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Biomedical subjects

W S Hu

Publications and source records attributed to W S Hu.

At least 73 records · Page 4Linked to original sources
PubMed1992

Primary culture of rat hepatocytes entrapped in cylindrical collagen gels: an in vitro system with application to the bioartificial liver. Rat hepatocytes cultured in cylindrical collagen gels.

A static culture model employing cylindrical collagen-hepatocyte gels is reported for large scale testing of conditions relevant to the three compartment hollow fiber bioartificial liver. High density hepatocyte cultivation was achieved by cell entrapment within the collagen-hepatocyte gel. Hepatocyte viability was assessed by vital staining, gel contraction, and insulin utilization. Measures of hepatocyte-specific function included albumin synthesis, ureagenesis, lidocaine biotransformation, and cholate conjugation. Although hepatocyte viability remained stable through the seven day incubation period, hepatocyte functions were not uniformly preserved. Albumin synthesis remained stable, while representative P-450 and conjugation activities decreased with time. This static culture system will facilitate the development of a hollow fiber bioartificial liver which utilizes cylindrical collagen-hepatocyte gels.

Albumins↗
PubMed1992

Identification of rate-limiting steps in cephalosporin C biosynthesis in Cephalosporium acremonium: a theoretical analysis.

A kinetic model describing the biosynthesis of cephalosporin C in Cephalosporium acremonium has been developed to identify the rate-limiting step(s). Using this model and in-vitro kinetic data of the biosynthetic enzymes, the production kinetics of cephalosporin C were examined theoretically. The predicted time profile of the specific production rate during batch culture is in good agreement with that of experimental results published previously. Sensitivity analysis indicates that delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine (ACV) synthetase is the rate-limiting enzyme. Our analysis also predicts that increasing ACV synthetase enhances the production rate initially until expandase/hydroxylase becomes rate-limiting. Furthermore, increasing expandase/hydroxylase reduces the accumulation of penicillin N, and thus, enhances the production of cephalosporin C. Based on our analysis, amplifying both ACV synthetase and expandase/hydroxylase concurrently should enhance the production rate to a great extent.

Acremonium↗
PubMed1992

Hepatocyte function in a hollow fiber bioreactor: a potential bioartificial liver.

We have developed a novel hepatocyte loaded hollow fiber bioreactor as a potential bioartificial liver. Freshly harvested rat hepatocytes were entrapped in a three-dimensional gel matrix within hollow fibers in a perfused bioreactor. Gel entrapment allowed cells to be cultured at high density while maintaining tissue-specific function. Hepatocyte function was evaluated in 10 bioreactors, each containing approximately 5 x 10(7) cells. Oxygen consumption averaged 0.32 pmole/cell/hr, albumin appearance averaged 0.60 pg/cell/hr, and lidocaine clearance (a measure of the P-450 activity) averaged 0.74 pg/cell/hr. Function persisted for the 7 days of the study. Electron microscopy at 7 days showed the distinctive ultrastructure of viable, differentiated hepatocytes: bile canaliculi, intercellular junctions, peroxisomes, abundant mitochondria, and glycogen granules. Maintenance of tissue specific function and ultrastructure suggests that this bioreactor configuration has potential as a device to support patients in liver failure, as well as to study hepatocytes in vitro.

Amino Acids↗
PubMed1992

Cultivation of mammalian cells on macroporous microcarriers.

Adherent cells can be cultivated in a stirred-tank bioreactor by attaching to microcarriers. Macroporous microcarriers, with their intraparticle space and surface area for cell growth, can potentially support a higher cell concentration than conventional microcarriers, which support cell growth only on the external surface. Chinese hamster ovary (CHO) cells and green monkey kidney (Vero) cells were cultivated on macroporous microcarriers, Cultispher-G. Cells attached to the microcarriers at a slow rate and grew to a high density. Thin sections of the microcarriers demonstrate that cells were initially on the exterior of the microcarriers and migrated into the interior as cell concentration increased. Vero cells cultivated on these microcarriers were successfully used for the production of vesicular stomatitis virus (VSV).

Animals↗
PubMed1992

Transesophageal echocardiography in adults with a continuous precordial murmur.

In order to assess the ability of echocardiography in the detection of intracardiac and extracardiac shunts, we studied 11 patients (aged 22-64 yr) with a continuous precordial murmur using transthoracic and transesophageal echocardiography, and correlated the results with the subsequent angiographic and surgical findings. We found that only in 5 of 6 patients with a patent arterial duct could the continuous flow pattern be detected in pulmonary artery using transthoracic echocardiography, whereas it could be readily and accurately identified by transesophageal echocardiography in all patients. The diameters of the patent arterial duct were also measured and found to be in good correlation with subsequent surgical findings (r = 0.98, p less than 0.05). In 2 patients with a ruptured aneurysm of sinus of Valsalva which originated from the right coronary sinus and perforated into the right ventricle, transesophageal echocardiography gave a better image than transthoracic echocardiography. In 2 patients with coronary artery fistula, the origin and site of drainage of the coronary artery could be imaged using transesophageal echocardiography, but the course of coronary artery fistula was more easily detected by transthoracic echocardiography. In one patient with aortopulmonary window, the defect between ascending aorta and main pulmonary artery could readily be imaged by transesophageal echocardiography. We therefore recommend transesophageal echocardiography when evaluating patients with precordial continuous murmur in whom intracardiac and extracardiac shunts or defects are suspected.

Adult↗
PubMed1992

Mammalian cell culture processes.

Over the past year, mammalian cell culture research has been aimed at investigating the influence of culture conditions on viability, productivity and the consistency of post-translational modifications. Studies of the effect of medium conditions and the development of kinetic models are being made in relation to current efforts to develop fed-batch strategies that will optimize recombinant protein production processes. Recent advances have included novel biosensor and bioreactor developments. New technologies have also been applied to investigate high cell density bioreactor and culture conditions.

Animals↗
PubMed1992

Hepatocyte culture systems for artificial liver support: implications for critical care medicine (bioartificial liver support).

OBJECTIVE: The primary purpose of this review article is to familiarize critical care practitioners with newly developing techniques of hybrid artificial liver support. Implantable and extracorporeal hepatocyte culture systems are emphasized based on their current experimental and clinical status. DATA SOURCES: Data used to prepare this document were obtained from the authors' personal files, as well as the computerized MEDLINE database. Medical headings used include: liver, artificial organs, cell culture, growth hormones, extracellular matrix, and transplantation. Only articles published in English have been cited. STUDY SELECTION: All studies are discussed in which hepatocyte culture systems have been used to support human patients with liver failure. All studies reported the patient's condition before therapy, duration of therapy, and outcome after therapy in order to be included in this review. Since the number of clinical trials is small at this time, animal studies were used to demonstrate application of other systems in the treatment of experimentally induced liver failure. Similar selection criteria were used to select animal studies for review. All initially identified human studies met these selection criteria. DATA EXTRACTION: Independent extraction by multiple observers. DATA SYNTHESIS: Liver failure, resulting from infection, drugs, or as a part of the multiple organ failure syndrome, remains a major cause of morbidity, mortality, and resource allocation. Current therapy is limited to supportive care, along with liver transplantation. Because of these therapeutic limitations, hybrid artificial liver systems have been proposed for temporary and long-term hepatic support. Several animal studies and a small number of preliminary human studies indicate that hepatocyte culture systems are capable of supporting nearly all essential hepatic functions and may supply biologically active substances that promote regeneration and repair of the damaged liver being supported. Hybrid systems may be constructed from materials that serve as immunoprotective barriers against host defenses. CONCLUSIONS: During the past decade, important progress has been made with hybrid artificial liver support systems. Cell culture technology has progressed sufficiently so that an artificial liver, composed of metabolically active hepatocytes, may be a potential reality in the foreseeable future. Both implantable and extracorporeal artificial liver support systems have been developed to provide metabolic support during acute liver failure, or to serve as a bridge to solid organ transplantation. Implantable hepatocyte systems, however, require a prolonged period for intraperitoneal engraftment and vascularization, not typically available to patients with acute liver failure. For this reason, extracorporeal hybrid designs offer the greatest hope for on-line treatment of acute liver failure. Such systems are entering the final stages of animal testing.

Animals↗
PubMed1992

Selective detection of Mycoplasma fermentans by polymerase chain reaction and by using a nucleotide sequence within the insertion sequence-like element.

A new assay using the polymerase chain reaction to amplify a 206-nucleotide specific gene sequence within the insertion sequence-like element of Mycoplasma fermentans has been developed. The unique insertion sequence-like element exists in multiple copies in the M. fermentans genome. The assay selectively amplifies DNA from all strains of M. fermentans tested. In contrast, DNA from other species of human and nonhuman mycoplasmas, common tissue culture-contaminating mycoplasmas, and bacteria, as well as human, monkey, and mouse tissues do not produce the amplified DNA products specific for M. fermentans.

Animals↗
PubMed1992

Effect of gamma radiation on retroviral recombination.

To elucidate the mechanism(s) of retroviral recombination, we exposed virions to gamma radiation prior to infecting target cells. By using previously described spleen necrosis virus-based vectors containing multiple markers, recombinant proviruses were studied after a single round of retrovirus replication. The current models of retroviral recombination predict that breaking virion RNA should promote minus-strand recombination (forced copy-choice model), decrease or not affect plus-strand recombination (strand displacement/assimilation model), and shift plus-strand recombination towards the 3' end of the genome. However, we found that while gamma irradiation of virions reduced the amount of recoverable viral RNA, it did not primarily cause breaks. Thus, the frequency of selected recombinants was not significantly altered with greater doses of radiation. In spite of this, the irradiation did decrease the number of recombinants with only one internal template switch. As a result, the average number of additional internal template switches in the recombinant proviruses increased from 0.7 to 1.4 as infectivity decreased to 6%. The unselected internal template switches tended to be 5' of the selected crossover even in the recombinants from irradiated viruses, inconsistent with a plus-strand recombination mechanism.

Base Sequence↗
PubMed1992

Entrapment of cultured pancreas islets in three-dimensional collagen matrices.

In vitro culture of islets of Langerhans decreases their immunogenicity, presumably by eliminating passenger leukocytes and other Ia+ presenting cells within the islets. Islets cultivated in petri dishes either at 37 degrees C or at 25 degrees C gradually disintegrate during culture in a time-dependent manner which is related to the free-floating condition of the islets. Also, a fraction of the islets disperse as single cells and beta-cell aggregates or adhere to the bottom of the culture dishes. Thus, the retrieval rate of transplantable islets is dampened due to their disintegration and spontaneous dispersion in conventional petri dish cultures. Entrapment of freshly harvested islets of Langerhans in a three-dimensional collagen matrix was studied as an alternative method for islet cultivation. The contraction of collagen fibrils during in vitro culture counteracts the dispersion of islets and helps in maintaining their integrity while in culture. It was observed that the entrapped islets maintain satisfactory morphology, viability, and capability of glucose-dependent insulin secretion for over 2 wk. The oxygen consumption rate and glucose metabolism of these islets was not deranged when entrapped in collagen. Also, the retrieval of islets is easier and more efficient than that observed in conventional culture systems. Our results indicate that culture of islets in three-dimensional collagen gels can potentially develop into an ideal system applicable to clinical transplantation of cultured islets or beta-cell aggregates.

Animals↗
PubMed1991

Cultivation of anchorage-dependent animal cells in microsphere-induced aggregate culture.

Diethylaminoethyl-derivatized dextran microspheres were used to cultivate Chinese hamster ovary, 293, Vero and swine testicular cells. Cells became attached to the microspheres but did not spread out. Instead, they grew in a more spherical shape and eventually formed multiple-cell-layer aggregates. Viability in these aggregates remained high after the cultures reached high cell concentrations. This cultivation method allows a high cell density to be achieved with a low microsphere concentration.

Animals↗
PubMed1991

Comparison of growth kinetics of producing and nonproducing hybridoma cells in batch culture.

Several clones of nonproducing cells were isolated from a continuous culture of hybridoma cells, which were originally producing antibody. Their behavior was compared to that of the producing cells in batch culture. The growth kinetics of five out of six clones exhibited higher specific growth rate, higher yield of cell mass on glutamine, and lower yields of lactate and ammonium. The implications of the comparisons for growth of hybridoma cultures are discussed.

Antibodies, Monoclonal↗
PubMed1991

Effect of gene amplification on mercuric ion reduction activity of Escherichia coli.

The mercury resistance (mer) operon of plasmid R100 was cloned onto various plasmid vectors to study the effect of mer gene amplification on the rate of Hg2+ reduction by Escherichia coli cells. The plasmids were maintained at copy numbers ranging from 3 to 140 copies per cell. The overall Hg2+ reduction rate of intact cells increased only 2.4-fold for the 47-fold gene amplification. In contrast, the rate of the cytoplasmic reduction reaction, measured in permeabilized cells, increased linearly with increasing gene copy number, resulting in a 6.8-fold overall amplification. RNA hybridizations indicated that mRNA of the cytoplasmic mercuric reductase (merA gene product) increased 11-fold with the 47-fold gene amplification, while mRNA of the transport protein (merT gene product) increased only 5.4-fold. Radiolabeled proteins produced in maxicells were used to correlate the expression levels of the mer polypeptides with the measured reduction rates. The results indicated that, with increasing gene copy number, there was an approximately 5-fold increase in the merA gene product compared with a 2.5-fold increase in the merT gene product. These data demonstrate a parallel increase of Hg2+ reduction activity and transport protein expression in intact cells with plasmids with different copy numbers. In contrast, the expression level of the mercuric reductase gene underwent higher amplification than that of the transport genes at both the RNA and protein levels as plasmid copy number increased.

Bacterial Proteins↗
PubMed1990

Retroviral recombination and reverse transcription.

Recombination occurs at a high rate in retroviral replication, and its observation requires a virion containing two different RNA molecules (heterodimeric particles). Analysis of retroviral recombinants formed after a single round of replication revealed that (i) the nonselected markers changed more frequently than expected from the rate of recombination of selected markers; (ii) the transfer of the initially synthesized minus strand strong stop DNA was either intramolecular or intermolecular; (iii) the transfer of the first synthesized plus strand strong stop DNA was always intramolecular; and (iv) there was a strong correlation between the type of transfer of the minus strand strong stop DNA and the number of template switches observed. These data suggest that retroviral recombination is ordered and occurs during the synthesis of both minus and plus strand DNA.

Biological Evolution↗
PubMed1990

Identification of an insertion-sequence-like genetic element in the newly recognized human pathogen Mycoplasma incognitus.

Cloned 2.2-kb DNA (plasmid psb-2.2) of Mycoplasma incognitus, a pathogen in AIDS and non-AIDS patients [Lo et al., Am. J. Trop. Med. Hyg. 41 (1989) 364-376; 601-616], contains a 1405-bp genetic element closely resembling bacterial insertion sequence (IS) elements. This IS-like element has 29-bp terminal inverted repeats with seven mismatches, is immediately flanked by 3-bp direct repeats, and has typical stem-and-loop structures at or near both the termini. Two potential open reading frames (ORF-1 and ORF-2) encode 143 amino acids (aa) and 103 aa, respectively, in this IS-like element. Part (57 aa) of the deduced aa sequence of ORF-2 has a significant homology (43%) with the putative transposase of Escherichia coli IS3. In this study, a series of synthetic oligodeoxyribonucleotides each containing a specific sequence of a selected segment in psb-2.2, have been used as probes which reveal that the IS-like element occurs more than ten times in the genome of M. incognitus. This potentially transposable element has many characteristic features in common with bacterial IS elements.

Amino Acid Sequence↗
PubMed1990

Detection of prosthetic mitral valve leak: a comparative study using transesophageal echocardiography, transthoracic echocardiography, and auscultation.

Paravalvular leakage is a major complication of prosthetic valve dysfunction. Sixty-one subjects with valvular heart disease who had received prosthetic mitral valve replacement 5 months to 5 years before (43 received a porcine prosthesis and 18 received Bjork-Shiley valve prostheses) were evaluated for this complication. Careful auscultation was performed by two experienced cardiologists followed by transthoracic and transesophageal echocardiography. Physiologic leaks were detected in all Bjork-Shiley valves, but in only 30% of porcine valves using transesophageal echocardiography. These regurgitant jets were flame-like, with mean low velocities of 50 +/- 12.3 cm/sec and 48 +/- 18.2 cm/sec in the two types of valves. Neither transthoracic echocardiography nor auscultation could detect physiological regurgitant jets. Ten cases with paravalvular leak were detected by transesophageal echocardiography and subsequently demonstrated by left ventriculography (7 porcine, 3 Bjork-Shiley valves). Pathologic regurgitant jets were seen as high-velocity, systolic-retrograde turbulent flow across the prosthesis. However, only 6 cases of prosthetic valve dysfunction were detected by transthoracic echocardiography, 4 cases of mild paravalvular leakage went undetected. Thirteen of the 61 subjects had an apical systolic murmur and suspected prosthetic valve leakage; in 10 of the 13 cases the findings corresponded to those obtained by transesophageal echocardiography. In 3 cases of double valve replacement with Bjork-Shiley valves the magnitude of the leakage was overestimated by auscultation.

Adult↗
PubMed1990

A two-compartment cell entrapment bioreactor with three different holding times for cells, high and low molecular weight compounds.

A new bioreactor for animal cell cultivation employs two compartments for cells and medium respectively. The two chambers are separated by an ultrafiltration membrane. Cells and solution of collagen or collagen/chitosan mixture were loaded to the cell chamber and were allowed to form gel inside. Contraction of the cell-laden gel occurred subsequently to create a new zone in the cell chamber. In such a bioreactor cells are retained in the reactor, the high molecular product(s) accumulate in the cell chamber, while the small molecular weight nutrients and metabolites are replenished and removed from the medium chamber. By adjusting the flow rates for cell and medium chambers, the resident time for cells, high and low molecular weight components of the system can be manipulated separately. The new bioreactor, in both flat-bed and hollow-fiber configurations, was used to cultivate recombinant human cell, 293, for Protein C production over 60 to 90 days.

Biological Products↗