gamma antibodies.

The crystal structure of the complex of an anti-Id Fab with an Fab specific for a Brucella polysaccharide antigen has previously been reported (Evans et al., 1994, J. Mol. Biol. 241, 691-705). To complement this study, the binding characteristics and immunological properties of this Ab2 and two others raised with a second anti-Brucella antibody were investigated, including quantitative kinetic measurements by surface plasmon resonance. The affinities of the Fabs from the Ab2s for the Ab1s were three orders of magnitude greater than those estimated for the antigen, but the Ab2s failed to induce antigen-binding Ab3s, that is, they were of the Ab2gamma type. The avidities of the Ab1s for antigen were however within one order of magnitude of their avidities for Ab2. Tests of 16 other anti-Brucella polysaccharide antibodies showed that the two idiotopes were not present in them, and in confirmation of the lack of a dominant idiotope, N-terminal sequencing of their H and L chains showed a wide variety of V genes were employed in the immune response to the Brucella polysaccharides. The failure of the Ab2 to induce antigen-reactive Ab3 thus appears to be due to neither intrinsic affinity nor idiotope frequency, but arises instead from structural reasons, for example, the incomplete penetration of the Ab2 into the binding-site cleft of the Ab1. The surface topography of polysaccharide antigens and their binding-sites thus appears to be especially difficult for Ab2s to mimic and will restrict their routine use as surrogates for T-cell independent polysaccharide antigens.

The amino acid sequences of jacalin and the Maclura pomifera agglutinin.

Amino acid sequences for the alpha-chains of the Moraceae lectins, jacalin and Maclura pomifera agglutinin, were determined by protein sequencing. Both are 133 residues long and contain several genetically variant positions; the overall homology is 85%. A possible site for the known glycopeptide of jacalin was located. The alpha-chains have a conserved tryptophan residue that may be part of the binding-site.

Post-translational processing of two alpha-amylase inhibitors and an arcelin from the common bean, Phaseolus vulgaris.

Mass spectrometric methods were used to investigate the proteolytic processing and glycopeptide structures of three seed defensive proteins from Phaseolus vulgaris. The proteins were the alpha-amylase inhibitors alphaAI-1 and alphaAI-2 and arcelin-5, all of which are related to the seed lectins, PHA-E and PHA-L. The mass data showed that the proteolytic cleavage required for activation of the amylase inhibitors is followed by loss of the terminal Asn residue in alphaAI-1, and in all three proteins, seven or more residues were clipped from the C-termini, in the manner of the seed lectins. In most instances, individual glycoforms could be assigned at each Asn site, due to the unique masses of the plant glycopeptides. It was found that alphaAI-1 and alphaAI-2 differed significantly in their glycosylation patterns, despite their high sequence homology. These data complement the previous X-ray studies of the alpha1-amylase inhibitor and arcelin, where many of the C-terminal residues and glycopeptide residues could not be observed.

Amino acid sequence and thermostability of xylanase A from Schizophyllum commune.

The amino acid sequence (197 residues) of xylanase A from the fungus, Schizophyllum commune, was determined by automated analysis of peptides from proteolytic and acid cleavage. The sequence is similar to two Trichoderma xylanases (approximately 56% identical amino acids), but also shows at least 40% identities with xylanases from Bacillus subtilis, B. pumilus and B. circulans. The conserved regions of the enzyme contain only two glutamic acid residues which implicates their possible involvement in catalysis. The disulfide bond in xylanase A is not conserved in this family. In spite of this, the B. subtilis xylanase was found to be more thermostable than xylanase A.

Sequential orthotopic heart transplantation in man.

Between January 1968 and March 1980, 202 hearts had been transplanted into 185 patients at Stanford University Medical Center. Occasionally, patients after transplantation develop myocardial failure which is amenable only to retransplantation. Sixteen patients underwent initial orthotopic allograft using standard techniques. Eight patients developed accelerated arteriosclerotic coronary disease, six had unrelenting rejection, and two had donor heart dysrrhythmia or right ventricular failure requiring retransplantation. One patient required a third transplant because of donor left ventricular ischemia. All sequential transplants were managed similarly to the primary transplant. Of the initial transplant hearts at risk, 60% survived for more than 1 year, and 57% survived for more than 2 years. These results are similar to those of patients not requiring retransplantation. Of the secondary transplant hearts at risk, 31% survived for more than 1 year and 29% survived for more than 2 years. The severity of infection and/or rejection contributed most significantly to secondary heart transplant mortality. Sequential orthotopic cardiac transplantation offers an acceptable alternative to patients with allograft failure. Survival is not as favorable as with initial transplantation because of the prolonged immunosuppression during sequential transplantation.

Deconvolution analysis in radionuclide quantitation of left-to-right cardiac shunts.

A poor bolus injection results in an unsatisfactory quantitative radionuclide angiocardiogram in as many as 20% of children with possible, left-to-right (L-R) cardiac shunts. Deconvolution analysis was applied to similar studies in experimental animals to determine whether dependence on the input bolus could be minimized. Repeated good-bolus, prolonged (greater than 2.5 sec), or multiple-peak injections were made in four normal dogs and seven dogs with surgically created atrial septal defects (ASD). QP/QS was determined using the gamma function. The mean QP/QS from ten good-bolus studies in each animal was used as the standard for comparison. In five trials in normal animals, where a prolonged or double-peak bolus led to a shunt calculation (QP/QS greater than 1.2 : 1), deconvolution resulted in QP/QS = 1.0. Deconvolution improved shunt quantitation in eight of ten trials in animals that received a prolonged bolus. The correlation between the reference QP/QS and the QP/QS calculated from uncorrected bad bolus studies was only 0.39 (p greater than 0.20). After deconvolution using a low pass filter, the correlation improved significantly (r = 0.77, p less than 0.01). The technique gave inconsistent results with multiple-peak bolus injections. Deconvolution analysis in these studies is useful in preventing normals from being classified as shunts, and in improving shunt quantitation after a prolonged bolus. Clinical testing of this technique in children with suspected L-R shunts seems warranted.

Anticoagulation therapy in children with mechanical prosthetic cardiac valves.

From 1980 through 1984, 28 children younger than 19 years (mean 7.9) underwent cardiac valve replacement with 30 mechanical prostheses. Patients were followed for a total of 471 months (mean 15.7) and received either warfarin (mean 0.16 mg/kg/day) or acetylsalicylic acid and dipyridamole (mean 6.1 and 1.9 mg/kg/day, respectively) as thromboembolism prophylaxis. The frequency and incidence of thromboembolism and hemorrhage were compared. Warfarin-treated patients were at increased risk of hemorrhage (5 of 20 [25%], or 22 per 100 patient-years, vs 0 of 10 [0%], or 0 per 100 patient-years, p less than 0.05). Three of the 5 hemorrhagic episodes were mild, and in no case was hemorrhage life-threatening. Patients who did not receive warfarin had a greater risk of thromboembolism (2 of 10 [20%], or 12 per 100 patient-years, vs 0 of 20 [0%], or 0 per 100 patient-years, p less than 0.05). Both episodes of thromboembolism were life-threatening and necessitated emergency valve replacement. Although warfarin is associated with greater risk of hemorrhage than is acetylsalicylic acid and dipyridamole, warfarin is better than antiplatelet drugs in thromboembolism prophylaxis and is indicated for anticoagulation therapy in children with mechanical cardiac prostheses.

Morphology of canine hearts after 24 hours' preservation and orthotopic transplantation.

Ten dogs underwent orthotopic cardiac transplantation after preservation of the donor heart for 24 hours in an oxygenated hypothermic, hypertonic, intracellular solution, either with (five dogs) or without (five dogs) continuous, oxygenated, low-pressure perfusion. Eight dogs survived for 24 hours after transplantation, at which time they were put to death. The two nonsurvivors were among the five with nonperfused hearts. Examination of all 10 donor hearts showed differences between the two groups: Four of five nonperfused hearts showed severe transmural myocardial coagulation necrosis but only small foci of contraction band necrosis (myofibrillar degeneration). The perfused hearts, however, showed more extensive subendocardial areas of contraction-band necrosis, but only minimal and focal coagulation necrosis, indicating less severe hypoxic damage. These results indicate that oxygenated perfusion with a hypothermic, hypertonic, intracellular solution may permit improved transplant survival after extended cardiac preservation.

Costs and results of cardiac operations in infants less than 4 months old. Are they worthwhile?

From 1979 through 1983, 328 of 1,388 pediatric cardiac operations involved patients undergoing their first procedure at less than 4 months of age. Of these, 220 patients had 265 nonductal procedures, and their case histories are reviewed for results and total hospital cost. Initial operative mortality was 20% (43 patients). Infants with lower operative age and operative weight tended to have closed procedures. Mortality and cure were not related to gestational age, birth weight, age at operation, number of operations, or type of operation. Lower operative weight was associated with a greater mortality. Evaluated survivors (142 patients) were followed for a mean of 24 months. Fifteen percent (33 patients) died during follow-up. Of survivors, 80% (114 patients) had optimized general health; a subset of 29% had normal cardiac function, and 17% were cured. Lower birth weight was associated with curable lesions and normalcy (p less than 0.04). Longer preoperative hospital stay and lower weight at operation were associated with higher hospital cost (p less than 0.05). Hospital cost was not related to type of operation, gestational age, birth weight, age at operation, mortality, cure, or normalcy. Acquired neurologic dysfunction and long-term disability were uncommon. The mean hospital cost for surviving infants was +80,000 (1984 dollars). Effective hospital cost per survivor was +110,000. Mortality, cure, and normal function after cardiac operations in infants less than 4 months of age were not related to gestational age, birth weight, or age at operation. Mortality was higher in patients with a lower weight at operation. Separation into distinct fiscal cost groups is not reasonable in this series. Because most survivors are in normal or optimized cardiac health, intensive cardiovascular care in this population is justified.

Translocation of the aortic valve for prosthetic valve endocarditis.

Aortic prosthetic valve endocarditis is frequently associated with a perivalvular ring abscess which destroys the normal annulus, so that it is difficult to seat a new prosthesis. Between November, 1974, and January, 1980, we treated four patients with aortic prosthetic endocarditis by translocation of the aortic valve, closure of the native coronary artery ostia, and placement of saphenous vein bypass grafts to the coronary arteries. In each case operation was undertaken because of progressive congestive heart failure resulting from aortic regurgitation; two patients had systemic emboli, and two patients had uncontrolled infection. Infection was due to Enterococcus in three instances and to an unknown organism in one. Total ischemic times averaged 2 hours, 15 minutes; a 25 mm Dacron graft containing a porcine valve was used to replace the ascending aorta and aortic valve, and two or three saphenous vein grafts were placed to distal coronary arteries. One patient died 40 days postoperatively of renal failure and Pseudomonas pneumonia with an intact repair. The other three patients were hospital survivors, with one doing well until dying of chronic active hepatitis 12 months postoperatively. The other two patients are alive at 4 months and 18 months with satisfactory hemodynamic function and are free of infection. Translocation of the aortic valve for prosthetic valve endocarditis is a useful alternative when conventional replacement techniques cannot be utilized.

Does open lung biopsy affect treatment in patients with diffuse pulmonary infiltrates?

The decision to perform open lung biopsy in the evaluation of a diffuse pulmonary infiltrate is based on the probability that this examination will yield specific information that may lead to a change in treatment. The role of this procedure remains controversial and many clinicians are reluctant to allow this invasive procedure without assurances that results will lead to a change in therapy for a significant number. To evaluate the impact of open lung biopsy on diagnosis and treatment of diffuse pulmonary infiltrates, we conducted a retrospective review of 61 patients undergoing this procedure at three university-affiliated hospitals during a recent 7-year period. There were 37 men and 24 women; average age was 57 years. Biopsy yielded a specific diagnosis in 21 (34%) patients and a change in therapy in 33 (54%) patients. A complication developed in 11 (18%) patients, directly related to the biopsy procedure in six (10%). Eight patients died. The immune status in 22 (36%) patients was compromised. A specific diagnosis was obtained in 13 (59%) immunocompromised patients and a change in therapy occurred in 17 (77%) of these patients after biopsy. A specific diagnosis was obtained in only eight (21%) of the 39 noncompromised patients and therapy was changed in 16 (41%) patients in this group (p less than 0.02 compromised versus noncompromised). Morbidity and mortality were not significantly different between the two groups. A nonspecific diagnosis led to a change in therapy as frequently as a specific diagnosis in both compromised and noncompromised groups. Open lung biopsy in the patient with a diffuse pulmonary infiltrate is an accurate diagnostic tool and frequently leads to a change in patient treatment. The procedure can be performed with acceptable morbidity and mortality in immunocompromised and noncompromised patients.

Early failures of Ionescu-Shiley bioprosthesis after mitral valve replacement in children.

Two infants, 101/2 and 11 1/2 months of age, underwent mitral valve replacement with Ionescu-Shiley bovine bioprosthesis for congenital cardiac defects. Both patients had early valve failure, 19 months and 4 months after implantation, due to the growth of excessive collagen on the ventricular surface of the bioprosthesis with adherence of the collagen primarily to the sewing rings and struts of the valves. The cause of this excessive collagen reaction is unknown. Further study is needed to document the true incidence of this problem.

Efficacy of and adherence to highly active antiretroviral therapy in children infected with human immunodeficiency virus type 1.

BACKGROUND: Clinical trials in adults have demonstrated the efficacy of highly active antiretroviral therapy (HAART) to suppress replication of HIV-1 to nondetectable levels, but lower success rates have been observed in practice. We sought to determine the efficacy of HAART in our population of HIV-1-infected children and to identify determinants of efficacy, especially the role of adherence to prescribed antiretrovirals. METHODS: The viral load and CD4+ T cell responses of 72 children with perinatally acquired HIV-1 treated with HAART including a protease inhibitor for at least 90 days were examined retrospectively in relation to adherence, as measured by pharmacy records for the first 180 days of HAART. RESULTS: Patients were defined as adherent if > or =75% of protease inhibitors and > or =75% of all antiretroviral prescriptions were filled. Of the 42 patients (58%) who were adherent, nondetectable viral loads were achieved and maintained in 22 (52%). A Kaplan-Meier plot showed a drop-off in patients maintaining a nondetectable viral load after 200 days. Higher initial viral load was the only pretreatment factor that identified adherent patients at risk for treatment failure. Only 3 (10%) nonadherent patients maintained a viral load of <400 copies/ml. The adherent group had a prompt and sustained increase in CD4+ T cell counts. CONCLUSIONS: HAART can achieve control of viral replication in HIV-1-infected children who adhere to therapy. However, treatment failure is likely unless there is a high level of adherence. Nonadherence to therapy is common and might be the major impediment to successful treatment of children infected with HIV-1.

Distant heart procurement for transplantation.

Between January 1, 1977, and September 15, 1978, 39 cardiac transplants were performed on 38 patients. Twenty donor hearts were removed at Stanford University Hospital, and 19 donor hearts were removed at distant hospitals. The characteristics of recipients and donors in both groups were similar. The only significant difference between donor hearts was the mean ischemia time (154 +/- 30 minutes in distant hearts and 52 +/- 12 minutes in local hearts, P less than 0.001). As of February 1, 1979, the total mortality rate was 32% for the distant heart donors and 40% for the local heart donors. No difference between the two groups was present in immediate myocardial function, the need for postoperative inotropic support, the mortality rate within the first 90 days after operation, the mean maximum serum enzyme levels, the occurrence of rejection or infection, and the histological appearance of the hearts, both early and late. The results of cardiac transplantation when hearts are removed at distant hospitals are entirely comparable to the results with hearts removed on site with a minimum ishchemic time. Distant heart procurement provides an expanded donor pool for potential cardiac recipients.

Construction of an aortic homograft conduit for right ventricle to pulmonary artery continuity.

Over the past decade the aortic homograft has become the extracardiac conduit of choice for repair of many congenital cardiac defects. Anastomosis of the homograft annulus to the right ventricle may distort the valve and render it incompetent. Prosthetic tube grafts have been used to bridge the right ventricle and distal homograft. We propose and have use successfully a method for establishing right ventricle to pulmonary artery continuity that is free of prosthetic materials, is technically simple, and places the homograft valve in the hilum, away from the distorting right ventricle free wall.