Retroviral display of antibody fragments; interdomain spacing strongly influences vector infectivity

Five different single-chain antibody fragments (scFv) against human cell-surface antigens were displayed on murine ecotropic retroviral vectors by fusing them to the Moloney SU envelope glycoprotein. The spacing between the scFv and the SU glycoprotein was varied by fusing the scFv to residue +7 or to residue +1 of Moloney SU and by inserting linker sequences of different lengths between the domains. All of the chimeric envelopes were efficiently incorporated into vector particles and could bind to human cells through their displayed antibody fragments, but did not infect them. The spacing between the scFvs and the SU glycoproteins had no significant effect on the efficiency of envelope expression or viral incorporation and did not affect the binding properties of the chimeric envelopes, nor did it influence the efficiency of targeted gene delivery to human cells by scFv-displaying vectors. However, on murine fibroblasts the infectivity of vectors incorporating the chimeric envelopes was strongly influenced by the length of the interdomain spacer. The titers were very low when the single-chain antibodies were fused through a tripeptide linker to SU residue +7 and were greatly enhanced (up to 10(5)-fold) when they were fused to SU residue +1 through a heptapeptide linker. These results point to the importance of steric interactions between the domains of chimeric envelope glycoproteins and may have implications for retroviral vector design for human gene therapy.     

There's more to the picture than meets the eye: young children's difficulty understanding biased interpretation

5 experiments investigated children's understanding that expectations based on prior experience may influence a person's interpretation of ambiguous visual information. In Experiment 1, 4- and 5-year-olds were asked to infer a puppet's interpretation of a small, ambiguous portion of a line drawing after the puppet had been led to have an erroneous expectation about the drawing's identity. Children of both ages failed to ascribe to the puppet an interpretation consistent with the puppet's expectation. Instead, children attributed complete knowledge of the drawing to the puppet. In Experiment 2, the task was modified to reduce memory demands, but 4- and 5-year-olds continued to overlook the puppet's prior expectations when asked to infer the puppet's interpretation of an ambiguous scene. 6-year-olds responded correctly. In Experiment 3, 4- and 5-year-olds correctly reported that an observer who saw a restricted view would not know what was in the drawing, but children did not realize that the observer's interpretation might be mistaken. Experiments 4 and 5 explored the possibility that children's errors reflect difficulty inhibiting their own knowledge when responding. The results are taken as evidence that understanding of interpretation begins at approximately age 6 years.     

Decreased baroreflex sensitivity in patients with stable coronary artery disease is correlated with the severity of coronary narrowing

Although studies have shown that arterial baroreflex sensitivity (BRS) is decreased in patients with acute myocardial infarction, BRS changes in patients with stable coronary artery disease (CAD) have not been studied extensively. We assessed BRS by the phenylephrine method in 55 normotensive and nondiabetic patients with chronic effort angina, old myocardial infarction, or both. The control group consisted of 24 age-matched patients without coronary lesions. To identify factors that determine BRS in stable CAD, we performed multivariate analysis using age, sex, left ventricular ejection fraction, pulmonary artery wedge pressure, resting systolic blood pressure, resting heart rate, the number of stenotic coronary arteries, history of myocardial infarction, and the presence or absence of angina pectoris as variables. BRS was significantly lower in patients with CAD than in control subjects (5.9 +/- 2.9 vs 6.9 +/- 2.4 ms/mm Hg, p < 0.05). In patients with CAD, BRS was inversely correlated with age, the resting heart rate, and the number of stenotic coronary vessels (p < 0.001, p < 0.005, and p < 0.005, respectively), but was independent of other clinical parameters, including the history of myocardial infarction. In control subjects, BRS was significantly correlated only with age. These results indicate that BRS is decreased in patients with stable CAD, and this decrease is correlated with the extent and severity of coronary narrowing.     

Genetic variants of human T-lymphotrophic virus type II in American Indian groups

The human T-lymphotropic virus type II (HTLV-II) is found in many New World Indian groups in North and South America and may have entered the New World from Asia with the earliest migration of ancestral Amerindians over 15,000 years ago. To characterize the phylogenetic relationships of HTLV-II strains infecting geographically diverse Indian populations, we used polymerase chain reaction to amplify HTLV-II sequences from lymphocytes of seropositive Amerindians from Brazil (Kraho, Kayapo, and Kaxuyana), Panama (Guaymi), and the United States (the Navajo and Pueblo tribes of the southwestern states and the Seminoles of Florida). Sequence analysis of a 780-base pair fragment (located between the env gene and the second exons of tax/rex) revealed that Amerindian viruses clustered in the same two genetic subtypes (IIa and IIb) previously identified for viruses from intravenous drug users. Most infected North and Central American Indians had subtype IIb, while HTLV-II infected members of three remote Amazonian tribes clustered as a distinct group within subtype IIa. These findings suggest that the ancestral Amerindians migrating to the New World brought at least two genetic subtypes, IIa and IIb. Because HTLV-II strains from Amazonian Indians form a distinct group within subtype HTLV-IIa, these Brazilian tribes are unlikely to be the source of IIa viruses in North American drug users. Finally, the near identity of viral sequences from geographically diverse populations indicate that HTLV-II is a very ancient virus of man.     

Low glutathione and high iron govern the susceptibility of oligodendroglial precursors to oxidative stress

We have previously shown, using qualitative approaches, that oligodendroglial precursors are more readily damaged by free radicals than are astrocytes. In the present investigation we quantified the oxidative stress experienced by the cells using oxidation of dichlorofluorescin diacetate to dichlorofluorescein as a measure of oxidative stress; furthermore, we have delineated the physiological bases of the difference in susceptibility to oxidative stress found between oligodendroglial precursors and astrocytes. We demonstrate that (a) oligodendroglial precursors under normal culture conditions are under six times as much oxidative stress as astrocytes, (b) oxidative stress experienced by oligodendroglial precursors increases sixfold when exposed to 140 mW/m2 of blue light, whereas astrocytic oxidative stress only doubles, (c) astrocytes have a three times higher concentration of GSH than oligodendroglial precursors, (d) oligodendroglial precursors have > 20 times higher iron content than do astrocytes, and (e) oxidative stress in oligodendroglial precursors can be prevented either by chelating intracellular free iron or by raising intracellular GSH levels to astrocytic values. We conclude that GSH plays a central role in preventing free radical-mediated damage in glia.     

CHESS: comprehensive health enhancement support system for women with breast cancer

The Comprehensive Health Enhancement Support System (CHESS) was developed to assist people dealing with health crises. Needs assessments with patients were conducted in its development and validation studies performed. CHESS provides information, social support, and decision-making assistance via a personal computer and modem that are placed in patients' homes. Women of all ages and varied socioeconomic backgrounds have successfully used this program to empower them to become active participants in their care following a diagnosis of breast cancer.     

Mechanism for improved cardiac performance with arteriolar dilators in aortic insufficiency

To determine how arteriolar dilation improves cardiac performance in aortic insufficiency, we evaluated the acute effects of hydralazine in 10 patients with chronic severe aortic insufficiency. Control measurements of intracardiac and intravascular pressures, cardiac output and left ventricular volumes were obtained at cardiac catheterization. Hydralazine, 0.3 mg/kg i.v. (maximal dose 20 mg), was administered and all measurements were repeated 30 minutes later. A reduction in systemic vascular resistance from 1264 to 710 dyn-sec-cm-5 was associated with significant increases in forward cardiac index (2.9 to 5.1 l/min/m2) and stroke volume index (37 to 55 ml/m2). Left ventricular end-diastolic pressure was reduced from 19 to 12 mm Hg. There was a significant reduction in mean arterial pressure (88 to 83 mm Hg) and a significant increase in heart rate (81 to 94 beats/min). Regurgitant stroke volume was reduced by more than 10 ml/m2 in seven patients and for the group was significantly reduced, from 65 to 53 ml/m2. Regurgitant fraction was reduced in all patients; the overall reduction from 0.64 to 0.48 was highly significant. Ejection fraction increased more than 0.10 in four patients, by 0.08 in an additional patient and for the group increased significantly from 0.50 to 0.57. Left ventricular end-diastolic volume decreased by more than 25 ml/m2 in four patients, by 19 ml/m2 in an additional patient and was decreased significantly, from 208 to 190 ml/m2, for the group. Arteriolar dilators improve cardiac performance in aortic insufficiency by reducing the amount of aortic regurgitation and, in some patients, by substantially improving systolic pump fraction. These data suggest a role for arteriolar dilators in the management of selected patients with aortic insufficiency.     

Cancer of the bladder in spinal cord injury patients

Since 1963, 10 cases of bladder carcinoma have been detected in 1,052 new admissions to our center. A high percentage of these patients had squamous cell carcinoma and/or squamous elements. This relatively high incidence stimulated a prospective study of 81 spinal cord injury patients either maintained on intraurethral catheter drainage for 10 years or an external appliance for 15 years. There were changes of squamous metaplasia in 19 per cent of the cases but no cancer was detected. It remains undetermined if squamous metaplasia is a pre-malignant lesion. However, the incidence of squamous metaplasia and squamous cell carcinoma in paraplegics with chronically infected bladders is not uncommon. Any spinal cord injury patient with hematuria needs a complete bladder evaluation and any long-term paraplegic with chronic infection should undergo cystoscopy and Papanicolaou smears as part of the yearly checkup.     

A confocal microscopic evaluation of the effects of insulin imprinting on the binding of Concanavalin A by Tetrahymena Pyriformis

With confocal microscopy it is possible to study the Concanavalin A (Con A) binding characteristics of the surface and interior of a single cell by viewing optical sections. It was observed in Tetrahymena pyriformis that Con A bound both to the plasma membrane and to intracellular structures. Incubation of cells with a competing sugar a-methylmannopyranoside, decreased binding. Hormonal imprinting with insulin resulted in an increase in binding of Con A to the cell surface and a decrease in intracellular binding. It is possible that the intracellular binding sites may migrate to the plasma membrane.