Biomedical subjects

D L Miller

Publications and source records attributed to D L Miller.

At least 19 recordsLinked to original sources

PubMed1999

Filtroporation: A simple, reliable technique for transfection and macromolecular loading of cells in suspension.

Cultured Chinese hamster ovary (CHO) cells suspended in their growth medium were forced by gas pressure through the uniformly sized micropores of filter membranes. This procedure caused transient damage to the plasma membrane, which increased the permeability of the cells to exogenous molecules. This "filtroporation" was indicated by uptake of fluorescent dextran molecules up to 500,000 MW in cells deemed viable by trypan blue dye exclusion. The macromolecular uptake was increased if the driving pressure was increased at constant micropore size, or if the micropore size was decreased at constant driving pressure. Larger membrane perturbations permitted uptake of a luciferase reporter plasmid, which resulted in transfection of the CHO cells with the surviving cells expressing luciferase activity after 2 days in culture. This simple and general new method of porating cells in suspension may be optimized to incorporate the desired macromolecules while retaining the maximum viability.

Animals↗

PubMed1999

Novel roles for saccharomyces cerevisiae mitotic spindle motors.

The single cytoplasmic dynein and five of the six kinesin-related proteins encoded by Saccharomyces cerevisiae participate in mitotic spindle function. Some of the motors operate within the nucleus to assemble and elongate the bipolar spindle. Others operate on the cytoplasmic microtubules to effect spindle and nuclear positioning within the cell. This study reveals that kinesin-related Kar3p and Kip3p are unique in that they perform roles both inside and outside the nucleus. Kar3p, like Kip3p, was found to be required for spindle positioning in the absence of dynein. The spindle positioning role of Kar3p is performed in concert with the Cik1p accessory factor, but not the homologous Vik1p. Kar3p and Kip3p were also found to overlap for a function essential for the structural integrity of the bipolar spindle. The cytoplasmic and nuclear roles of both these motors could be partially substituted for by the microtubule-destabilizing agent benomyl, suggesting that these motors perform an essential microtubule-destabilizing function. In addition, we found that yeast cell viability could be supported by as few as two microtubule-based motors: the BimC-type kinesin Cin8p, required for spindle structure, paired with either Kar3p or Kip3p, required for both spindle structure and positioning.

Dyneins↗

PubMed1999

Editing of cytochrome b mRNA in physarum mitochondria.

The reading frame in the mRNA for the cytochrome b apoprotein in mitochondria of Physarum polycephalum is created by the insertion of 43 nucleotides in the mRNA relative to the mtDNA sequence encoding it (RNA editing). Most of these insertions (31) are single cytidines; however, single uridines are inserted at six sites, and the dinucleotides, CU and GC, are inserted at two sites and one site, respectively. These insertions create a 392-codon reading frame in the mature mRNA. The amino acid sequence inferred from this reading frame has similarity to cytochrome b apoproteins encoded by other mtDNAs. The insertions are quite evenly distributed throughout the length of the reading frame with an average spacing of 27 nucleotides. This mRNA has the highest percentage (23%) of noncytidine insertions of any Physarum RNA characterized to date. cDNAs corresponding to partially edited RNAs can be enriched by selective amplification. Some cDNAs that lack the GC dinucleotide insertion are fully edited at sites flanking the GC dinucleotide insertion site. Similarly some cDNAs lack the CT dinucleotide insertion or have a CC or TT insertion flanked by a fully edited sequence. These results imply that dinucleotide editing occurs by a process separate from the global insertion of cytidines.

Amino Acid Sequence↗

PubMed1999

Clinical progression of transplanted large granular lymphocytic leukemia in Fischer 344 rats exposed to 60 Hz magnetic fields.

The purpose of this study was to determine if 60 Hz magnetic fields can alter the clinical progression of leukemia in an animal model. Large granular lymphocytic (LGL) leukemia cells from spleens of leukemic rats were transplanted into young male Fischer 344 rats, producing signs of leukemia in approximately 2-3 months. The animals were randomly assigned to 4 treatment groups (108/group) as follows: 1) 10 G (1.0 mT) linearly polarized 60 Hz magnetic fields, 2) sham exposed [null energized unit with residual 20 mG (2 microT) fields], 3) ambient controls [<1 mG (0.1 [microT)], and 4) positive controls (a single 5 Gy whole body exposure to 60Co 4 days prior to initiation of exposure). All rats were injected intraperitoneally (ip) with 2.2 x 10(7) LGL leukemic cells at the initiation of exposure or sham exposure. The magnetic fields were activated for 20 h/day, 7 days/week, allowing time for animal care. The experimental fields were in addition to natural ambient magnetic fields. Eighteen rats from each treatment group were bled, killed, and evaluated at 5, 6, 7, 8, 9, and 11 weeks of exposure. Peripheral blood hematological endpoints, changes in spleen growth, and LGL cell infiltration into the spleen and liver were measured to evaluate the leukemia progression. No significant or consistent differences were detected between the magnetic field exposed groups and the ambient control group, although the clinical progress of leukemia was enhanced in the positive control animals. These data indicate that exposure to sinusoidal, linearly polarized 60 Hz, 10 G magnetic fields did not significantly alter the clinical progression of LGL leukemia. Furthermore, the data are in general agreement with previous results of a companion repeated-bleeding study in which animals were exposed for 18 weeks.

Animals↗

PubMed1999

Automated bolus chase peripheral MR angiography: initial practical experiences and future directions of this work-in-progress.

Bolus chase 3-dimensional MR angiography (3D MRA) is a recent development that extends the effective field of view for arterial imaging from the typical single 40-50 cm to over 100 cm. This technique is well suited for imaging long vascular territories such as the lower extremity. Bolus chase peripheral 3D MRA is achieved with overlapping 3D gradient-echo scans during the arterial transit of a single intravenous injection of gadolinium-chelate contrast media. This technique can depict the arteries from the infrarenal aorta to the ankles in less than 2 minutes. The initial experiences with bolus chase peripheral MRA using an automated algorithm that controls both table translation and 3D data acquisition are described. Suggestions for future refinements to the technique are also discussed.

Algorithms↗

PubMed1999

Pulmonary artery occlusion and reperfusion causes microvascular constriction in the rabbit lung.

INTRODUCTION: Reperfusion injury of the lung after ischemia is associated with altered alveolar blood flow and ventilation-perfusion mismatch, which is a significant cause of morbidity and mortality after lung transplantation. We examined the effect of ischemia and reperfusion on the tone of individual subpleural arterioles in the pulmonary circulation by using video microscopy with polarized epiillumination. METHODS: In 11 open-chested rabbits anesthetized with pentobarbital (2.3 to 2.5 kg), we ventilated the lungs through the lower trachea (air or 50% oxygen) and examined the response of subpleural arterioles (diameter 75 +/- 13 microm) to ischemia (76 +/- 32 min) of the right lung caused by occluding the right main pulmonary artery. Observations were made during baseline, occlusion, and during early (20 to 32 min) and late (48 to 63 min) reperfusion using a long working distance lens (550x) with a dipping cone held at the pleural surface while the lungs were statically inflated (10 cm H2O) with oxygen for brief periods. Data are expressed as mean +/- standard deviation. RESULTS: Arteriolar diameter was decreased 57% +/- 19% during early reperfusion. There was a decrease in blood flow and alveolar walls were pale, indicating reduced capillary perfusion. During late reperfusion, arteriolar diameter was diminished (19% +/- 26%); flow was still reduced. Overall pulmonary vascular resistance increased during early reperfusion but returned to baseline level during late reperfusion. Arterial partial pressure of oxygen averaged 200 mm Hg during ischemia and reperfusion. CONCLUSIONS: Constriction of small arterioles by ischemia and reperfusion can have a significant effect on the early phase of ventilation-perfusion mismatch and pulmonary dysfunction by altering alveolar perfusion. This response does not appear to be mediated by hypoxia because it was not prevented by ventilation with oxygen.

Animals↗

PubMed1999

Inflammatory pseudotumors of the lung.

BACKGROUND: Inflammatory pseudotumors of the lung are rare and often present a dilemma for the surgeon at time of operation. We reviewed our experience with patients who have this unusual pathology. METHODS: Between February 1946 and September 1993, 56,400 general thoracic surgical procedures were performed at the Mayo Clinic. Twenty-three patients (0.04%) had resection of an inflammatory pseudotumor of the lung. There were 12 women and 11 men. Median age was 47 years (range, 5 to 77 years). Six patients (26%) were less than 18 years old. All pathologic specimens were re-reviewed, and the diagnosis of inflammatory pseudotumor was confirmed. Eighteen patients (78%) were symptomatic which included cough in 12, weight loss in 4, fever in 4, and fatigue in 4. Four patients had prior incomplete resections performed elsewhere and underwent re-resection because of growth of residual pseudotumor. Wedge excision was performed in 7 patients, lobectomy in 6, pneumonectomy in 6, chest wall resection in 2, segmentectomy in 1, and bilobectomy in 1. Complete resection was accomplished in 18 patients (78%). Median tumor size was 4.0 cm (range, 1 to 15 cm). There were no operative deaths. Follow-up was complete in all patients and ranged from 3 to 27 years (median, 9 years). RESULTS: Overall 5-year survival was 91%. Nineteen patients are currently alive. Cause of death in the remaining 4 patients was unrelated to pseudotumor. The pseudotumor recurred in 3 of the 5 patients who had incomplete resection; 2 have had subsequent complete excision with no evidence of recurrence 8 and 9 years later. CONCLUSIONS: We conclude that inflammatory pseudotumors of the lung are rare. They often occur in children, can grow to a large size, and are often locally invasive, requiring significant pulmonary resection. Complete resection, when possible, is safe and leads to excellent survival. Pseudotumors, which recur, should be re-resected.

Adolescent↗

PubMed1999

Ischemic preconditioning limits infarct size following regional ischemia-reperfusion in in situ mouse hearts.

OBJECTIVE: Ischemic preconditioning has been demonstrated in a wide variety of animals, from dogs to rats. Experimentally-induced murine myocardial ischemia-reperfusion has been described, but ischemic preconditioning has not been reported in mouse hearts. To test the hypothesis that mouse hearts exhibit preconditioning-induced protection, experiments were conducted in anesthetized open chest mice subjected to regional myocardial ischemia-reperfusion. METHODS: Following barbiturate anesthesia the FVB and C57BL/6J mice underwent a tracheostomy and were mechanically ventilated. The heart was exposed via a left thoracotomy performed with the aid of a dissecting microscope. A 7-0 silk suture on a curved taper needle was passed under the proximal left anterior descending coronary artery to form a snare. Mice were then randomly assigned to receive either no preconditioning or preconditioning. All mice were subjected to 60 min regional myocardial ischemia followed by 2.5 h of reperfusion. Ischemic preconditioning (IP) was induced with two (FVB mice) or three (C57BL/6J mice) cycles of 5 min coronary occlusion and 5 min reperfusion. Control animals did not receive preconditioning ischemia. Area-at-risk was assessed with fluorescent particles. Infarct size was assessed with triphenyl tetrazolium chloride, and is expressed below as a percentage of the area-at-risk. RESULTS: In FVB mice preconditioning reduced infarct size 49%, from 36.7 +/- 4.5% to 18.9 +/- 2.8% (P < 0.05). In C57BL/6J mice preconditioning reduced infarct size by 66%, from 56.4 +/- 8.3% to 18.9 +/- 4.2% (P < 0.05). CONCLUSION: From these data we conclude that the infarct limiting effect of ischemic preconditioning is demonstrable in murine hearts.

Animals↗

PubMed1999

Early and long-term results of prosthetic chest wall reconstruction.

OBJECTIVE: The purpose of this report is to evaluate our results in patients who underwent prosthetic bony reconstruction after chest wall resection. METHODS: We retrospectively reviewed all patients who underwent chest wall resection and reconstruction with prosthetic material at the Mayo Clinic. RESULTS: From January 1, 1977, to December 31, 1992, 197 patients (109 male patients and 88 female patients) underwent chest wall resection and reconstruction with prosthetic material. Median age was 59 years (range, 11-86 years). The indication for resection was recurrent chest wall malignancy in 65 patients (33.0%), primary chest wall malignancy in 62 patients (31.5%), contiguous lung or breast carcinoma in 58 patients (29.4%), and other reasons in 12 patients (6.1%). Three patients (1.5%) each had an open draining wound. This review covers 2 time periods. Sixty-four patients (32.5%) underwent reconstruction with polypropylene mesh during the period from 1977 to 1986. One hundred thirty-three patients (67.5%) underwent reconstruction with polytetrafluoroethylene from 1984 to 1992. Soft tissue coverage was achieved with transposed muscle in 116 patients (58.9%), local tissue in 81 patients (41.1%), and omentum in 3 patients (1.5%). There were 8 deaths (operative mortality rate, 4.1%). Ninety-one patients (46.2%) experienced complications. Seromas occurred in 14 patients (7.1%). Wound infections occurred in 9 patients (4.6%; 5 patients with polypropylene mesh and 4 patients with polytetrafluoroethylene). The prosthesis was removed in all 5 patients with polypropylene mesh and in none of the patients with polytetrafluoroethylene. Follow-up was complete in 179 operative survivors (94.7%) and ranged from 1 to 204 months (median, 26 months). A well-healed asymptomatic wound was present in 127 patients (70.9%). CONCLUSIONS: Chest wall resection and reconstruction with prosthetic material will yield satisfactory results in most patients. Little difference exists between polypropylene mesh and polytetrafluoroethylene.

Adolescent↗

PubMed1999

Results of reoperation on the upper esophageal sphincter.

OBJECTIVE: Reoperation on the upper esophageal sphincter is infrequent. We reviewed our experience in patients who underwent reoperation on the upper esophageal sphincter. METHODS: This is a retrospective report of accumulative series from 2 separate institutions. RESULTS: From September 1, 1976, to February 28, 1997, 37 patients underwent reoperation on the upper esophageal sphincter for recurrent or persistent obstructive symptoms. There were 29 men and 8 women. The median age was 69 years (range, 38-87 years). The original indication for the operation was a pharyngoesophageal (Zenker's) diverticulum in 33 patients (89.2%), oculopharyngeal dystrophy in 3 patients (8.1%), and muscular dystrophy in 1 patient (2.7%). One prior upper esophageal sphincter operation had been performed in 26 patients (70.3%), two operations in 9 patients (24. 3%), and three operations in 2 patients (5.4%). All patients were symptomatic; 35 patients (94.6%) had dysphagia; 23 patients (62.2%) had regurgitation; and 12 patients (32.4%) had episodes of aspiration. Thirty of the patients (91.0%) with Zenker's diverticulum were found to have a recurrent or persistent diverticulum at reoperation. A diverticulectomy and cricopharyngeal myotomy were performed in 23 patients (62.2%); cricopharyngeal myotomy alone, in 7 patients (18.9%); diverticulopexy and cricopharyngeal myotomy, in 6 patients (16.2%); and diverticulectomy alone, in 1 patient (2.7%). There were no operative deaths. Complications developed in 10 patients (27.0%). Follow-up was complete in 34 patients (91.9%) and ranged from 2 to 149 months (median, 39 mo). Thirty-two patients (94.1%) were improved. Functional results were classified as excellent in 26 patients (76. 5%), good in 2 patients (5.9%), fair in 4 patients (11.7%), and poor in 2 patients (5.9%). CONCLUSIONS: Reoperation for patients who have persistent or recurrent symptoms after an operation on the upper esophageal sphincter is associated with acceptable morbidity and mortality rates. Resolution of symptoms occurs in most patients.

Adult↗

PubMed1999

Sonoporation of cultured cells in the rotating tube exposure system.

Suspensions of Chinese hamster ovary cells were exposed to ultrasound in the presence of fluorescent dextran to determine the conditions needed for sonoporation with uptake of the large molecules. Albunex, a gas-body- based ultrasound contrast agent, was added to enhance cavitation. Ultrasound was continuous wave at frequencies of 1.0, 1.68, 2.25, 3.3, 5.3, and 7.15 MHz. Sterile 4.5-mL exposure chambers were rotated at 60 rpm to promote cavitation activity during the 1-min exposures. After exposure, cells were tested for sonoporation by counting fluorescent cells and for cell lysis by counting cells stained by trypan blue. Sonoporation was a sensitive bioeffects indicator that was detected at pressure amplitudes lower than were needed for transient cavitation or cavitation-induced cell lysis. For 10% Albunex, apparent thresholds for sonoporation, which were comparable to the levels required to perturb the gas bodies, were 0.084 MPa (spatial peak negative pressure amplitude) from 1.0-3.3 MHz and 0.27 MPa at 5.3 and 7.15 MHz. Sonoporation decreased slightly if the tube was not rotated. The effects increased for increasing Albunex concentration (with rotation). The plating efficiency of cells exposed to 0.2 MPa at 2.25 MHz and sorted by a flow cytometer was 19% (3.6% standard deviation [SD]) for fluorescent cells, compared to 67% (1% SD) for nonfluorescent exposed cells and 62% (6% SD) for sham-exposed cells. The reduced viability represents an important consideration for possible applications of sonoporation.

Albumins↗

PubMed1999

Heating vs. cavitation in the induction of mouse hindlimb paralysis by ultrasound.

Grip strength tests were performed on hairless mice before and after various ultrasound exposures in a temperature-controlled water bath at 37 degrees C. Lithotripter exposure of 800 shock waves produced no effect on hindlimb function. In contrast, 1.09-MHz exposures at 1 MPa with 10:100 ms burst mode did produce a statistically significant reduction in grip strength of about 60%. The exposure duration was important for the 1.0-MPa burst mode exposure, with grip-strength reductions appearing after 150 s or longer exposures. Continuous exposure at 3.3 W cm(-2) (0.32 MPa peak) for 200 s produced the same effect as burst mode exposure at 3.3 W cm(-2) (1 MPa peak) for 200 s, which implicates the temporal average intensity as an important factor. The temperature elevations for 1-MPa burst mode was estimated from thermocouple measurements in the spine to be 12 degrees C after 200-s exposure. Although tests of exposures in cool (32 degrees C) and warm (42 degrees C) baths produced inconclusive results in regard to the thermal mechanism, the effects observed appear to result from ultrasonic heating (rather than from cavitation). Thus, any potentially harmful consequences associated with the effects examined might be related more, for example, to ultrasonic hyperthermia therapy than to shock-wave lithotripsy.

Animals↗

PubMed1999

Popliteal artery entrapment syndrome: arteriographic findings and thrombolytic therapy.

PURPOSE: To review the arteriographic appearance of popliteal artery entrapment syndrome (PAES) and functional popliteal artery entrapment, and determine the role of thrombolysis in the treatment of these disorders. MATERIALS AND METHODS: Retrospective review of hospital records from 1991 to 1998. RESULTS: Seven patients with PAES and one with functional entrapment were identified. The popliteal artery was occluded in two limbs and compressed in 13. Active plantar flexion was necessary to demonstrate impingement in nine limbs. Medial deviation of the popliteal artery was evident in six of 14 patent popliteal arteries, and lateral deviation was observed in one limb. "Classic" abrupt medial angulation of the popliteal artery was observed in one limb. Both limbs were involved in all six patients who underwent bilateral popliteal exploration. Thrombolytic therapy was performed in three limbs. In two instances, it permitted a less extensive surgical procedure than would otherwise have been required. CONCLUSIONS: There is considerable variability in the arteriographic appearance of PAES, which is arteriographically indistinguishable from functional entrapment. It is frequently bilateral. Thrombolytic therapy does not obviate surgery but may permit a less extensive procedure to be performed.

Adult↗

PubMed1999

Hypoxic regulation of vascular endothelial growth factor mRNA stability requires the cooperation of multiple RNA elements.

Vascular endothelial growth factor (VEGF) is a key regulator of developmental, physiological, and tumor angiogenesis. Upregulation of VEGF expression by hypoxia appears to be a critical step in the neovascularization of solid cancers. The VEGF mRNA is intrinsically labile, but in response to hypoxia the mRNA is stabilized. We have systematically analyzed the regions in the VEGF mRNA that are responsible for its lability under normoxic conditions and for stabilization in response to hypoxia. We find that the VEGF mRNA not only contains destabilizing elements in its 3' untranslated region (3'UTR), but also contains destabilizing elements in the 5'UTR and coding region. Each region can independently promote mRNA degradation, and together they act additively to effect rapid degradation under normoxic conditions. Stabilization of the mRNA in response to hypoxia is completely dependent on the cooperation of elements in each of the 5'UTR, coding region, and 3'UTR. Combinations of any of two of these three regions were completely ineffective in responding to hypoxia, whereas combining all three regions allowed recapitulation of the hypoxic stabilization seen with the endogenous VEGF mRNA. We conclude that multiple regions in the VEGF mRNA cooperate both to ensure the rapid degradation of the mRNA under normoxic conditions and to allow stabilization of the mRNA in response to hypoxia. Our findings highlight the complexity of VEGF gene expression and also reveal a mechanism of gene regulation that could become the target for strategies of therapeutic intervention.

3' Untranslated Regions↗

PubMed1999

Lack of effect of a 60 Hz magnetic field on biomarkers of tumor promotion in the skin of SENCAR mice.

It has been proposed that extremely low frequency magnetic fields may enhance tumorigenesis through a co-promotional mechanism. This hypothesis has been further tested using the two-stage model of mouse skin carcinogenesis, i.e. 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced promotion of skin carcinogenesis in mice initiated by a single subcarcinogenic dose of 7,12-dimethylbenz[a]anthracene. Experimentation utilized three different doses of TPA within its dose-response range (0.85, 1.70 or 3.40 nmol) and examined the following early biomarkers of tumor promotion after 1, 2 and 5 weeks of promotion: increases in epidermal thickness and the labeling index of epidermal cells, induction of epidermal ornithine decarboxylase activity and down-regulation of epidermal protein kinase C activity. Mice exposed to a 60 Hz magnetic field having a flux density of 2 mT for 6 h/day for 5 days/week were compared with mice exposed to an ambient magnetic field. Within the sensitivity limits of the biomarker methodology and the exposure parameters employed, no consistent, statistically significant effects indicative of promotion or co-promotion by the magnetic field were demonstrated.

Animals↗

PubMed1999

What is the cost of getting the price wrong?

BACKGROUND: The objective of this study was to compare differences in cost estimates for paediatric HIV hospital service provision based on hospital prices with cost estimates obtained through a research-based service-specific costing exercise. METHODS: Activity data on the use of hospital services of children by stage of HIV infection were collected from case-notes for 118 HIV antibody positive children, managed at St Mary's Hospital NHS Trust, London, 1 January 1986-31 December 1994. Hospital unit prices were obtained from the Hospital Trust Finance Department; unit cost estimates were obtained from relevant hospital departments through a research-based service-specific costing exercise. Financial data related to the 1993-1994 financial year, and were indexed to 1995-1996 prices. The main outcome measures were cost estimates per patient-year by stage of HIV infection. Three cost scenarios were calculated: first by linking activity data with hospital prices (Trust Prices); second by linking activity data with routinely available hospital prices plus units costs from the costing exercise where no relevant hospital prices existed (Supplemented Trust Prices); third, by linking activity data exclusively with unit costs from the hospital-specific costing exercise (Unit Costs). RESULTS: There were substantial differences between unit cost estimates per patient-year based on Trust Prices and Supplemented Trust Prices compared with those based on Unit Costs. Differences increased with more intense use of services. The deficit based on Trust Prices compared with Unit Costs ranged from Pound Sterling 432 per patient-year for HIV negative children, Pound Sterling 574 for asymptomatic HIV-infected children, Pound Sterling 1288 for indeterminate children, Pound Sterling 1814 for children with symptomatic non-AIDS to Pound Sterling 7418 per patient-year for children with AIDS. CONCLUSIONS: In this hospital, reliance on generic hospital prices to derive cost estimates for paediatric HIV services produced considerable underestimates of the cost of service provision compared with data derived through the costing exercise. If this occurs across all or most areas of service provision, this can lead to substantial financial deficits, which in turn may mean that the needs of specific client populations may not be met.

Bias↗

PubMed1999

Molecular analysis of the 18S rRNA gene of Cryptosporidium serpentis in a wild-caught corn snake (Elaphe guttata guttata) and a five-species restriction fragment length polymorphism- based assay that can additionally discern C. parvum from C. wrairi.

An adult wild-caught corn snake (Elaphe guttata guttata) was presented for humane euthanasia and necropsy because of severe cryptosporidiosis. The animal was lethargic and >5% dehydrated but in good flesh. Gastric lavage was performed prior to euthanasia. Histopathologic findings included gastric mucosal hypertrophy and a hemorrhagic erosive gastritis. Numerous 5- to 7-microm-diameter round extracellular organisms were associated with the mucosal hypertrophy. A PCR, acid-fast stains, Giemsa stains, and an enzyme immunoassay were all positive for Cryptosporidium spp. PCR and restriction fragment length polymorphism (RFLP) analysis on gastric lavage and gastric mucosal specimens, and subsequent sequencing of the 18S rRNA gene, enabled a distinct molecular characterization of the infecting organism as Cryptosporidium serpentis. Until recently, studies on snake Cryptosporidium have relied on host specificity and gross and histopathologic observations to identify the infecting species. A multiple alignment of our sequence against recently published sequences of the 18S rRNA gene of C. serpentis (GenBank accession no. AF093499, AF093500, and AF093501 [L. Xiao et al., unpublished data, 1998]) revealed 100% homology with the C. serpentis (Snake) sequence (AF093499) previously described by Xiao et al. An RFLP method to differentiate the five presently sequenced strains of Cryptosporidium at this locus was developed. This assay, which uses SpeI and SspI, complements a previously reported assay by additionally distinguishing the bovine strain of Cryptosporidium from Cryptosporidium wrairi.

Animals↗

PubMed1999

Quantification of sub-femtomole amounts of Alzheimer amyloid beta peptides.

We evaluated methods for the quantitative Western blot analysis of A beta 1-40 and A beta 1-42. Both chromogenic and chemiluminescent detection methods gave similar sensitivities (0.15 fmol of A beta 1-40 and 0.3 fmol of A beta 1-42); however, the chromogenic method was more rapid, simpler, less expensive and gave fewer background problems; consequently, it yielded more reliable results. Adsorption to various types of laboratory plasticware can greatly interfere with the accurate measurement of A beta, but this can be prevented by the addition of SDS or bovine serum albumin. Among several methods for concentrating A beta from biological materials, immunoadsorption to Sepharose-bound antibodies was the most efficient. It yielded 50% recovery of 1 pM A beta 1-42 or A beta 1-40 and so was a suitable method to measure A beta levels in human plasma. Through combined immunoadsorption and Western blotting we could determine the amounts of A beta isoforms secreted from 1 x 10(6) cells after a culture period as short as 1 h. This eliminates the need to use radiolabelling or over-expression to study A beta precursor processing Bovine serum contains subnanomolar A beta levels, similar to those that reportedly stimulate cell proliferation. That cultured cells quickly secrete these levels of A beta suggests that the peptide might exert an autocrine effect.

Alzheimer Disease↗