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PubMed2004

Polyhydroxyalkanoate (PHA) accumulation in sulfate-reducing bacteria and identification of a class III PHA synthase (PhaEC) in Desulfococcus multivorans.

Seven strains of sulfate-reducing bacteria (SRB) were tested for the accumulation of polyhydroxyalkanoates (PHAs). During growth with benzoate Desulfonema magnum accumulated large amounts of poly(3-hydroxybutyrate) [poly(3HB)]. Desulfosarcina variabilis (during growth with benzoate), Desulfobotulus sapovorans (during growth with caproate), and Desulfobacterium autotrophicum (during growth with caproate) accumulated poly(3HB) that accounted for 20 to 43% of cell dry matter. Desulfobotulus sapovorans and Desulfobacterium autotrophicum also synthesized copolyesters consisting of 3-hydroxybutyrate and 3-hydroxyvalerate when valerate was used as the growth substrate. Desulfovibrio vulgaris and Desulfotalea psychrophila were the only SRB tested in which PHAs were not detected. When total DNA isolated from Desulfococcus multivorans and specific primers deduced from highly conserved regions of known PHA synthases (PhaC) were used, a PCR product homologous to the central region of class III PHA synthases was obtained. The complete pha locus of Desulfococcus multivorans was subsequently obtained by inverse PCR, and it contained adjacent phaE(Dm) and phaC(Dm) genes. PhaC(Dm) and PhaE(Dm) were composed of 371 and 306 amino acid residues and showed up to 49 or 23% amino acid identity to the corresponding subunits of other class III PHA synthases. Constructs of phaC(Dm) alone (pBBRMCS-2::phaC(Dm)) and of phaE(Dm)C(Dm) (pBBRMCS-2::phaE(Dm)C(Dm)) in various vectors were obtained and transferred to several strains of Escherichia coli, as well as to the PHA-negative mutants PHB(-)4 and GPp104 of Ralstonia eutropha and Pseudomonas putida, respectively. In cells of the recombinant strains harboring phaE(Dm)C(Dm) small but significant amounts (up to 1.7% of cell dry matter) of poly(3HB) and of PHA synthase activity (up to 1.5 U/mg protein) were detected. This indicated that the cloned genes encode functionally active proteins. Hybrid synthases consisting of PhaC(Dm) and PhaE of Thiococcus pfennigii or Synechocystis sp. strain PCC 6308 were also constructed and were shown to be functionally active.

Acyltransferases↗
PubMed1990

[Inhibition of PHA-stimulated T cell conditioned medium (PHA-TCM) on the growth of human myeloid progenitor cells (CFU-C)].

Cord blood T cells were enriched by nylon wool colomn, and effects of PHA-stimulated T cell supernatant collected from 18 h to 7 days on the proliferation of CFU-c were studied. The results showed that the supernatant collected at 18 h (PHA-TCM) could significantly inhibit the growth of CFU-c and the inhibition was PHA-TCM dose dependent, suggesting there is a CFU-c inhibitory activity in PHA-TCM. Kinetic studies demonstrated that the activity was decreased in the supernatant collected at 48 h and disappeared at 7 days. On the other hand, unstimulated T cell supernatant and PHA alone had no inhibitory effect on CFU-c growth. Indomethacin did not affect the production of the inhibitory activity and no interferon activity could be detected in PHA-TCM. These suggested that the inhibition was mediated by a non-interferon, non-prostaglandin suppressor. Further studies revealed that the suppressor was a protein stable at 56 degrees C and lost in pH 2 and pH 11 for 3 h, its molecular weight was large than 10,000 dolton.

Adult↗
PubMed1986

Different pathways of human T-cell activation revealed by PHA-P and PHA-M.

Antigen-specific T-cell activation is mediated via the CD3-Ti (antigen receptor) complex, and monoclonal antibodies to both CD3 and Ti cause a rapid rise in intracellular Ca2+. This calcium mobilization is not inhibited by monoclonal antibodies to CD2. The rise in calcium mobilization induced by purified PHA (PHA-P) does not occur in a cell line which lacks CD2 expression, and can be blocked in other T cells by anti-CD2 antibodies. A combination of monoclonal antibodies to different epitopes of CD2 causes calcium mobilization and mitogenesis. Reagent grade PHA (PHA-M) induces calcium moblization in cells that lack CD2, and its effects in other T cells cannot be blocked by anti-CD2 antibodies. The effects of PHA-P and PHA-M are thus mediated predominantly through different activation pathways.

Antibodies, Monoclonal↗
PubMed1989

[Effect of PHA-T cell conditioned medium (PHA-TCM) on leukemic cells].

Cord blood T cells, purified by nylon wool column, were incubated with PHA for 18 hours, and the supernatant were harvested as PHA-TCM. The results demonstrated that PHA-TCM could significantly inhibit the growth of U937 cells and reduce their 3H-TdR incorporation and spontaneous colony formation. After 3-6 days of treatment with PHA-TCM in culture, part of U937 cells became macrophage-like, latex-phagocytic, and capable of reducing NBT dye. On the contrary, HL-60 cells did not respond to PHA-TCM inhibition, indicating the selective effect of PHA-TCM.

Cell Differentiation↗
PubMed2007

Analysis of polyhydroxyalkanoate (PHA) synthase gene in activated sludge that produces PHA containing 3-hydroxy-2-methylvalerate.

The identification of phaC which encodes PHA synthase, that is involved in the formation of polyhydroxyalkanoate (PHA) containing 3-hydroxy-2-methylvalerate (3H2MV), was attempted. As of now, PHA containing 3H2MV has been reported to be produced only by mixed microbial cultures in activated sludge, but no pure bacterial cultures. A laboratory-scale activated sludge process was operated for 67 days. During the operation of the activated sludge process, its capacity to produce PHA containing 3H2MV, and the diversity of the partial phaC genes in the activated sludge microorganisms were monitored periodically. Analysis of the partial phaC genes was conducted by PCR followed by cloning and DNA sequencing, or by PCR followed by terminal-restriction fragment length polymorphism (T-RFLP). The cloning-sequencing of the 263 clones gave 11 distinct genetic groups (GGs). All of the genetic groups had similarities to known phaC higher than 48%, and one of them had similarity as high as 96% to that of Alcaligenes sp. The behavior of each of the genetic groups during the operation of the activated sludge process was monitored by the T-RFLP method. The restriction enzyme AccII, with the help of MboI, enabled the monitoring of each of the genetic groups. One of the genetic groups was found to have a strong correlation with the capability of the activated sludge to produce PHA containing 3H2MV, and its DNA sequence together with its amino acid sequence are reported.

Acyltransferases↗
PubMed2005

Expression of PHA polymerase genes of Pseudomonas putida in Escherichia coli and its effect on PHA formation.

Poly-3-hydroxyalkanoates (PHAs) are synthesized by many bacteria as intracellular storage material. The final step in PHA biosynthesis is catalyzed by two PHA polymerases (phaC) in Pseudomonas putida. The expression of these two phaC genes (phaC1 and phaC2)was studied in Escherichia coli, either under control of the native promoter or under control of an external promoter. It was found that the two phaC genes are not expressed in E. coli without an external promoter. During heterologous expression of phaC from Plac on a high copy number plasmid, a rapid reduction of the number of colony forming units was observed, especially for phaC2. It appears that the plasmid instability was partially caused by high-level production of PHA polymerase. Subsequently, tightly regulated phaC2 expression systems on a low copy number vector were applied in E. coli. This resulted in PHA yields of over 20 of total cell dry weight, which was 2 fold higher than that obtained from the system where phaC2 is present on a high copy number vector. In addition, the PHA monomer composition differed when different gene expression systems or different phaC genes were applied.

Acyltransferases↗
PubMed2000

Studies on polyhydroxyalkanoate (PHA) accumulation in a PHA synthase I-negative mutant of Burkholderia cepacia generated by homogenotization.

In the genome of Burkholderia cepacia strain IPT64, which accumulates a blend of the two homopolyesters poly(3-hydroxybutyrate), poly(3HB), and poly(3-hydroxy-4-pentenoic acid), poly(3H4PE), from sucrose or gluconate as single carbon source, the polyhydroxyalkanoate (PHA) synthase structural gene was disrupted by the insertion of a chloramphenicol-resistant gene cassette (phaC1::Cm). The suicide vector pSUP202 harboring phaC1::Cm was transferred to B. cepacia by conjugation. The inactivated gene was integrated into the chromosome of B. cepacia by homologous recombination. This mutant and also 15 N-methyl-N'-nitrosoguanidine (NMG)-induced mutants still accumulated low amounts of PHAs and expressed low PHA synthase activity. The analysis of the mutant phaC1::Cm showed that it accumulated about 1% of PHA consisting of 68.2 mol% 3HB and 31.8 mol% 3H4PE from gluconate. The wild-type, in contrast, accumulated 49.3% of PHA consisting of 96.5 mol% 3HB and 3. 5 mol% 3H4PE. Our results indicated that the genome of B. cepacia possesses at least two PHA synthase genes, which probably have different substrate specificities.

3-Hydroxybutyric Acid↗
PubMed1993

Therapy of recurrent high grade gliomas with surgery, and autologous mitogen activated IL-2 stimulated killer (MAK) lymphocytes: I. Enhancement of MAK lytic activity and cytokine production by PHA and clinical use of PHA.

Nineteen patients with recurrent high grade gliomas were treated in a phase I/II trial with aggressive debulking of the tumor, mitogen activated IL-2 stimulated peripheral blood lymphocytes (MAK cells), and rIL-2. Phytohemagglutin (PHA) was introduced into the tumor site in 16 patients prior to implanting MAK cells and IL-2 in an attempt to trigger more effective lysis of the tumor in vivo. In vitro both TNF bioactivity and cytolytic activity of long term cultured MAK (LMAK) cells were dramatically enhanced by adding PHA to the cultures of these activated PBL. Three of eleven patients (27%) had a decrease in size of the enhancing lesion on CT and/or MRI. Seven (37%) patients clinically improved. Median survival after therapy was 30 weeks. PHA was shown to be safe in vivo and more effective than IL-2 triggering enhanced effector function in vitro.

Adrenal Cortex Hormones↗
PubMed1983

Inhibitory effect of a low dose of prednisone on PHA-induced Ia antigen expression by human T cells and on proliferation of T cells stimulated with autologous PHA-T cells.

Administration of a small dose of prednisone markedly reduced (1) the PHA-induced expression of Ia antigens by T cells, (2) the stimulatory activity of Ia antigen-bearing T cells in autologous and allogeneic mixed lymphocyte reactions (MLRs), and (3) the proliferative response of T cells stimulated with autologous PHA-activated T cells or autologous or allogeneic non-T cells. The inhibitory effects of prednisone are reversible and are not detectable on T cells isolated from blood drawn 24 hr following prednisone administration. The kinetics of the prednisone-mediated inhibition of MLRs with autologous PHA-T cells is different from that of MLRs with autologous non-T cells. These data in conjunction with the information available in the literature suggest that the mechanisms underlying these two types of autologous MLRs are different.

Adult↗
PubMed2002

Enhanced accumulation and changed monomer composition in polyhydroxyalkanoate (PHA) copolyester by in vitro evolution of Aeromonas caviae PHA synthase.

By in vitro evolution experiment, we have first succeeded in acquiring higher active mutants of a synthase that is a key enzyme essential for bacterial synthesis of biodegradable polyester, polyhydroxyalkanoate (PHA). Aeromonas caviae FA440 synthase, termed PhaC(Ac), was chosen as a good target for evolution, since it can synthesize a PHA random copolyester of 3-hydroxybutyrate and 3-hydroxyhexanoate [P(3HB-co-3HHx)] that is a tough and flexible material compared to polyhydroxybutyrate (PHB) homopolyester. The in vitro enzyme evolution system consists of PCR-mediated random mutagenesis targeted to a limited region of the phaC(Ac) gene and screening mutant enzymes with higher activities based on two types of polyester accumulation system by using Escherichia coli for the synthesis of PHB (by JM109 strain) (S. Taguchi, A. Maehara, K. Takase, M. Nakahara, H. Nakamura, and Y. Doi, FEMS Microbiol. Lett. 198:65-71, 2001) and of P(3HB-co-3HHx) [by LS5218 [fadR601 atoC(Con)] strain]. The expression vector for the phaC(Ac) gene, together with monomer-supplying enzyme genes, was designed to synthesize PHB homopolyester from glucose and P(3HB-co-3HHx) copolyester from dodecanoate. Two evolved mutant enzymes, termed E2-50 and T3-11, screened through the evolution system exhibited 56 and 21% increases in activity toward 3HB-coenzyme A, respectively, and consequently led to enhanced accumulation (up to 6.5-fold content) of P(3HB-co-3HHx) in the recombinant LS5218 strains. Two single mutations in the mutants, N149S for E2-50 and D171G for T3-11, occurred at positions that are not highly conserved among the PHA synthase family. It should be noted that increases in the 3HHx fraction (up to 16 to 18 mol%) were observed for both mutants compared to the wild type (10 mol%).

Acyltransferases↗
PubMed1985

Neuroanatomical tracing by use of Phaseolus vulgaris-leucoagglutinin (PHA-L): electron microscopy of PHA-L-filled neuronal somata, dendrites, axons and axon terminals.

Following iontophoretic application of the plant lectin Phaseolus vulgaris-leucoagglutinin (PHA-L) to brain areas of rats, and subsequent immunohistochemistry, reaction product can be observed with the light microscope to fill neurons completely, including their somata, dendrites, dendritic appendages and axons. Moreover, axons often show profuse collateralization and indications of termination, including numerous en passant and terminal varicosities. The present report describes a protocol for combining light microscopic examination of PHA-L-stained neurons and electron microscopy of details of their processes, including the axonal varicosities. The results support the hypothesis that axonal varicosities are the light microscopic representations of synaptic axon terminals seen in electron microscopic preparations.

Agglutinins↗
PubMed2003

Polyhydroxyalkanoate (PHA) hemeostasis: the role of PHA synthase.

Polyhydroxyalkanoates (PHAs) are biodegradable polymers with properties of thermoplastics Production of these polymers in an economically competitive fashion via bioengineering requires an understanding of the biosynthetic pathway and its regulation. This review summarizes our current knowledge of the mechanism of the class I and III PHA synthases: the initiation, elongation and termination processes. It also summarizes our current understanding of the phase transition from soluble substrates (coenzyme A esters of beta-hydroxyalkanoates) to insoluble granules and our understanding of the requirement for a transcription factor, phasin proteins, and depolymerases in PHA homeostasis.

Acyltransferases↗
PubMed1995

Cloning, sequencing and expression in Escherichia coli of Pha a 1 and four isoforms of Pha a 5, the major allergens of canary grass pollen.

BACKGROUND: The pollen of canary grass, which was introduced as a pasture grass from Europe, is a major allergen source in the external environment of southern Australia. This study was performed to characterize the major recombinant allergens of canary grass pollen. It is anticipated that recombinant allergens may be useful in diagnosis and immunotherapy of grass pollen induced allergies. OBJECTIVE: To clone major canary grass pollen allergens and assess their nucleotide and amino acid sequence homologies with other grass pollen allergens. This sequence information may then be useful in T and B cell epitope mapping studies. METHODS: A canary grass pollen lambda gt11 cDNA expression library was constructed and screened with sera of grass-pollen-sensitive patients. IgE-reactive clones were isolated, sub-cloned into Escherichia coli, sequenced and, along with the deduced amino acid sequences, compared with other sequences in nucleotide and amino acid databases. RESULTS: One of the clones encoded the group 1 allergen of canary grass pollen, Pha a 1, with a deduced amino acid sequence identity of 88.8% with Lol p 1, from rye-grass pollen, 88.1% with Hol l 1, from velvet grass pollen and 86.6% with Phl p 1, from timothy grass pollen. The other clones (e.g. clones, 5, 14, 28, 29) encoded polymorphic forms of Pha a 5. These polymorphic forms showed between 60.6-95.5% nucleotide and 40.1-81.7% deduced amino acid sequence identities with each other. Moreover, they shared significant sequence identity with other group 5 allergens from rye-grass, timothy and Kentucky bluegrass pollens. CONCLUSIONS: Group 1 and four isoforms of group 5 allergens of canary grass pollen have been cloned and upon sequencing demonstrated strong nucleotide and amino acid sequence identities with other group 1 and 5 grass pollen allergens.

Allergens↗
PubMed1986

Peripheral blood lymphocyte response to exogenous interleukin 2 by PHA-prestimulated and non-PHA-prestimulated cells in patients with cancer.

The study aims were to assess the response of peripheral blood lymphocytes (PBL) of cancer patients to exogenous Interleukin 2 (IL 2) either by PHA-prestimulated or non PHA-prestimulated PBL, and to carry out preliminary experiments for a direct quantitative evaluation of endogenous IL 2 production by PBL cultures of cancer patients in order to define the actual role of IL 2 in the disease. Analysis of PBL subsets was also carried out with monoclonal antibodies in a selected group of patients. A total of 134 patients entered the study. Cancer sites were: larynx 32, breast 36, lung (NSC) 24, colorectal 17 and gynecologic 25. In the former 3 cancer sites staging showed localized or only locally advanced disease, and in the last 2 sites disseminated disease. Our results provided evidence that cancer patients exhibit a T-cell functional immunodepression, which progresses during tumor growth, so that the localized disease shows a low-grade defect, and advanced disease a high-grade defect. Our data also clearly suggested that the factor involved with a primary role in this functional immune impairment is the IL 2 deficiency. A perspective may be drawn on the therapeutic administration in vivo of IL 2 and IL 2-activated lymphokine-activated killer cells in controlled clinical trials of selected groups of cancer patients.

Cells, Cultured↗
PubMed1987

Interleukin 2 (IL 2) relationships with the cancer-related immunodeficiency: in vitro response to exogenous IL 2 by PHA-activated and non PHA-activated peripheral blood mononuclear cells from cancer patients.

The aims of the investigation were: 1) to determine if there are defects in interleukin 2 (IL 2) regulation either on phytohemagluttinin (PHA)-activated or non PHA-activated peripheral blood mononuclear cells (PBMC) in cancer patients, in order to ascertain the role of IL 2 in this disease, and 2) to carry out preliminary experiments for a direct quantitative evaluation of endogenous IL 2 production by PBMC cultures of cancer patients. An assessment of lymphocytes subsets was also performed with monoclonal antibodies in a selected group of patients. A total of 159 patients entered the study. Cancer sites were: larynx, 49; breast, 42; lung (NSC), 25; colorectal, 18; and gynecologic, 25. In the former 3 cancer sites, staging showed localized or only locally advanced disease and in the later 2 sites it showed disseminated disease. Our results provided evidence that the cancer patients exhibit a T cell functional immunodepression, which progresses during tumor growth, so that the localized disease shows a low-grade defect and advanced disease shows a high-grade defect. Our data also clearly suggested that the factor involved with a primary role in this functional immune impairment is the IL 2 deficiency. In our study we have not found a substantial difference of activity between recombinant and nonrecombinant IL 2, although the comparison of the relative activities of the two types of IL 2 is not easy to make, since they are expressed in different ways; however the recombinant one appeared to be slightly more active, probably for the higher purity. Our data also seem to support the perspective of the in vivo therapeutic administration of IL 2 in cancer patients.

Breast Neoplasms↗
PubMed2001

Estimation of the number of polyhydroxyalkanoate (PHA)-degraders in soil and isolation of degraders based on the method of most probable number (MPN) using PHA-film.

A new method to estimate the number of polyhydroxyalkanoates (PHA)-degraders in soil and to isolate degraders, called the film-MPN method, is proposed. The incubation time was measured by the first order reaction (FOR) model. This method was used to estimate numbers of poly(3-hydroxybutyrate-co-3-hydroxyvalerate)[P(3HB-co-3HV)]- and poly(3-hydroxyvalerate-co-4-hydroxybutyrate)[P(3HB-co-4HB)]-degraders in garden soil (4.30 x 10(5) and 2.15 x 10(5) aerobic degraders per gram of dry soil, respectively). The number of P(3HB-co-3HV)-degraders in paddy field soil was 5.06 x 10(5) aerobic degraders per gram dry soil. Also, several P(3HB-co-3HV)-degraders were isolated directly from positive-growth tubes of high dilution.

Biodegradation, Environmental↗
PubMed1976

A rapid HLA-D matching method using PHA blasts as responding cells (preliminary data on PHA blasts HLA-D typing).

Day 3-4 PHA stimulated lymphocytes were found to respond against HLA-D without any period of latency, thus allowing a discriminative MLR reading as early as 24-48 h. The test is therefore characterized by the minute number of responding cells necessary (5 X 10(3)), a one-way reaction without additional treatment of the stimualtion normal lymphocytes (5 x 10(4)), and the rapid obtaining of data. We found the preliminary data testing its concordance with classical HLA-D typing encouraging and worthy of being tested on a larger scale. Using mitogen stimulated recipient cells stored frozen, HLA-D matching with potential donors was found to be feasible within 24-48 h. The mechanism of such a prompt response by mitogen stimulated lymphocytes is unknown but appears to be independent of the nature and dose of a given mitogen.

HLA Antigens↗