Automated parallel DNA sequencing on multiple channel microchips.

We report automated DNA sequencing in 16-channel microchips. A microchip prefilled with sieving matrix is aligned on a heating plate affixed to a movable platform. Samples are loaded into sample reservoirs by using an eight-tip pipetting device, and the chip is docked with an array of electrodes in the focal plane of a four-color scanning detection system. Under computer control, high voltage is applied to the appropriate reservoirs in a programmed sequence that injects and separates the DNA samples. An integrated four-color confocal fluorescent detector automatically scans all 16 channels. The system routinely yields more than 450 bases in 15 min in all 16 channels. In the best case using an automated base-calling program, 543 bases have been called at an accuracy of >99%. Separations, including automated chip loading and sample injection, normally are completed in less than 18 min. The advantages of DNA sequencing on capillary electrophoresis chips include uniform signal intensity and tolerance of high DNA template concentration. To understand the fundamentals of these unique features we developed a theoretical treatment of cross-channel chip injection that we call the differential concentration effect. We present experimental evidence consistent with the predictions of the theory.

Healing response associated with balloon-dilated ePTFE.

Deployment of endovascular grafts composed of a metallic stent surrounded by expanded polytetrafluoroethylene (ePTFE) stretches the polymer beyond its original dimensions, altering the structural characteristics of the ePTFE. We hypothesized this structural modification would alter the healing response associated with the implant. In this study, 4 mm i.d. of ePTFE (30 microns internodal distance) vascular grafts were balloon dilated using angioplasty balloons having final diameters of 6 (1.5X), 8 (2X), 10 (2.5X), 12 (3X), and 18 (4.5X) mm. Following balloon dilatation of the ePTFE, a circular punch (6 mm in diameter) was used to prepare polymer samples for implantation. The ePTFE circular patches were implanted within subcutaneous tissue and epididymal fat pads of male Sprague-Dawley rats. After 5 weeks, the implants were removed and analyzed for fibrous capsule formation, inflammation, and neovascularization associated with the material. Histological analysis revealed the formation of fibrous capsules only with control subcutaneous implants. The inflammatory response associated with subcutaneously implanted ePTFE was decreased significantly following balloon dilatation to at least 2.5 times the original diameter of the graft. In contrast, ePTFE implanted within adipose tissue demonstrated a significantly greater inflammatory response following balloon dilatation when compared to control implants. Only ePTFE balloons dilated to 6 mm and implanted within adipose tissue demonstrated neovascularization to any extent. These data suggest the structural modifications incurred by ePTFE following balloon dilatation dramatically affect the inflammatory response associated with an implant. Therefore, polymeric materials used for endovascular graft technology require designs that consider changes in polymer healing inherent to device design.

Stent versus endovascular graft healing characteristics in the porcine iliac artery.

PURPOSE: To evaluate the healing characteristics of stents versus endovascular grafts in the porcine iliac artery. MATERIALS AND METHODS: A total of 20 iliac arteries in 10 domestic swine were used to evaluate the healing characteristics of stents versus endovascular grafts. Each animal received one stent and one endovascular graft in opposite iliac arteries. The endovascular grafts were constructed with use of 6 cm of expanded polytetrafluoroethylene (ePTFE) (3 mm inner diameter, 30 microm internodal distance) and Palmaz stents (P204 or P188) secured at each end of the graft. A solitary Palmaz stent (P308 or P294) was used on the opposite side. The devices were explanted at 1, 5, and 12 weeks. RESULTS: One of three endovascular grafts and two of three stents were patent at 1 week. Two of three endovascular grafts and all three stents were patent at 5 weeks. All three endovascular grafts and stents were patent at 12 weeks. Gross examination, histologic, and scanning electron microscopy demonstrated differences in the healing response of the two devices. A marked abluminal inflammatory response to the graft material was observed. This resulted in neovascularization of the tissue along the abluminal surface of the graft. In addition, marked neointimal thickening at the unsupported section of the endovascular graft resulted in significant luminal narrowing. CONCLUSION: The porcine model may be used for evaluating the healing characteristics of endovascular grafts. Intravascular placement of ePTFE prosthetic graft material dramatically alters the healing of this type of graft material. The graft material did not prevent the formation of a progressively thickening neointima.

Effects of balloon dilatation on ePTFE structural characteristics.

The search for less invasive treatments for cardiovascular disease has lead to the development of endovascular stent grafts, metallic and alloy stents surrounded by prosthetic vascular graft material. Introduced intravascularly, the deployment of stent grafts requires balloon dilatation of the device which results in expansion of the stent along with the vascular graft material. We hypothesized that balloon dilatation of stent grafts would alter the physical structure of the prosthetic graft material. In this study, noncompliant angioplasty balloons were used to dilate expanded polytetrafluoroethylene (ePTFE), a material commonly used for endovascular stent-graft technology. The maximal outer diameter (inflated balloon within the lumen) and the recoiled outer diameter (balloon removed) of two types of ePTFE, 3-mm inside diameter (i.d.) thin wall (30-micron internodal distance) and 4-mm i.d. standard wall (30-micron internodal distance), were measured to compare material recoil. Following balloon dilatation, ePTFE samples were prepared for scanning electron microscopic examination and the following parameters were measured: wall thickness, internodal distance, nodal width, interfiber distance, and fiber width. Following primary dilatation, both types of ePTFE recoiled approximately 20% regardless of inflated balloon diameter. However, following eight repetitive balloon dilatations, recoil decreased to approximately 10%. Scanning electron microscopic analysis revealed variations in internodal distance and significant decreases in wall thickness, nodal thickness, and interfiber distance. Fiber width was significantly decreased following dilatation of 3 mm, but not 4 mm ePTFE. Our data support our initial hypothesis that balloon dilatation alters the structure of ePTFE.

Human chorionic gonadotrophin-beta gene sequences in women with disorders of HCG production.

Women with recurrent abortion, primary unexplained infertility, and gestational trophoblastic neoplasia (GTN) manifest disordered human chorionic gonadotrophin (HCG) secretion. Mutations in the HCG beta/luteinizing hormone (LH) beta gene complex could cause aberrant HCG production in these disorders. The purpose of this study was to determine whether HCG beta gene deletions occur in women with recurrent abortion or primary unexplained infertility, and whether HCG beta gene duplications are present in women with GTN. DNA was extracted from 10 patients with unexplained recurrent abortion, 10 patients with unexplained primary infertility, 12 patients with GTN, three partners of women with GTN, and 30 controls. Southern blots were constructed and hybridized with DNA probes for HCG beta-5 and the LH beta gene. No gene deletions were identified in patients with recurrent abortion or primary unexplained infertility. Likewise, no gene duplications were identified in women with GTN. A previously described Mbol restriction fragment length polymorphism (RFLP) was identified in both patients and controls. A new Pstl RFLP was also characterized, but was present in patients and controls. Deletion/duplication mutations in the HCG beta/LH beta gene complex do not appear to be common causes of aberrant HCG production in humans with these disorders.

Hypoechoic fat: a sonographic pitfall.

Fat has classically been described as hyperechoic on sonograms because of its acoustic impedance relative to surrounding tissue, although certain types of fat in certain anatomic locations can be hypoechoic. Examples in the literature include hypoechoic fat in and around the kidneys as well as in ovarian neoplasms [1-3]. We present several cases of hypoechoic fat collections in various anatomic locations to illustrate the importance of its presence and to minimize the number of erroneous diagnoses.

Identification of restriction-fragment length polymorphisms for the human chorionic gonadotropin-beta/luteinizing hormone-beta gene cluster.

OBJECTIVE: To determine whether restriction fragment length polymorphisms are present using a deoxyribonucleic acid (DNA) probe for human luteinizing hormone beta subunit (hLH-beta). If the gene for hLH-beta is polymorphic, genetic diagnosis of disorders of luteinizing hormone (hLH) and human chorionic gonadotropin (hCG) production could become possible. DESIGN: Study of genomic DNA from controls with a variety of restriction enzymes to identify polymorphisms. SETTING: Laboratories of the Department of Obstetrics and Gynecology, Department of Oral Biology, Medical College of Georgia, Augusta, Georgia. PATIENTS: Unrelated control men and women seen in clinics at the Medical College of Georgia. INTERVENTIONS: Genomic DNA was extracted from patients and digested with eight different restriction enzymes for the study of the hLH-beta gene by Southern analysis. MAIN OUTCOME MEASURE: Fragment (band) sizes on radiographs from Southern blots were compared with those from molecular weight standards. CONCLUSIONS: Restriction fragment length polymorphisms were identified for four of the restriction enzymes, DraI, HincII, MboI, and KpnI. These polymorphisms may be useful in the diagnosis of disorders of hLH and hCG production.

Sonoelasticity imaging: results in in vitro tissue specimens.

The authors present a method for imaging tissue stiffness (sonoelasticity) that has been developed and tested in a laboratory setting by using in vitro canine and human prostate glands. A low-frequency acoustic source was used to induce vibration in tissue under examination, and a color Doppler ultrasound (US) instrument was modified to detect vibration amplitude. The resulting image is a color "map" of tissue vibration superimposed on conventional gray-scale US images. Stiffer tissues vibrated less in response to audible sound, regardless of echogenicity. Normal human and canine prostate glands demonstrated a uniform vibration pattern. Four of four human prostatic adenocarcinomas and two stiff inclusions injected into canine prostate glands demonstrated a lack of vibration in comparison with normal surrounding tissue. The authors conclude that while further study is necessary, sonoelasticity imaging may enhance the detection of neoplasms by enabling their identification solely on the basis of stiffness.

The detection of Hind III restriction-fragment length polymorphisms using a deoxyribonucleic acid probe for the beta subunit of follicle-stimulating hormone.

Molecular diagnosis of disorders of follicle-stimulating hormone (FSH) production may become possible now that the gene for FSH beta has been characterized. Restriction-fragment length polymorphism (RFLP) analysis provides a means of organized search for molecular variants of FSH. The purpose of this study was to screen controls for the presence of RFLPs using the deoxyribonucleic acid (DNA) probe pFSH beta -1.4. Genomic DNA was digested with 12 different restriction endonucleases; Southern blots were constructed and hybridized to pFSH beta -1.4. No polymorphisms were identified with 11 enzymes. Three of 24 (12.6%) Hind III digests demonstrated a polymorphic fragment of either 5.2, 4.7, or 4.3 kb. These are the first RFLPs identified for the FSH beta gene with pFSH beta -1.4. RFLPs for FSH beta constitute the first step in the molecular analysis of disorders of FSH production.

Review of biotechnology applications to nuclear waste treatment.

This paper gives an overview of the feasibility of the application of biotechnology to nuclear waste treatment. The contents are based on a report which PA Technology carried out for the Department of the Environment (DoE Reference: DoE/RW/88.008 Sector No 2.3). Many living and dead organisms accumulate heavy metals and radionuclides. The controlled use of this phenomenon forms the basis for the application of biotechnology to the removal of radionuclides from nuclear waste streams. Indeed, biotechnology offers a series of new opportunities for removal of radionuclides from dilute aqueous process effluents. Such technology is already used for heavy metal removal on a commercial basis and could be optimised for radionuclide removal. An overview of biotechnology areas, namely the use of biopolymers and biosorption using biomass applicable to the removal of radionuclides from industrial nuclear effluents is given. The potential of biomagnetic separation technology, genetic engineering and monoclonal antibody technology is also to be examined. The most appropriate technologies to develop for radionuclide removal in the short term appear to be those based on biosorption of radionuclides by biomass and the use of modified and unmodified biopolymers in the medium term.

Interaction of ribulose bisphosphate carboxylase/oxygenase with 2-carboxyhexitol 1,6-bisphosphates.

2-C-Carboxy-D-glucitol 1,6-bisphosphate (CGBP) and 2-C-carboxy-D-mannitol 1,6-bisphosphate (CMBP) have been synthesized, isolated, and the structures of these compounds and the derived lactones elucidated by NMR spectroscopy and periodate oxidation. Both carboxyhexitol bisphosphates, which are homologs of the transition state analog 2-C-carboxy-D-arabinitol 1,5-bisphosphate, exhibit competitive inhibiton of ribulose bisphosphate carboxylase/oxygenase (EC 4.1.1.9) isolated from spinach (Spinacia oleracea), with respect to ribulose 1,5-bisphosphate. CMBP was a more potent inhibitor (100-fold) displaying an inhibition constant (Ki at pH 8.0 and 30 degrees C) of 1-2 microM with enzymes from spinach, barley (Hordeum vulgare), and Chromatium vinosum. In contrast the Rhodospirillum rubrum enzyme was inhibited about 40-fold more weakly (Ki = 53 microM at pH 8.0 and 30 degrees C). Both CGBP and CMBP potentiated activation of RuBP carboxylase from spinach and R. rubrum.

The response to ribulose bisphosphate(4-) (RuBP (4-)) and RuBP-Mg (2-) in catalysis by structurally divergent RuBP carboxylase/oxygenases.

Free ribulose hisphosphate (RuBP(4-)) rather than its magnesium complex (RuBP-Mg(2-)) was the apparent substrate for spinach ribulose bisphosphate carboxylase/oxygenase. The apparent Km for total RuBP (pH 8.0 at 30° C) increased with increasing Mg(2+) concentrations from 11.6 µM at 13.33 mM Mg(2+) to 32.6 µM at 40.33 mM Mg(2+). Similarly the apparent Km for RuBP-Mg(2-) complex increased with increasing Mg(2+) from 9.4 µM at 13.33 mM Mg(2+) to 29.7 µM at 40.33 mM Mg(2+). However, the Km values for uncomplexed RuBP(4-) were independent of the (saturating) concentration of Mg(2+) (Km=2.2 µM). The Vmax did not vary with the changing concentrations of Mg(2+).In contrast, the Km for total RuBP remained constant with varying Mg(2+) concentrations (Km=59.5 µM) for the enzyme from R. rubrum. The apparent Km for the RuBP-Mg(2-) complex decreased with increasing Mg(2+) concentrations from 16.0 µM at 7.5 mM Mg(2+) to 5.9 µM at 27.5 mM Mg(2+). The initial velocity for the C. vinosum enzyme was also found to be independent of the (saturating) concentration of Mg(2+) when total RuBP was varied in the assay. Thus the response to total RuBP by these two bacterial enzymes, which markedly differ in structure, was closely similar.

The response to ribulose bisphosphate(4-) (RuBP (4-)) and RuBP-Mg (2-) in catalysis by structurally divergent RuBP carboxylase/oxygenases.

Free ribulose bisphosphate (RuBP(4-)) rather than its magnesium complex (RuBP-Mg(2-)) was the apparent substrate for spinach ribulose bisphosphate carboxylase/oxygenase. The apparent Km for total RuBP (pH 8.0 at 30° C) increased with increasing Mg(2+) concentrations from 11.6 µM at 13.33 mM Mg(2+) to 32.6 µM at 40.33 mM Mg(2+). Similarly the apparent Km for RuBP-Mg(2-) complex increased with increasing Mg(2+) from 9.4 µM at 13.33 mM Mg(2+) to 29.7 µM at 40.33 mM Mg(2+). However, the Km values for uncomplexed RuBP(4-) were independent of the (saturating) concentration of Mg(2+) (Km=2.2 µM). The Vmax did not vary with the changing concentrations of Mg(2+).In contrast, the Km for total RuBP remained constant with varying Mg(2+) concentrations (Km=59.5 µM) for the enzyme from R. rubrum. The apparent Km for the RuBP-Mg(2-) complex decreased with increasing Mg(2+) concentrations from 16.0 µM at 7.5 mM Mg(2+) to 5.9 µM at 27.5 mM Mg(2+). The initial velocity for the C. vinosum enzyme was also found to be independent of the (saturating) concentration of Mg(2+) when total RuBP was varied in the assay. Thus the response to total RuBP by these two bacterial enzymes, which markedly differ in structure, was closely similar.

Dehydrogenation by gluconate 6-phosphate dehydrogenase of an isosteric phosphonate substrate analogue.

6,7-Dideoxy-D-gluco-heptonic-7-phosphonic acid, the isosteric phosphonate analogue of gluconate 6-phosphate, was prepared by incubation of the corresponding analogue of glucose 6-phosphate with glucose 6-phosphate dehydrogenase and NADP+ in the presence of an enzyme NADPH-NADP+ recycling system. The analogue of gluconate 6-phosphate is a substrate for yeast gluconate 6-phosphate dehydrogenase, showing Michaelis-Menten kinetics at pH 7.5 and 8.0. At both pH values the Km values are approx. 3-fold higher and the Vmax. values approx. 7-fold lower than those of the natural substrate.