Biomedical subjects

R Tom

Publications and source records attributed to R Tom.

12 recordsLinked to original sources

PubMed1999

On-line monitoring of the progress of infection in Sf-9 insect cell cultures using relative permittivity measurements.

The use of on-line relative permittivity (epsilon') measurements for monitoring cultures of Sf-9 cells was evaluated in a batch culture and a batch infected with a baculovirus expressing beta-galactosidase. It was found that viable cell density and volume essentially accounted for all the variation in epsilon' in both non-infected and synchronously infected cultures, indicating that the epsilon' of a cell suspension was sensitive only to changes in the viable cell population. Additionally the parameter provided clearly defined signposts of the progress of the infection.

Animals↗

PubMed1998

Identification and crystallization of a protease-resistant core of calnexin that retains biological activity.

Calnexin is a molecular chaperone that facilitates folding of glycoproteins in the endoplasmic reticulum (ER). The cloned lumenal domain of canine calnexin, cnxDeltaTMC, retains its biological activity without the transmembrane and cytosolic region. For the purpose of structure determination we generated a crystallizable core by mild proteolysis and identified its termini by N-terminal sequencing and molecular mass determination. A truncated gene was cloned accordingly. Its product, cnxDeltaN25C15, was purified to apparent homogeneity and crystallized. This truncated variant remains biologically active as shown by its binding to monoglucosylated oligosaccharides and functional interaction with ERp57. A heavy atom derivative was identified.

Amino Acid Sequence↗

PubMed1994

Maximization of recombinant protein yield in the insect cell/baculovirus system by one-time addition of nutrients to high-density batch cultures.

Suspension cultures of Sf-9 cells at different stages of growth were infected with a recombinant baculovirus expressing beta-galactosidase, using a range of multiplicities of infection (MOI) of 0.05 to 50. Following infection, the cells were resuspended either in the medium in which they had been grown or in fresh medium. Specific beta-galactosidase yields were not markedly affected by either MOI or medium change in cultures infected in early exponential phase (< or = 3 x 10(6) cells mL-1). In cultures infected at later growth stages, beta-galactosidase yields could only be maintained by medium replacement. The possibility that this requirement for medium replacement is due either to the accumulation of an inhibitory byproduct or nutrient limitation was examined. Alanine, a major byproduct of cultured insect cell metabolism, did not significantly reduce recombinant protein yield when added to infected cultures in concentrations of up to 40 mM. Following a factorial design, various nutrient concentrates were added alone or in combination to cultures infected in late exponential phase. Additions that included both yeastolate ultrafiltrate and an amino acid mixture restored specific beta-galactosidase yields to levels observed at earlier growth stages or in late stages with medium replacement; the addition of these concentrates, by permitting production at higher cell density, led to increases in the volumetric yield of recombinant protein. Together or separately, the concentrates when added to uninfected late exponential phase cultures, lead to a doubling of the maximum total cell protein level normally supported by unamended medium.

Alanine↗

PubMed1993

Effect of platelet-activating factor (PAF) on human spermatozoa-oocyte interactions.

The effect of exogenous platelet-activating factor (PAF) on human spermatozoa was examined by monitoring acrosomal status and hamster oocyte penetration. Induction of the acrosome response by PAF was dose, and time, dependent and required capacitated spermatozoa. Addition of 10(-14) mol PAF l-1 enhanced the acrosome reaction eightfold compared with controls treated with human serum albumin (HSA). Similar concentrations of lyso-PAF failed to induce acrosomal loss and preincubation of spermatozoa with CV3988, an inhibitor of PAF, prevented PAF induction of the acrosome reaction. PAF significantly increased the number of zona-free hamster oocytes penetrated compared with controls (9.8 +/- 0.5 of 25 oocytes were penetrated by control spermatozoa compared with 23.3 +/- 0.8 out of 25 oocytes penetrated after incubation of spermatozoa with 10(-14) mol PAF l-1; 93% of all oocytes were penetrated by at least one spermatozoon following incubation with PAF), and also increased the number of decondensed spermatozoa found per egg during the sperm penetration assay (from 1.7 +/- 0.3 spermatozoa/egg with control spermatozoa to 3.3 +/- 0.5 spermatozoa/egg with PAF-treated spermatozoa). PAF-induced increases in acrosome reaction and sperm penetration assay values were similar to effects obtained with human follicular fluid and were calcium dependent. Induction of the acrosome reaction by physiological concentrations of PAF appeared to be morphologically similar to the response induced by follicular fluid as assessed by electron microscopy.

Acrosome↗

PubMed1991

Effect of culture process on alkaloid production by Catharanthus roseus cells. I. Suspension cultures.

The processes for production of indole alkaloids in shake flask suspension cultures of Catharanthus roseus cells using Zenk's alkaloid production medium (APM) were evaluated. The 1-stage process consisted of inoculating APM and incubating for 15 days. The 2-stage process involved 6 d of cultivation in growth medium followed by 15 d of incubation in APM. Growth, main nutrient consumption and alkaloid production were monitored. Both culture processes produced approximately 20 g dw per 1 biomass. However, 2-stage cultures yielded an inorganic nutrient richer and more active plant cell biomass, richer in inorganic nutrients, as indicated by higher (greater than 70%) nutrient availability and consumption. Total and individual indole alkaloid production were 10 times higher (740 mg l-1 and 25 to 4000 micrograms per g dw, respectively) for 2-stage than for 1-stage cultures. For both processes, highest alkaloid productivity coincided with complete extracellular consumption of major inorganic nutrients, especially nitrate, by the cells. Complete carbohydrate consumption in 2-stage cultures resulted in a 40% decline in production. Small but significant (approximately 10%) product release was observed for both culture regimes, which seemed not to be related to cell lysis.

Alkaloids↗

PubMed1991

Effect of culture process on alkaloid production by Catharanthus roseus cells. II. Immobilized cultures.

Two processes for the production of indole alkaloids 2 l surface-immobilized bioreactor cultures of Catharanthus roseus cells using Zenk's Alkaloid Production Medium (APM) were evaluated. The 1-stage process consisted of inoculating APM containing bioreactors and incubating for 15 d. The 2-stage process involved inoculating growth medium-containing bioreactors, growing the immobilized cultures for a certain period of time and subsequently replacing this medium with APM. The production stage which lasted for 15 d. High production in 2-stage cultures required the replacement of the growth regulator 2,4-dichlorophenoxyacetic acid by indole-3-acetic acid in the growth medium and a growth stage of 6 d (late exponential phase) before production initiation. Growth, main nutrient consumption and alkaloid production were monitored. Both culture regimes resulted in similar biomass production, dw (10-13 g l-1). The 2-stage cultures yielded biomass richer in organic nutrients (200-300%) and with higher respiratory activity (approximately 250%), indicated by their lower biomass-to-carbohydrate yields (31% and 26%), as compared to 1-stage cultures (41%). Two-stage cultures produced more known products (10 as compared to 6) at yields (5 to 4800 micrograms g-1) 3 to 5 times higher than 1-stage cultures. More alkaloids were alkaloids released in the medium of 2-stage cultures, under non-lysing conditions, (20 to 4700 micrograms l-1) than in 1-stage cultures (20 to 460 micrograms l-1). These results were compared to those obtained from shake flask cultures performed at the same time, with the same C. roseus cell line and under similar regimes and reported previously. Suspension and immobilized cultures performed according to the 1-stage regime showed similar total production. However, release of known alkaloids was 2 to 3 times higher in immobilized than in suspension cultures. Total alkaloid production of 2-stage suspension cultures was 3.8-fold higher than 2-stage immobilized cultures. Two stage immobilized cultures released 4 more known alkaloids than the 2-stage suspensions. Lower oxygen availability in the 2 l immobilized cultures may explain lower specific growth rates (0.15-0.22 d-1) and total alkaloid production levels, compared to 200 ml suspension cultures (0.2-0.4 d-1) reported in our previous paper.

Alkaloids↗

PubMed1991

Stimulated indole alkaloid release from Catharanthus roseus immobilized cultures. Initial studies.

Vacuolar sequestration of valuable secondary metabolites remains the major limitation to the use of immobilization technology for large scale plant-cell-based bioprocesses, which otherwise may be a more efficient culture system than suspension for this biomass. In this initial study, the release of indole alkaloids produced by immobilized Catharanthus roseus cells cultured in Zenk's Alkaloid Production Medium was evaluated. Unstimulated alkaloid release in immobilized cultures reached levels of 10 to 50% of total production or 3 to 100% of known alkaloid content (30 to 4700 micrograms l-1), which was higher than that found for suspension cultures of the cell line used (10 to 25% of total production) without apparent cell lysis. Modifications of the medium pH value of immobilized cultures were explored in order to improve this release. Periodical additions of acid (HCl 0.1 N) or base (KOH 0.1 N) solutions (2% v/v) to different cultures resulted in rapid (less than 3 h) and transient variations in extracellular pH value from 5.5 to 4.3, and 5.8 to 8.5, respectively. In both cases, these variations provoked significant increase in total alkaloid (from approximately 5-10 mg l-1 to 15 mg l-1), ajmalicine (from 0 to approximately 0.29 mg l-1) and serpentine (from 0 to approximately 0.20 mg l-1) release, without apparent cell lysis or decrease in the culture viability. This product release was estimated to represent 100% of alkaloids produced.

Alkaloids↗

PubMed1991

Platelet-activating factor in sperm from fertile and subfertile men.

STUDY OBJECTIVE: To examine the proposed role of platelet-activating factor (PAF) as a mediator of sperm function first by quantitating the concentration of PAF in sperm and second by incubating sperm with PAF and assessing the change in sperm motion parameters with a computerized motion analysis system. DESIGN: Retrospective, case-control study. SETTING: University hospital urology clinic. PATIENTS: Concentrations of PAF were measured in sperm from 6 randomly chosen normal donors and 8 asthenozoospermic patients. The change in sperm motion parameters was assessed in sperm from 10 randomly chosen normal donors and 7 asthenozoospermic patients. RESULTS: Platelet-activating factor concentrations were significantly higher (P less than 0.05) in spermatozoa from asthenozoospermic patients (149 +/- 18.0 fmol/10(6) sperm) than in sperm from normal donors (91.9 +/- 13.0 fmol/10(6) sperm). Exogenous PAF did not enhance the percent motility of precapacitated or capacitated sperm from either patients or donors, and computerized semen analysis revealed no enhancement of sperm motion parameters in asthenozoospermic patients after addition of PAF to either precapacitated or capacitated sperm. CONCLUSION: Platelet-activating factor does not appear to have therapeutic value as a means of enhancing sperm motility.

Case-Control Studies↗

PubMed1989

Semen manipulation: improved sperm recovery and function with a two-layer Percoll gradient.

The authors compared a simple, two-layer Percoll density gradient technique with the swim-up technique for semen preparation in 128 men. In samples from normospermic (n = 55), oligospermic (n = 26), and asthenospermic (n = 29) men, the Percoll technique significantly improved yield, percent motility, and absolute number of motile sperm recovered, but in samples from oligoasthenospermic men (n = 18), only percent motility was improved. The Percoll density gradient also selected sperm with markedly improved function as assessed by both the sperm penetration assay and the fertility index. In 37 samples negative on the sperm penetration assay when processed with the swim-up technique, 19 (51%) became positive when processed with the Percoll technique. The Percoll density gradient is an improved method for semen manipulation as it allows greater recovery of sperm with higher motility and improved sperm function.

Adult↗

PubMed1989

Immunological status of patients before and after vasovasostomy as determined by the immunobead antisperm antibody test.

With the immunobead antisperm antibody test a prospective study was conducted to evaluate the immune status of 55 men before and after vasectomy reversal. A third of the vasectomy patients (19 of 55) had significant serum-sperm antibodies (20 per cent binding or more) detected by the indirect immunobead antisperm antibody test. Of 31 vasovasostomy patients 12 (38 per cent) had significant sperm-surface antibodies (20 per cent binding or more) by the direct immunobead antisperm antibody test. Preoperative serum assays correctly classified the antibody status of 69 per cent of the vasectomy patients. The immunological impact of spermatic granuloma formation, duration of vasal obstruction, patient age and presence of sperm in the vasal fluid at operation also were assessed. An inverse relationship between the proportion of antibody-bound sperm and the percentage of motile sperm in the ejaculate of vas reversal patients was found with videomicrographic semen analysis. The percentage motility was significantly lower among patients with greater quantities of sperm-surface antibodies. No other parameter of semen analysis showed this difference when compared for positive or negative immunobead antisperm antibody test results.

Adult↗

PubMedDate not supplied

Growth, nutrient consumption, and end-product accumulation in Sf-9 and BTI-EAA insect cell cultures: insights into growth limitation and metabolism.

Growth, nutrient consumption, and end-product accumulation were quantitated in shake-flask cultures of two insect cell lines, Sf-9 and BTI-EAA, in three different serum-supplemented media. Per cell consumption or production rates were calculated for most medium components analyzed. Glucose was growth-limiting in TNM-FH medium and was the most important single source of organic-C for the cells in all cultures. Cells utilized fructose and maltose but not sucrose. alpha-Ketoglutarate and malate contributed significantly to the carbon budget of cells in TNM-FH. Lactate generally did not accumulate during growth. Most of the amino acids were consumed by the cells, with the exception of alanine which was produced. Most of the amino acids appeared to be present in adequate supply in the cultures. Glutamate was generally the most rapidly consumed of the amino acids, followed closely by glutamine. Alanine accumulation was correlated with glucose consumption. In Sf-9 cultures, ammonia accumulated only slightly or not at all as long as glucose was present in the medium, and uric acid was detectable at the end of growth and in the stationary phase. Added ammonia up to a concentration of 10 mM did not affect the growth of either cell line. Ammonia and lactate may be of less importance in limiting growth in insect cell cultures than in mammalian cell cultures. A hypothetical outline of the major metabolic pathways of the cultured insect cells is presented on the basis of information obtained here and in the literature.

Amino Acids↗

PubMedDate not supplied

Human sperm hypo-osmotic swelling test: relationship to sperm fertilizing ability.

The simple hypo-osmotic swelling test (HOS) has been proposed as a replacement for the labor-intensive sperm penetration assay (SPA), but its reliability is still controversial. To investigate the clinical correlation between results of the HOS and the SPA, we studied the subpopulation of washed "motile" and capacitated spermatozoa as well as sperm in the original sample in both a heterogeneous patient population being evaluated for infertility (n = 47) and a donor population (n = 13). In our patient population, the correlation between HOS results and conventional semen parameters, i.e., motility and vitality, was poor. Although the mean HOS values of the donor and patient populations differed significantly, the overlap in individual variation was too great for practical application. Using motile sperm tested two hours after separation from the seminal plasma, the correlation between a positive HOS and a positive SPA was good, with only a 4% false-negative rate. However, the correlation in capacitated sperm (20 hours) or semen itself was far less reliable. Although HOS tests correlated well with the SPA in individuals with normal sperm, in those with negative results on SPA the predictive value was extremely poor: divergent findings ranged from 32% using semen samples to 74% in the two-hour motile population. The events in the fertilizing process measured by the sperm penetration assay appear to be different from those assessed by the hypo-osmotic test. The HOS test, therefore, could not be used to screen or predict results of the SPA.

Female↗