Concerted action of RAS and G proteins in the sexual response pathways of Schizosaccharomyces pombe.

We have shown that the expression of mam2, the gene encoding the Schizosaccharomyces pombe P-factor pheromone receptor, is dependent upon components of the pheromone signal transduction pathway, including Ras1, Gpa1, Byr1 and Byr2, each of which is required for both conjugation and sporulation. Studies of the expression of mam2 in mutant S. pombe cells confirm previous conclusions, based on the ability of cells to sporulate, that the Byr1 protein kinase acts downstream of the Byr2 protein kinase and that both act downstream of Ras1, the S. pombe RAS homolog, and Gpa1, the G alpha component that mediates the occupancy of the mam2 receptor. In addition, our present studies show that Ras1 and Gpa1 each act downstream from the other and hence act in concert. The Spk1 kinase, which is required for conjugation and sporulation and which is a structural and functional homolog of the vertebrate MAP kinases, is not required for mam2 expression.     

Preliminary validation of an activation assay for ex vivo activated T cells utilized in cancer immunotherapy

An in vitro assay that measures the activation level of ex vivo activated (EVA) T cells currently being used in the adoptive immunotherapy of metastatic renal cell carcinoma has been developed. This assay is based on the ability of activated, but not resting. T cells to proliferate in response to the protein kinase C activator, phorbol myristate (PMA). To utilize this assay for in-process monitoring and control, we have begun an initial validation of the overall reproducibility of this assay. The proliferation of activated T cells in response to PMA, as measured by the mean cpm values of (3)H-thymidine incorporated, was demonstrated to have intra-assay coefficients of variation (cv's) for individual analysts that were typically less than 10% and rarely exceeded 20%. Activated T cells could be frozen and stored for at least 6 weeks with little or no deterioration in their ability to proliferate in response to PMA. Using these cells, inter-assay cv's that were typically less than 15% were obtained by individual analysts, and overall cv's of 10% to 25% were obtained for different samples assayed by different analysts at different times. This level of variability is very reasonable for a cellular assay. Furhter validation of this assay will address the issues of sensitivity, linearity and selectivity. To date, this assay has been used to analyze over 90 patient EVA cell samples and has revealed a broad range of proliferative responses to PMA. Taken together, these results suggest that this assay may be useful in defining the potency of the activated T cell used therapeutically.     

A protein involved in co-ordinated regulation of inorganic carbon and glucose metabolism in the facultative photoautotrophic cyanobacteriumSynechocystis PCC6803

The involvement of a gene ofSynechocystis PCC6803,icfG, in the co-ordinated regulation of inorganic carbon and glucose metabolism, was established. TheicfG gene codes for a 72 kDa protein, which shows no homology with those registered in data libraries. Expression oficfG required glucose, the actual inducer probably being glucose-6-phosphate, and was independent of light and of the external inorganic carbon concentration. Mutants carrying an inactivated copy oficfG were constructed. Their growth characteristics were identical to those of the wild type under all regimes except in limiting inorganic carbon with glucose being present either before or after the transfer to the limiting conditions. These conditions completely prevented growth, both in the light and in the dark. The inhibition could be relieved by several intermediates of the tricarboxylic acid cycle. Assays of various enzymic activities related to inorganic carbon uptake and to its assimilationvia either the Calvin cycle or phosphoenolpyruvate carboxylase did not reveal the level of action of IcfG. Possible models include a blockage of the assimilation of both carbon sources in the absence of IcfG, or the inhibition of Ci incorporation route(s) essential under limiting inorganic carbon conditions, even when glucose is present, and even in the dark.     

The effect of monoterpenes on astaxanthin production by a mutant ofPhaffia rhodozyma

Summary The total pigment and astaxanthin content ofPhaffia rhodozyma increased with increasing concentrations -pinene up to 500 l -pinene/l. Above this concentration the total pigment and astaxanthin content as well as the biomass production decreased. The addition of 500 l -pinene/l increased the total pigment content from 1652 g/g to 2201 g/g and the astaxanthin content from 1554 g/g to 1883 g/g. A sharp decrease in maximum specific growth rate occurred above 150 l -pinene/l.     

Overview of a quality assurance/quality control compliance program consistent with FDA regulations and policies for somatic cell and gene therapies: a four year experience

Somatic cell and gene therapy involve the application of biological technologies to an individual patient through the use of living cells which provide a therapeutic benefit (Aliski, 1991). Various forms of cellular and gene therapies are being developed and evaluated in an increasing number of clinical trials for congential and acquired disorders. The potential and progress of these therapeutic applications have resulted in an increasing effort by the Food and Drug Administration (FDA) to develop the regulatory framework under which these therapeutic approaches would insure safety and efficacy, the primary mandate of the FDA.Over five years ago Cellcor began to define the parameters, specifications, and conditions relevant to a Quality Assurance/Quality Control (QA/QC) program that has evolved to insure safety and maximize the efficacy of applications of the company'sex vivo technology, autolymphocyte therapy. Autolymphocyte therapy is an outpatient form of somatic cell immunotherapy based upon the infusion of T cells that have been activatedex vivo using a combination of previously generated autologous cytokines and an anti-CD3 monoclonal antibody.We have been able to demonstrate the feasibility for the safe, controlled, and consistent preparation and delivery of a cellular therapy by application of relevant GMP regulations. This presentation reviews aspects of this program and chronicles our experience which at present amounts to over 4400 infusions for over 700 patients. This program provides a high degree of assurance that a cellular therapy program can be carried out in a multisite mode involving hundreds of patients through the strict adherence to cGMP as set forth in existing regulations. It would be prudent that developers of cellular andex vivo gene therapies establish a similar cell processing and QA/QC infrastructure at an early developmental stage to optimize safety and reproducibility and facilitate regulatory review.     

Assessment of lead availability in soils contaminated by mine spoil

The uptake of lead by two contrasting plant species, radish and red fescue, grown in soils contaminated by mine spoil was investigated. Uptake was found to be poorly correlated either with pH or total Pb concentration in the soils. By contrast, a good correlation was obtained, particularly for red fescue, between Pb uptake and Pb concentration in the solution of equilibrated soil suspensions over a wide range of soil pH, total soil Pb and soil solution Pb concentration.Calculations suggested a similar order of magnitude in the total amounts of Pb taken up by the plants and Pb in the soil solution of the root zone, justifying the latter as a good index of Pb-availability. ei]Section editor: A C Borstlap     

Effect of acetic acid on astaxanthin production by Phaffia rhodozyma

Summary Low concentrations of acetic acid decreased the growth rate of and astaxanthin production by Phaffia rhodozyma on glucose, with growth completely inhibited by 2 g acetic acid/l. Using H₂SO₄ for pH control after sugar depletion caused a decline in the biomass concentration, whereas using acetic acid as titrant resulted in an increase in the biomass with a high astaxanthin content of 1430 g/g cells. An extended culture with a continuous glucose feed failed to maintain a high astaxanthin content.     

Polystomatidae (Monogenea) of Anura in southern Africa: Polystoma testimagna n. sp. parasitic in Strongylopus f. fasciatus (Smith, 1849)

Polystoma testimagna n. sp. is described as a new species of the Polystomatidae, parasitic in the urinary bladder of the striped stream frog Strongylopus f. fasciatus collected in the Vernon Crookes Nature Reserve, Natal, South Africa. Parasite prevalence was found to be 50.0% and 27.7% in two successive years, and the mean intensity was 1.5 and 1.6, respectively. The species occurs together with another Polystoma species in the same water body and within one kilometre from a third species. Aspects of host specificity are discussed and data on the ecology and distribution of the host presented.