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PubMed1982

Discovery of a high affinity of Phaseolus vulgaris agglutinin (PHA) with gastrin-secreting cells.

A lectin, Phaseolus vulgaris agglutinin (PHA), isolated from the red kidney bean, was found to have a very high affinity with gastrin-secreting cells (G cells). The unique PHA-G cell interaction is confirmed by (1) G cell reaction only with PHA, not with other lectins, and (2) PHA reaction with G cells but not with other endocrine cells. The authors concluded that gastrin-secreting cells are composed of a large quantity of galactose-N-acetyl-glucose-manose-containing glycoconjugates. The significance of this glyco-conjugate in G cells is not ascertained. The use of a PHA histochemical technic may well be suitable to study the carbohydrate (or pathophysiological) changes in G cells.

Endocrine Glands↗
PubMed1998

pha-4, an HNF-3 homolog, specifies pharyngeal organ identity in Caenorhabditis elegans.

To build complex organs, embryos have evolved mechanisms that integrate the development of cells unrelated to one another by cell type or ancestry. Here we show that the pha-4 locus establishes organ identity for the Caenorhabditis elegans pharynx. In pha-4 mutants, pharyngeal cells are transformed into ectoderm. Conversely, ectopic pha-4 expression produces excess pharyngeal cells. pha-4 encodes an HNF-3 homolog selectively expressed in the nascent digestive tract, including all pharynx precursors at the time they are restricted to a pharyngeal fate. We suggest that pha-4 is a key component of a transcription-based mechanism to endow cells with pharyngeal organ identity.

Animals↗
PubMed1981

Screening and use of high titered anti-HLA-DR sera in PHA-blast complement fixation and B-lymphocytotoxicity techniques.

Screening for anti-HLA-DR sera was performed by complement fixation on PHA stimulated peripheral blood lymphocytes (PHA-CF), or cultured B lymphoid cell lines. Out of 1,350 sera from multiparous women, multitransfused patients, and patients transfused during extra-corporal circulation (ECC), 219 contained anti-HLA-DR activity (16.2%). Anti-HLA-DR antibodies developed after ECC were often high titered (1:10 to 1:100). In half of these sera anti-HLA-A, B antibodies were weak or absent, making it possible to use then as anti-HLA-DR reagents without platelet absorption. Of the 219 positive sera 51 contained defined anti-DR antibodies (20 monospecific and 31 bi- or multispecific). The 13 best sera recognized DR1 and DR7 specificities with r values from 0.83 to 1. Twenty-four sera selected by CF were also studied by lymphocytotoxicity technique against peripheral blood B lymphocytes (B-LCT). Both PHA-CF and B-LCT techniques gave similar results, detecting the same specificities and showing comparable sensitivity. The advantages of CF are: easy storage of target cells at -80 degrees C or + 4 degrees C, and fast reading. For these reasons PHA-CF or CF on cultured B lymphoid cell lines can be proposed for large scale screening of anti-HLA-DR sera. The sera thus screened can be used for HLA-DR typing either by PHA-CF or B-LCT.

Antibodies↗
PubMed1990

Hydatidiform mole pregnancy trophoblast extracts differentially suppress interleukin-2-induced proliferation of human T-lymphocytes and PHA-blasts.

Immunoregulatory factors of trophoblast origin may partially abrogate maternal immune responses to the fetus during pregnancy. We have previously shown that soluble factors extracted from hydatidiform mole trophoblast suppress interleukin-2 (IL-2)-dependent proliferation of a cloned murine cytotoxic T cell line (CTLL-2). To characterize human T cell responses to this trophoblast extract, we measured the effects of molar tissue extracts (HME) on IL-2-stimulated proliferation of human T-lymphocytes and mitogen (PHA) transformed T-cell blasts (PHA-blasts). HME significantly (P less than 0.05) suppressed T-lymphocyte proliferation in response to 5 and 10 units/ml of IL-2 at 500 and 250 micrograms/ml, while no effect was observed at the 100 micrograms/ml concentration. Suppression by HME of IL-2-stimulated T-cell proliferation was partially overcome by the addition of excess IL-2. HME also suppressed (P less than 0.05) IL-2-stimulated proliferation of PHA-blasts at 500 and 250 micrograms/well at both 5 and 10 units/ml of IL-2. As observed with resting T-cell responses, no suppression of PHA-blast proliferation was observed using 100 micrograms/ml of HME. In contrast to the response of the resting T-cells to excess IL-2, HME suppression of IL-2-stimulated blast proliferation was not affected by increasing the concentration of IL-2. These results indicate that extracts from hydatidiform mole trophoblast contain immunosuppressive factors that block human T-cell clonal expansion by inhibiting the utilization and/or production of IL-2. Furthermore, the effects of HME are not reversed by excess IL-2 when PHA-blasts are reacted compared to resting T-cell responses, which are partially reversed in the presence of excess IL-2.(ABSTRACT TRUNCATED AT 250 WORDS)

Cell Survival↗
PubMed1997

Direct cytotoxic response of human lymphocytes to porcine PHA-lymphoblasts and lymphocytes.

Transplantation of cells and organs from pigs to human beings offers potential treatment for medical conditions such as diabetes, kidney and heart failure, and Parkinson's disease. When the antibody-mediated hyperacute rejection barrier is overcome, a xenograft may not be treated as an allograft by the human immune system. Without prior culture with porcine cells, human lymphocytes are cytotoxic to some porcine cells. Our aim was to functionally characterize this direct cytotoxic response to porcine PHA-lymphoblasts and lymphocytes. Peripheral blood mononuclear cells from seven of eight human beings were cytotoxic to porcine PHA-lymphoblasts in bulk chromium-release assays, but not to the porcine lymphocytes from which the PHA-lymphoblasts were derived. The NK cell-sensitive cell line K562 only partly blocked the response to the PHA-lymphoblasts. IL-2-expanded clones of human lymphocytes were able to discriminate between PHA-lymphoblasts from two pigs and unable to lyse K562. When using IL-2 to make the anti-porcine cells proliferate under limiting dilution conditions, the proliferation and/or function of these cells did not conform to single-hit kinetics. All the observations from experiments with cells in bulk cultures and as short-term clones suggest that the direct cytotoxic response of human lymphocytes to porcine cells is heterogeneous and composed of a small population of in vivo-activated T cells as well as NK cells.

Animals↗
PubMed1999

PhaF, a polyhydroxyalkanoate-granule-associated protein of Pseudomonas oleovorans GPo1 involved in the regulatory expression system for pha genes.

The phaC1 gene codes for the medium-chain-length polyhydroxyalkanoate (mcl PHA) synthase of Pseudomonas oleovorans GPo1, which produces mcl PHA when grown in an excess of carbon source and under nitrogen limitation. In this work, we have demonstrated, by constructing a recombinant P. oleovorans strain carrying a phaC1::lacZ reporter system, that the phaC1 gene is expressed efficiently in the presence of octanoic acid while its expression is repressed when glucose or citrate is used as the carbon source. Moreover, a P. oleovorans GPo1 mutant (strain GPG-Tc6) expressing higher levels of the reporter gene than the wild-type strain in the presence of glucose or citrate has been generated by mini-Tn5 insertional mutagenesis. Characterization of this mutant allowed us to conclude that phaF, a gene located downstream of the pha gene cluster, was knocked out in this strain. P. oleovorans GPG-Tc6 regained the ability to control phaC1 gene expression when complemented with the phaF wild-type gene. Sequencing data revealed the presence of three complete open reading frames (ORFs) in this region: ORF1 and phaI and phaF genes. The amino acid sequences of the phaI gene product and the N-terminal half of the PhaF protein showed a significant degree of similarity. Furthermore, the primary structure of the PhaF C terminus identifies this protein as a member of the histone H1-like group of proteins. Northern blot analysis showed two transcription units containing phaF, i.e., phaF and phaIF transcripts. Expression of the phaIF operon is more efficient in the presence of octanoic acid and is enhanced by the lack of the PhaF protein. In addition, it has also been demonstrated that both PhaF and PhaI proteins are bound to PHA granules produced by P. oleovorans. A model for the role of PhaF in regulating PHA synthesis is presented.

Acyltransferases↗
PubMed1999

Analyses of a polyhydroxyalkanoic acid granule-associated 16-kilodalton protein and its putative regulator in the pha locus of Paracoccus denitrificans.

The polyhydroxyalkanoic acid (PHA) granule-associated 16-kDa protein (GA16 protein) of Paracoccus denitrificans was identified, and its corresponding gene was cloned and analyzed at the molecular level. The N-terminal amino acid sequence of GA16 protein revealed that its structural gene is located downstream from the PHA synthase gene (phaCPd) cloned recently (S. Ueda, T. Yabutani, A. Maehara, and T. Yamane, J. Bacteriol. 178:774-779, 1996). Gene walking around phaCPd revealed two new open reading frames (ORFs) possibly related to PHA synthesis, one of which was the phaPPd gene, encoding GA16 protein, and the other was the phaRPd gene, encoding a protein that is putatively involved in the regulation of the expression of phaPPd. Overproduction of PhaPPd was observed in Escherichia coli carrying phaPPd, but the overproduction was not observed in the presence of phaRPd. Coexpression of phaPPd and PHA biosynthesis genes in E. coli caused increases in both the number of poly-(3-hydroxybutyric acid) (PHB) granules and PHB content and caused decreases in both the size of the granules and the molecular weight of PHB. GA16 protein was considered a phasin protein. The phaRPd gene had significant similarities to stdC, a possible transcriptional factor of Comamonas testosteroni, as well as to other ORFs of unknown function previously found in other PHA-synthetic bacteria.

Acyltransferases↗
PubMed1978

Dynamics of respiratory VT response to isocapnic pHa forcing in chemodenervated cats.

Arterial blood pH (pHa) was continuously monitored in decerebrate or pentobarbital-anesthetized cats with a rapidly responding hydrogen ion sensor inserted into the aorta. Alveolar carbon dioxide partial pressure and pHa were controlled independently during infusions of 1 N NaHCO3 or 0.5 N HCl into the inferior vena cava. Shifts in pHa up to 0.3 unit were effected isocapnically within 2.5-20 s over a working pHa range of 6.9-7.7. Before carotid sinus neurotomy, average onset latency of the tidal volume (VT) response to acid and alkaline pHa shifts was less than 5 S and the average VT response half time was less than 8.5 s regardless of whether the vagus nerves had been interrupted. After carotid sinus neurotomy, the deltaVT onset latency was approximately doubled, whereas the response half time was prolonged about eightfold on the average. Subsequent vagotomy tended further to increase the responding time lag. Nevertheless, the minimum response latency after peripheral chemodenervation was less than the deltapHa forcing rise time. It is concluded that the central chemoreceptors promptly sense pH change in the arterial blood and that neural processes adjust the time course of the respiratory response through the VT controller.

Animals↗
PubMed1977

Effects of irradiation, anti-thymocyte serum and corticosteroids on PHA and LPS responsive cells of the mouse.

Peripheral lymphocytes responsive to stimulation by phytohaemagglutinin (PHA) and bacterial lipopolysaccharide (LPS) in culture have been quantitated following treatment of mice with anti-thymocyte serum (ATS), total body irradiation or corticosteroids. The ATS reduced the number of PHA-responsive cells in both blood and spleen, and induced splenomegaly, but it had little deleterious effect on spleen-borne LPS-responsive cells. In contrast, the spleens of mice treated with hydrocortisone acetate were atrophied and the remaining cells had a reduced LPS response and an enhanced PHA response. Total body irradiation impaired both PHA and LPS responsiveness in the spleen. Recovery of PHA responsiveness after either irradiation or ATS treatment was prolonged and was dependent on the presence of an intact thymus; recovery of LPS responsiveness after corticosteroid treatment was more rapid and was thymus-independent.

Animals↗
PubMed2006

Sequence analysis and structure prediction of type II Pseudomonas sp. USM 4-55 PHA synthase and an insight into its catalytic mechanism.

BACKGROUND: Polyhydroxyalkanoates (PHA), are biodegradable polyesters derived from many microorganisms such as the pseudomonads. These polyesters are in great demand especially in the packaging industries, the medical line as well as the paint industries. The enzyme responsible in catalyzing the formation of PHA is PHA synthase. Due to the limited structural information, its functional properties including catalysis are lacking. Therefore, this study seeks to investigate the structural properties as well as its catalytic mechanism by predicting the three-dimensional (3D) model of the Type II Pseudomonas sp. USM 4-55 PHA synthase 1 (PhaC1P.sp USM 4-55). RESULTS: Sequence analysis demonstrated that PhaC1P.sp USM 4-55 lacked similarity with all known structures in databases. PSI-BLAST and HMM Superfamily analyses demonstrated that this enzyme belongs to the alpha/beta hydrolase fold family. Threading approach revealed that the most suitable template to use was the human gastric lipase (PDB ID: 1HLG). The superimposition of the predicted PhaC1P.sp USM 4-55 model with 1HLG covering 86.2% of the backbone atoms showed an RMSD of 1.15 A. The catalytic residues comprising of Cys296, Asp451 and His479 were found to be conserved and located adjacent to each other. In addition to this, an extension to the catalytic mechanism was also proposed whereby two tetrahedral intermediates were believed to form during the PHA biosynthesis. These transition state intermediates were further postulated to be stabilized by the formation of oxyanion holes. Based on the sequence analysis and the deduced model, Ser297 was postulated to contribute to the formation of the oxyanion hole. CONCLUSION: The 3D model of the core region of PhaC1P.sp USM 4-55 from residue 267 to residue 484 was developed using computational techniques and the locations of the catalytic residues were identified. Results from this study for the first time highlighted Ser297 potentially playing an important role in the enzyme's catalytic mechanism.

Acyltransferases↗
PubMed1994

The pha-4 gene is required to generate the pharyngeal primordium of Caenorhabditis elegans.

In the 4-cell Caenorhabditis elegans embryo, two blastomeres are destined to generate pharyngeal cells, each by a distinct developmental strategy: one pathway is inductive, while the other is autonomous. Here, we identify the pha-4 locus. In animals lacking pha-4 activity, an early step in pharyngeal organogenesis is blocked: no pharyngeal primordium is formed and differentiated pharyngeal cells are absent. Most other tissues are generated normally in pha-4 mutants, including cells related to pharyngeal cells by cell lineage and position. Thus, pha-4 activity is required to form the pharyngeal primordium. We propose that pha-4 marks a convergence of the inductive and autonomous pathways of pharyngeal development and suggest that establishment of pharyngeal organ identity is a crucial step for pharyngeal organogenesis.

Animals↗
PubMed1993

The essentiality of folate for the maintenance of deoxynucleotide precursor pools, DNA synthesis, and cell cycle progression in PHA-stimulated lymphocytes.

The fidelity and progression of DNA synthesis is critically dependent on the correct balance and availability of the deoxynucleoside triphosphate (dNTP) precursors for the polymerases involved in DNA replication and repair. Because folate-derived one-carbon groups are essential for the de novo synthesis of both purines and pyrimidines, the purpose of this study was to determine the effect of folate deprivation on deoxynucleotide pool levels and cell cycle progression. Primary cultures of phytohemagglutin (PHA)-stimulated splenocytes were used as the cellular model. T-cells and macrophages were purified from spleen cell suspensions obtained from F344 rats and recombined in culture. The cells were harvested after a 66-hr incubation with PHA and analyzed for nucleotide levels by reverse-phase HPLC with diode array detection. The proportion of cells in the different phases of the cell cycle was determined by bivariate flow cytometric measurement of bromodeoxyuridine (BrdU) incorporation and DNA content (propidium iodide staining). PHA-stimulated T-cells cultured in medium lacking folate and methionine manifested significant decreases in the deoxynucleotides dCTP, dTMP, dGTP, and dATP relative to cells cultured in complete medium. The reduction in dNTP pools was associated with a decrease in the corresponding ribonucleotide pools. Flow cytometric analysis revealed a 2-fold increase in S and G2/mitosis (G2/M) DNA content in PHA-stimulated cells cultured in the medium lacking folate and methionine, which suggests a delay in cell cycle progression. These alterations in DNA content were accompanied by a 5-fold decrease in BrdU incorporation relative to PHA-stimulated cells cultured in complete medium.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed2004

Disappearance of the PHA-E lectin binding site on the surface of ejaculated sperm and sperm capacitation in the dog.

Ejaculated semen and cross sections of the cauda epididymides collected from 3 normal dogs were smeared or stamped on glass slides, and the sperm on the slides were stained with 7 different FITC-lectins (Con A, DBA, GS-1, PHA-E, PSA, UEA-1, WGA) to examine the relation between sperm-binding glycoprotein derived from the canine prostate and sperm capacitation. The only lectin that stained the ejaculated sperm but not the cauda epididymal sperm was PHA-E. The sperm ejaculated by 5 other dogs were incubated for 4 hr in fluid flushed from the uterine horns or oviducts of estrous bitches, and then the percentages of actively motile sperm and hyperactivated sperm (HA-sperm) were determined. The percentages of PHA-E-labeled sperm and sperm labeled with fluoresceinated Ca indicator to assess the influx of Ca into the sperm were also evaluated. The mean percentages of actively motile sperm, HA-sperm, and Ca-labeled sperm after 4 hr of incubation in the uterine flush fluid and oviductal flush fluid were significantly higher than in control medium (P<0.05, 0.01), but the mean percentages of PHA-E-labeled sperm were lower (both P<0.01). The percentages of PHA-E-unlabeled sperm correlated with the percentages of both HA-sperm and Ca-labeled sperm (r(2)=0.787 and 0.812, respectively). The results indicate that loss of the glycoprotein secreted by the canine prostate on the sperm surface induces the influx of Ca into the sperm, and then hyperactivation of the sperm.

Acrosome Reaction↗
PubMed1975

Release of mitogenic factor by mouse lymph node cells stimulated with PHA in vitro. Inhibition of this phenomenon by the addition of thymocytes.

Thymidine uptakes of mouse lymph node cells and thymocytes in response to phytomitogen lectins were investigated in vitro. It was observed that mixtures of lymph node cells and thymocytes yielded higher 3H-TdR incorporations than expected when exposed to concanavalin A (Con A) or pokeweed mitogen (PWM), but lower incorporations than anticipated with phytohemagglutinin (PHA) as a stimulant. The observed enhancement of the response could be explained by factors which are stimulatory for thymocytes released by the lymph node cells. However, such mitogenic factors (MF) were released not only by Con A and PWM exposed cells but also by PHA expose lymph node cells. Further experiments showed that the admixture of thymocytes inhibited the release of MF by lymph node cells exposed to PHA but not to Con A or PWM. Since medullary thymocytes did not exhibit any inhibitory activity, it is likely that cortical thymocytes are responsible for this effect. The mechanism by which thymocytes inhibit MF production of lymph node cells exposed to PHA is not clear. MF was efficiently produced by lymph node cells cultured with PHA in "thymocyte-conditioned" medium and the MF activity was only marginally decreased by absorption with thymocytes. The results are discussed in the light of recent findings showing that thymocytes may both enhance and depress the immune response of other lymphocytes.

Animals↗
PubMed1975

Clonal proliferation of PHA-stimulated human lymphocytes in soft agar culture.

The purpose of this investigation was the induction of clonal proliferation of PHA-stimulated normal human lymphocytes using a two-layer soft agar technique. Essential conditions for colony formation include preceding sensitization of lymphocytes with PHA, and continuous presence of PHA in the soft agar culture. Two types of colonies developed: large colonies which appeared 3-4 days after seeding and comprised, after 5-6 days, 200-500 cells, and small colonies which were seen after 6-7 days of culture, resulting in production of 50-150 cells. Morphological study showed that all cells were blast-like and the mitotic index exceeded that in liquid medium by a factor of 50. Comparison between the number of colonies developing from cultured bone marrow and spleen cells with those from peripheral blood showed that, in proportion to the number of lymphocytes seeded, a larger number of colonies developed from bone marrow cells and a lower number of colonies developed from spleen cells. The time required for sensitization of lymphocytes in liquid medium with PHA was found to be no less than 12 hours. The greatest number of colonies appeared when the optimal concentration of PHA was placed in the lower agar layer. A linear relation between the number of cells seeded and the number of resulting colonies was found. One out of 2 X 10(3) or 3 X 10(3) lymphocytes in peripheral blood has the potential to develop as colony. The rosette-forming ability and morphological identification of the cells suggest that the colonies are composed of T lymphocytes.

Agar↗
PubMed1975

Correlations among cutaneous reactivity to DNCB, PHA-induced lymphocyte blastogenesis and peripheral blood E rosettes.

Comparisons of the results obtained in a study of fifty-two patients with genitourinary malignancies using three assays to monitor the thymus-dependent immune system (delayed cutaneous hypersensitivity to DNCB, PHA-induced lymphocyte blastogenesis, and peripheral blood E rosette-forming lymphocyte counts) yielded statistically significant positive correlations between the DNCB and PHA assays in twenty-one of twenty-eight instances, the DNCB and E rosette assays in thirteen of sixteen instances, the PHA and E rosette assays in twenty-eight of thirty-six instances, and among the DNCB, PHA and E rosette assays in ten of fourteen instances. The results suggest that both PHA-stimulated lymphocyte blastogenesis and peripheral blood E rosette-forming lymphocyte levels provide meaningful in vitro correlates of cell-mediated immunity.

Aged↗
PubMed1992

In vitro proliferative and differentiated responses of lymphoma cells to PHA and IL-4 in a patient with intermediate lymphocytic lymphoma.

We carried out in vitro B cell colony assays, with phytohemagglutinin-P (PHA-P) and IL-4, on B cells from one intermediate lymphocytic lymphoma (ILL) patient and four chronic lymphocytic leukemia (CLL) patients. Peripheral blood B cells from the ILL patient responded to PHA and IL-4, they proliferated, and differentiated into cells with plasmacytoid cell morphology. They lost the CD19 surface antigen after 10 day co-culture with PHA and IL-4. The bone marrow of this ILL patient contained atypical plasma cells with multiple nuclei. Peripheral blood B cells from the four CLL patients responded to PHA, but IL-4 did not increase PHA-induced B cell colony formation in these cells. The CLL cells did not differentiate into plasma cells, and they were clearly different from the ILL cells in morphology, as shown by scanning electron microscope examination. Since this study was performed on cells from only one ILL patient, further examination of cells from many patients might be necessary to confirm the difference between ILL and B cell-type CLL.

Antigens, Surface↗
PubMed2003

Polyhydroxyalkanoate (PHA) production from waste.

PHA (polyhydroxyalkanoate) production was attempted with SBRs from food waste. Seed microbes were collected from a sewage treatment plant with a biological nutrient removal process, and acclimated with synthetic substrate prior to the application of the fermented food waste. Laboratory SBRs were used to produce PHA with limited oxygen and nutrients. The maximum content of 51% PHA was obtained with an anaerobic/aerobic cycle with P limitation, and the yield was estimated to be about 0.05 gPHA(produced)/gCOD(applied) or 25 kg PHA/dry ton of food waste, assuming more than 40% of the PHA contents were recoverable. PHB/PHA ratios were 0.74 to 0.77 due to the higher acetate concentrations. Economical analysis seemed to suggest the PHA produced from the food waste could be an alternative material to produce the biodegradable plastic to be used for the collection bags for solid waste.

Acetates↗