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PubMed2000

Cell death in regenerating populations of neurons in BDNF mutant mice.

There are two populations of neurons which are continually renewed in the adult, the dentate gyrus granule neurons and the olfactory bulb granule and periglomerular neurons. In the dentate gyrus, a secondary proliferative zone termed the subgranular zone is established along the interface between the dentate gyrus and the hilus where granule cells are born throughout life. Olfactory bulb neurons are generated in the anterior subventricular zone of the lateral ventricle and migrate via the rostral migratory stream to the olfactory bulb. We examined animals lacking brain-derived neurotrophic factor (BDNF) in order to establish whether this neurotrophin could be involved in the generation and/or survival of these neurons in vivo. We find that cells in nestin-positive regions of both the subgranular layer of the dentate gyrus and the subventricular zone of the olfactory bulb undergo apoptosis starting 2 weeks after birth in the absence of BDNF. However, increased apoptosis was not limited to precursors, as apoptotic cells were also found in the granule cell layer of the dentate gyrus and in the granule and periglomerular layers of the olfactory bulb. The excessive cell death was limited to these populations of neurons as no excessive cell death was detected in other forebrain areas. We conclude that BDNF is essential for the survival of neurons specifically in populations which are continuously being regenerated in the brain.

Aging↗

PubMed2001

Pretransplant donor-specific helper T cell reactivity as a tool for tailoring the individual need for immunosuppression.

BACKGROUND: A reliable immunological assay for quantification of donor-specific alloreactivity to identify patients at risk for future allograft rejection would be a helpful tool in organ transplantation. Therefore, we questioned whether the T cell reactivity in patients measured before transplantation was predictive for the occurrence of acute rejection during the first year after kidney transplantation. METHODS: The pretransplant T cell reactivity of peripheral blood mononuclear cells to donor and third-party antigens was tested in mixed lymphocyte cultures, and to tetanus toxoid. In addition, we measured the frequency of donor and third-party reactive helper T lymphocyte precursor and cytotoxic T lymphocyte precursors using limiting dilution analysis. RESULTS: Patients who experienced acute rejection had significantly higher donor-specific mixed lymphocyte cultures responses (n=38; median stimulation index): 113 vs. 15, P=0.005) and helper T lymphocyte precursor frequency (n=37; median 194/106 vs. 62/106, P=0.009) measured before transplantation compared to patients without acute rejection. All patients with a low mixed lymphocyte culture response (stimulation index</=20; 13/13 vs. 12/25, P=0.001) and an undetectable helper T lymphocyte precursor frequency (<10/106 peripheral blood mononuclear cells; 7/7 vs. 17/30, P=0.04) before transplantation did not experience acute rejection. The donor-specific cytotoxic T lymphocyte precursor frequency (n=34; median 53/106 vs. 28/106, P=0.58) and tetanus toxoid-reactivity (n=38; median stimulation index: 53 vs.16, P=0.56) measured before transplantation did not correlate with acute rejection. No correlation between third-party reactivity and acute rejection was observed. CONCLUSIONS: From these results we conclude that despite the current HLA matching criteria, undetectable helper T lymphocyte precursor frequency and low mixed lymphocyte culture responses against donor antigens measured before transplantation are predictive for a rejection-free first posttransplant year. These in vitro assays can be used to identify patients who require less immunosuppression after transplantation.

Acute Disease↗

PubMed2000

Three biotechnical processes using Ashbya gossypii, Candida famata, or Bacillus subtilis compete with chemical riboflavin production.

Chemical riboflavin production, successfully used for decades, is in the course of being replaced by microbial processes. These promise to save half the costs, reduce waste and energy requirements, and use renewable resources like sugar or plant oil. Three microorganisms are currently in use for industrial riboflavin production. The hemiascomycetes Ashbya gossypii, a filamentous fungus, and Candida famata, a yeast, are naturally occurring overproducers of this vitamin. To obtain riboflavin production with the gram-positive bacterium Bacillus subtilis requires at least the deregulation of purine synthesis and a mutation in a flavokinase/FAD-synthetase. It is common to all three organisms that riboflavin production is recognizable by the yellow color of the colonies. This is an important tool for the screening of improved mutants. Antimetabolites like itaconate, which inhibits the isocitrate lyase in A. gossypii, tubercidin, which inhibits purine biosynthesis in C. famata, or roseoflavin, a structural analog of riboflavin used for B. subtilis, have been applied successfully for mutant selections. The production of riboflavin by the two fungi seems to be limited by precursor supply, as was concluded from feeding and gene-overexpression experiments. Although flux studies in B. subtilis revealed an increase both in maintenance metabolism and in the oxidative part of the pentose phosphate pathway, the major limitation there seems to be the riboflavin pathway. Multiple copies of the rib genes and promoter replacements are necessary to achieve competitive productivity.

Bacillus subtilis↗

PubMed2005

Activated marrow-infiltrating lymphocytes effectively target plasma cells and their clonogenic precursors.

A major limitation of adoptive immunotherapy is the availability of T cells specific for both terminally differentiated tumor cells and their clonogenic precursors. We show here that marrow-infiltrating lymphocytes (MILs) recognize myeloma cells after activation with anti-CD3/CD28 beads with higher frequency than activated peripheral blood lymphocytes from the same patients. Furthermore, activated MILs target both the terminally differentiated CD138+ plasma cells and the myeloma precursor as shown by profound inhibition in a tumor clonogenic assay. The presence of antigen in the marrow microenvironment seems to be important for the maintenance of tumor specificity. Taken together, these results highlight the intrinsic tumor specificity of MILs and describe a novel approach for the generation of tumor-specific T-cell populations suitable for adoptive immunotherapy of multiple myeloma.

Adult↗

PubMed1995

Interleukin-1 beta secretion. A possible multistep process that is regulated in a cell type-specific manner.

In a prior study, we found that the processed form of human interleukin-1 beta (mature IL-1 beta) is secreted to a significantly greater extent than the precursor form of the protein, indicating that the precursor domain acts in some manner to reduce the secretory potential of the protein. In view of this observation, we sought to define the sequence(s) in the IL-1 beta precursor that limit the secretion of the protein as well as the sequences in the mature protein that promote secretion. The P388D1 murine macrophage cell line and the Jurkat human T-cell line were transiently transfected with cDNA expression vectors encoding truncated forms of human precursor IL-1 beta proteins, lacking either the first 76, 94, 99, or 104 amino acids. The removal of increasing numbers of precursor amino acid residues resulted in a graded increase in the secretion of the truncated precursor IL-1 beta proteins from both cell lines. The minimal region of the precursor sequence required to inhibit the optimal secretion of IL-1 beta occurs between amino acids 100 and 104 for P388D1 cells and 95-99 for Jurkat cells. Deletion of the amino acids within these regions increased the secretion level of the truncated proteins to that of mature IL-1 beta. Mutagenesis of the mature IL-1 beta sequence revealed that a region of basic amino acids may play an important role in the optimal secretion of mature IL-1 beta in P388D1 cells, but not in Jurkat cells. Based on the differences in the structural requirements for IL-1 beta secretion in P388D1 and Jurkat cell lines, it is likely that the secretion of IL-1 beta may be subject to multiple levels of regulation that are differentially operative in different cell types.

Amino Acid Sequence↗

PubMedDate not supplied

Regulation of natural killer cell production in bone marrow of mice: no evidence for negative feedback control.

The possible presence of a negative feedback control mechanism regulating natural killer (NK) cell production in the bone marrow of B6 mice was investigated by depleting NK cells with a single intravenous injection of anti-ASGM1 antibody. At times ranging from 1 to 21 days following injection, the response of the bone marrow cells to this depletion was assessed by measuring both NK activity against YAC-1 target cells in a 51Cr assay and the frequency of NK precursors by limiting dilution analysis. For comparison, measurements of lytic activity were also done in the peripheral blood and spleen. Anti-ASGM1 injection resulted in a depletion of mature NK cell activity in all compartments tested as expected, as well as the depletion of NK precursors from the bone marrow. Clearance of the anti-ASGM1 from the circulation of recipient mice was biphasic. More than 99% of the antibody was eliminated in the first 4 min, while the remaining 0.24% had a half-life in the serum of 6 days. Bone marrow was able to produce new lytic NK cells which were detectable between days 6 and 10 after depletion. The bone marrow of the NK cell-depleted mice did not show an 'overshoot' (compensatory increase) in NK cell production through day 21 after depletion. It therefore appears that the NK cell production in the bone marrow is independent of the activity of the peripheral NK cell pool.

Animals↗

PubMed1995

Accurate quantitation of residual B-precursor acute lymphoblastic leukemia by limiting dilution and a PCR-based detection system: a description of the method and the principles involved.

The detection of residual leukemia cells in the bone marrow of patients during morphologic remission has been greatly facilitated by use of the polymerase chain reaction (PCR) to amplify leukemia-specific sequences. While the current PCR strategies for estimating the amount of residual leukemia claim a detection sensitivity of one leukemia cell amongst 10(5) or 10(6) normal cells, a rigorous assessment of the relative error associated with these techniques has not been presented. We have developed a method of estimating the amount of residual leukemia in remission marrows that is analogous to the limiting dilution assays used to determine the frequency of immunocompetent cells in a responder cell population. Using this method we measured the fraction of all-or-none (i.e. positive or negative) reactions of the PCR amplification of the leukemia-specific IgH gene rearrangement in replicate samples of serial dilutions of DNA obtained from diagnostic bone marrow specimens from 15 children with B-precursor acute lymphoblastic leukemia (ALL). A sigmoid curve representing the fraction of positive PCR reactions at a given dilution of leukemia DNA was found to be the best fit to the data. The narrowness of the log-linear region of this curve prevents the direct application of the analysis methodology that has previously been described for limiting dilution assays. However, the residual leukemia burden during morphological remission in these 15 patients and in two additional patients who experienced relapse could be estimated by the described dilution analysis method using the best-fit equation. Furthermore, the data generated for diagnostic, remission and relapse marrow samples exhibited a small interspecimen variation. The results suggest that this method can reliably estimate residual leukemia over a range of five orders of magnitude. Although the PCR reaction appears to be one of the most sensitive methods for detecting residual leukemia, all techniques based on this procedure, including our own, must exhibit limitations inherent to the amplification process. Our estimates or relative error suggest that a realistic limit for the PCR estimation of residual leukemia lies in the range of one leukemia cell per 10(5) normal cells. The suggested method is rapid, technically simple and relatively inexpensive. Furthermore, the principles that it is based upon can be applied to any PCR-based strategy.

Bone Marrow Examination↗

PubMed2005

Effects of later-occurring nonlinguistic sounds on speech categorization.

Nonspeech stimuli influence phonetic categorization, but effects observed so far have been limited to precursors' influence on perception of following speech. However, both preceding and following speech affect phonetic categorization. This asymmetry raises questions about whether general auditory processes play a role in context-dependent speech perception. This study tested whether the asymmetry stems from methodological issues or genuine mechanistic limitations. To determine whether and how backward effects of nonspeech context on speech may occur, one experiment examined perception of CVC words with [ga]-[da] series onsets followed by one of two possible embedded tones and one of two possible final consonants. When the tone was separated from the target onset by 100 ms, contrastive effects of tone frequency similar to those of previous studies were observed; however, when the tone was moved closer to the target segment assimilative effects were observed. In another experiment, contrastive effects of a following tone were observed in both CVC words and CV nonwords, although the size of the effects depended on syllable structure. Results are discussed with respect to contrastive mechanisms not speech-specific but operating at a relatively high level, taking into account spectrotemporal patterns occurring over extended periods before and after target events.

Acoustic Stimulation↗

PubMed1983

Cellular events in tolerance IX: maintenance of immunological tolerance in the presence of normal b-cell precursors and in the absence of demonstrable suppression.

The cellular events involved in immunological tolerance to fluoresceinated sheep gammaglobulin (FL-SGG) were analyzed at the level of hapten-specific B cells. One single iv injection of FL-SGG induced tolerance as measured by challenge with thymus-dependent (FL-KLH) or thymus-independent (FL-Ficoll) antigens in vivo or thymus-independent (FL-LPS) antigen in vitro. As noted earlier, unresponsiveness was maintained until 6-8 weeks after tolerance induction. Limiting-dilution precursor analysis demonstrated a reduction in B-cell precursors on Day 7 after tolerogen treatment; precursor frequencies returned to control levels by 3-4 weeks. This recovery of precursors in the presence of stable tolerance was not due to suppressor activity. Rather, results show that tolerant hapten-specific B cells are clonally anergic and display a reduced burst size in response to antigen. Hence, unresponsiveness is maintained in the presence of apparently normal precursor levels by an intrinsic defect in antigen-specific B cells.

Animals↗

PubMed1999

Expression of Flt3 and c-kit during growth and maturation of human CD34+CD38- cells.

Studies of murine stem cells suggest that the cytokine receptors Flt3 and c-kit are expressed differentially on the earliest reconstitutional cells, such that Flt3 is not expressed until after stem cell activation. Much less is known about the expression of Flt3 and c-kit on primitive human cells, especially those mobilized into circulation for transplantation. In this study, early circulating precursors were analyzed for expression of Flt3 at the gene and protein levels. Flow cytometric studies showed that >90% of CD34+CD38- cells expressed Flt3 antigen (CD135). The proportion of fresh CD34+ cells expressing Flt3 decreased as CD38 staining increased. These results were confirmed by reverse transcriptase polymerase chain reaction (RT-PCR) analyses, which showed that Flt3 gene expression generally was limited to the CD34+CD38- population. Because Flt3 ligand (FL) enhances the growth and/or maintenance of primitive cells, it was important to know how long early cells retain Flt3 receptor expression in expansion culture. Both RT-PCR analyses and functional tests demonstrated that primitive cells are capable of expressing Flt3 for as long as 2 weeks in liquid medium. During the first week of culture, FL enhanced the generation of cells and progenitors without causing a loss of primitive CD34+CD38-Flt3+ cells. Flt3 expression in cell cultures was limited to precursors retaining a CD34+CD38(-/lo) phenotype. Because the most primitive human precursors are believed to express c-kit at a low level, we examined the FL responsiveness of CD34+CD38-c-kit(-/lo) cells and CD34+CD38-c-kit+ cells. CD34+CD38-c-kit(-/lo), cells constituted a small fraction (12%) of the CD34+CD38- population. Whereas both c-kit(-/lo) and c-kit+ subsets were stimulated by FL, cell expansion (p < 0.01) and colony formation (p < 0.01) were greater and maintained longer with CD34+CD38-c-kit(-/lo) cells. Furthermore, the rapid response to FL suggests that primitive CD34+CD38-c-kit(-/lo) cells express Flt3 at the time of isolation or shortly thereafter. These results demonstrate the presence of Flt3 on CD34+CD38 blood cells and suggests that Flt3 also may be present on a c-kit(-/lo) subset, among the most primitive in circulation. Flt3 is lost during maturation to committed (CD34+CD38+) lineages. Addition of FL to primitive cell cultures stimulates cell expansion while maintaining early CD34+CD38-Flt3+ precursors for at least 7 days. The possible existence of a more primitive CD34+CD38-c-kit(-/lo) Flt3(-/lo) precursor remains to be determined.

ADP-ribosyl Cyclase↗

PubMed1999

Giardia induces proliferation and interferon gamma production by intestinal lymphocytes.

BACKGROUND: Murine intraepithelial lymphocytes kill Giardia lambia; responses of human intestinal lymphocytes to this parasite are unknown. AIMS: To examine giardia induced proliferation, interferon gamma production, migration, and cytotoxicity by lymphocytes from the human intestine and peripheral blood. METHODS: Giardia were added to intraepithelial lymphocytes, lamina propria lymphocytes, and peripheral blood lymphocytes, obtained from jejunal mucosa and blood of otherwise healthy patients undergoing gastric bypass surgery for morbid obesity. Proliferation was measured by 3H-thymidine incorporation; frequency of proliferation precursors, by limiting dilution analysis; interferon gamma production, by ELISA; cytotoxicity, by 51Cr release of radiolabelled giardia and by release of serine esterases by effector lymphocytes that mediate cytotoxicity. RESULTS: The CD4+ T lymphocytes from intestine and blood proliferated in response to giardia. The stimulus by the parasite was mitogenic rather than antigenic due to the fact that the peak response was on day 3 rather than day 6, and the large number of precursors was in the range of that for mitogens. CD4+ T lymphocytes from both sites produced interferon gamma in response to giardia. Lymphocytes did not migrate towards or kill the parasite. CONCLUSIONS: Giardia induced the same degree of proliferation and interferon gamma production by CD4+ T lymphocytes in intestine and blood, but did not trigger cytotoxicity or migration.

Animals↗

PubMed1999

Design of limiting dilution analysis experiments for helper T lymphocyte precursor frequency determination in the context of allogeneic bone marrow transplantation.

Helper, interleukin 2 (IL-2) producing, T lymphocyte precursor (HTLp) frequency determination by limiting dilution analysis (LDA) is of value for quantifying alloreactivity in allogeneic bone marrow transplantation (BMT). LDA assays are labour-intensive and time-consuming to perform and the numbers of donor and recipient cells available are limited. It is therefore important that the design of the experiment yields reliable frequencies with a minimum of effort and a realistic cell requirement. We have critically evaluated the methods proposed for LDA design by Strijbosch et al. [Strijbosch, L.W., Buurman, W.A., Does, R.J., Zinken, P.H., Groenewegen, G., 1987. Limiting dilution assays. Experimental design and statistical analysis. J. Immunol. Methods 97, 133] and by Blackett and Gordon [Blackett, N.M., Gordon, M.Y., 1996. Optimizing limiting dilution assays: frequency and 'ability' measurements of haemopoietic progenitor cells. Br. J. Haematol. 92, 507 (see comments)] and found them inadequate for this application. The estimation of the HTLp frequency is traditionally based on the single-hit Poisson model and the adequacy of this model was compared with that of a double-hit model. The results were in favour of the single-hit model. Ten different LDA experimental designs were explored by Monte Carlo simulations. The optimal design exploits the maximal numbers of cells that can be obtained for analysis to estimate HTLp frequencies in the range 1:1,000,000-1:20,000 with a coefficient of variation of 10-20% and with a minimum of manual labour.

Bone Marrow Transplantation↗

PubMed1994

Limiting dilution analysis reveals the precursors of interleukin-4-producing CD4+ cells induced by protein immunization.

Although cytokine-producing T cells play a key role in the response to vaccination, they are not always revealed by antigen stimulation of primed lymphoid cells in vitro. In this study, mice were immunized subcutaneously with alum-precipitated keyhole limpet haemocyanin (KLH) in adjuvant to activate interleukin-4 (IL-4)-producing CD4+ T cells. IL-4 mRNA was the dominant cytokine mRNA species found in draining lymph nodes (LN) 7 days after immunization and its levels were increased after in vitro stimulation with KLH for 24 hr. IL-4 protein, on the other hand, was not detected in the supernatants of such antigen-stimulated cultures. The presence of T cells primed for IL-4 production was nevertheless suggested by the findings that primed LN cells produced low IL-4 titres in response to anti-CD3 antibody, whereas normal LN cells did not, and primed CD4+ LN cells produced readily detectable IL-4 levels in response to antigen after one or more cycles of in vitro restimulation. Culture at limiting dilution showed that 1-2% of 7 day KLH-primed CD4+ LN cells were clonogenic and specific for KLH without prior expansion in vitro, and that this frequency was markedly increased by repeated stimulation in bulk culture. Most clonogenic cells in primed LN gave rise to IL-4-secreting clones and a smaller number gave rise to interferon-gamma (IFN-gamma)-producing clones. The precursor frequency of IL-4-producing CD4+ cells in primed LN and the average IL-4 titre per cloned cell support the conclusion that these two parameters account for the low levels of IL-4 produced in bulk culture by LN cells from immunized mice.

Animals↗

PubMed1991

Optimising a limiting dilution culture system for quantitating frequencies of alloreactive cytotoxic T lymphocyte precursors.

We have developed a limiting dilution assay for estimating the frequencies of allo-MHC reactive cytotoxic T lymphocyte precursors (CTL-p) in peripheral blood. The culture conditions were optimised in order to give maximum sensitivity while at the same time maintaining a high degree of specificity. While the addition of filler cells, interleukin-1 and interleukin-4 were unnecessary, the addition of interleukin-2 (IL-2) was crucial for optimal expansion of the CTL-ps. However, excess exogenous IL-2 was found to give rise to nonspecific cytotoxicity, and 5 U/ml final concentration of recombinant IL-2 added on Days 3 and 6 was optimal. The establishment of baseline control cultures for determining positivity and negativity of the test cultures is discussed. Using this modified assay system, we demonstrated that the culture conditions fulfil "single hit" criteria and that the assay is highly specific.

Cell Differentiation↗

PubMed1989

[The structural organization of interferon molecules as precursors of immuno- and neuroactive oligopeptides].

The oligopeptides effecting signalling functions are postulated to emerge from protein precursors by limited proteolytic cleavage in close vicinity to appropriate cell receptors. A variety of immunological activities of interferons are probably mediated by this mechanism. The C-terminal tripeptides liberated from IFN-alpha in limited proteolysis reactions may result in a variety of polarins eliciting a wide spectrum of immunoregulatory effects, which in combination with the antiviral activity of IFN are responsible for effective defense mechanisms in the organism.

Animals↗

PubMed1980

Apparent involvement of ribonuclease D in the 3' processing of tRNA precursors.

Escherichia coli RNase D and RNase II have been purified to homogeneity and compared for their ability to remove extra nucleotides following the -C-C-A sequence in tRNA precursors. RNase D and RNase II are single-chain proteins with molecular weights of 38,000 and 78,000, respectively. Both enzymes require a divalent cation for activity on tRNA precursors, but, in addition, RNase II is stimulated by monovalent cations. RNase D specifically removes mononucleotide residues from a mixture of tRNA precursors to generate amino acid acceptor activity for essentially all amino acids. Although RNase II can also remove precursor-specific residues, no amino acid acceptor activity is recovered. Similarly, RNase D action on the E. coli tRNATyr precursor is limited, whereas RNase II causes extensive degradation. In contrast to the processive mode of hydrolysis by RNase II, RNase D removes nucleotides randomly and slows down greatly at the -C-C-A sequence, thereby allowing the tRNA to be aminoacylated and protected from further degradation. These results suggest that RNase D is the 3'-processing nuclease in vivo and that RNase II is a nonspecific degradative enzyme. The importance of RNA conformation for correct processing is also discussed.

Escherichia coli↗

PubMed2003

Genome expression analysis in yeast reveals novel transcriptional regulation by inositol and choline and new regulatory functions for Opi1p, Ino2p, and Ino4p.

In Saccharomyces cerevisiae, genes encoding phospholipid-synthesizing enzymes are regulated by inositol and choline (IC). The current model suggests that when these precursors become limiting, the transcriptional complex Ino2p-Ino4p activates the expression of these genes, whereas repression requires Opi1p and occurs when IC are available. In this study, microarray-based expression analysis was performed to assess the global transcriptional response to IC in a wild-type strain and in the opi1delta, ino2delta, and ino4delta null mutant strains. Fifty genes were either activated or repressed by IC in the wild-type strain, including three already known IC-repressed genes. We demonstrated that the IC response was not limited to genes involved in membrane biogenesis, but encompassed various metabolic pathways such as biotin synthesis, one-carbon compound metabolism, nitrogen-containing compound transport and degradation, cell wall organization and biogenesis, and acetyl-CoA metabolism. The expression of a large number of IC-regulated genes did not change in the opi1delta, ino2delta, and ino4delta strains, thus implicating new regulatory elements in the IC response. Our studies revealed that Opi1p, Ino2p, and Ino4p have dual regulatory activities, acting in both positive and negative transcriptional regulation of a large number of genes, most of which are not regulated by IC and only a subset of which is involved in membrane biogenesis. These data provide the first global response profile of yeast to IC and reveal novel regulatory mechanisms by these precursors.

Basic Helix-Loop-Helix Proteins↗

PubMedDate not supplied

Metabolic inhibitors, elicitors, and precursors as tools for probing yield limitation in taxane production by Taxus chinensis cell cultures.

Inhibition of biosynthetic enzymes and translation and translocation processes, elicitation, and precursor feeding were used to probe biosynthetic pathway compartmentation, substrate-product relationships, and yield limitation of the diterpenoid taxanes in cell cultures of Taxus chinensis (PRO1-95). The results suggest the following: (i) the source of isopentenyl pyrophosphate in taxane production is likely plastidic rather than cytoplasmic; (ii) baccatin III may not be a direct precusor of Taxol (Taxol is a registered trademark of Bristol-Myers Squibb for paclitaxel); (iii) baccatin III appears to have cytoplasmic and plastidic biosynthetic components, while Taxol production is essentially plastidic; and (iv) arachidonic acid specifically stimulates Taxol production but does not have a significant effect on baccatin III yield. Semiempirical mathematical models were used to describe these results and predict potential yield-limiting steps. Model simulations suggest that, under current operating conditions, Taxol production in Taxus chinensis (PRO1-95) cultures is limited by the ability of the cells to convert phenylalanine to phenylisoserine rather than by the branch-point acyl transferase. This result is supported by the lack of improvement of Taxol yield by feeding phenylalanine or benzoylglycine. The methods described in this article, while specifically expanding our knowledge of taxane production in PRO1-95 cultures, could be generally useful in investigating complex aspects of secondary metabolic pathways in plant cell cultures, especially when details of the pathway and compartmentation are sparse.

Alkaloids↗